2-heptylcyclopentanone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fleuramone was tested in an OECD TG 422 and the NOAEL was >= 800 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 dec 2011 - 27 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods of Testing New Chemical Substances (PFSB Notification No. 0331-7 on March 31, 2011; MIB Notification No. 5 on March 29, 2011; EPPB Notification No. 110331009)

Version / remarks:

March 2011

Deviations:

no

Principles of method if other than guideline:

An additional group of 10/sex was not mated in the control and 800 mg/kg bw/day groups Of these after completing the 42-day repeat dose administration, 5 cases each of the males and non-mating females in the control and 800 mg/kg bw/day groups were assigned to the recovery-study group to examine recovery from the toxicities through 14 days of drug administration.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)[SPF]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center of Charles River Laboratories Japan, Inc.,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old
- Weight at study initiation: males: 323 to 381 g; females: 204 to 248 g
- Fasting period before study: no
- Housing: The animals were placed individually in rearing cages covered in the front and bottom with stainless nets and reared with washable rearing machines. However, successfully mated female animals were reared from Day 18 of gestation to Day 4 of nursing period in Ekonkeji with nesting materials.
- Diet: The animals were fed freely with radiation-sterilized solid feeds (CRF-1, lot no.: 110106, Oriental Yeast).
- Water: Tap-water (Iwata City Waterworks) was taken freely as the drinking water by the animals through automatic water nozzles or the water bottles.
- Acclimation period: 8 days (after a 5 day quarantine period)

DETAILS OF FOOD AND WATER QUALITY: Analysis of contaminations in the feeds was conducted by Eurofin Scientific, verifying that they were within the acceptable range drafted by Japan Experimental Animal Feed Association, and the analysis results were retained by BSRC. Inspection of water quality of the tap water based on Water Supply Act was conducted in October 2011 by EcoPro Research Company Limited, and it was verified that the analysis results meet the water quality standards for drinking water (MHLW Ordinance No. 101 on May 30, 2003). Moreover, bacteriological tests (general bacteria and coliform tests) were conducted by BSRC in November and December 2011 and January 2012, verifying that bacteria are not detected in the water. The test results (certificate of
water quality test: No. 113689-3, bacterial tests: No. GT11-11, GT11-12, and GT12-01) were retained by BSRC. Contaminations that could have affected the study were not present in the nesting materials, feeds, and water used. Therefore, there were no changes in environmental factors with any potential impacts on the reliability of the data during the rearing period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 to 24.4
- Humidity (%): 41.0 to 60.5
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 November 2011 To: 27 December 2011

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

See repeated dose sectionDetails on route of administration
The volume of administration, 0.5 mL per 100 g of body weight was adopted, and the amounts of solutions to be administered to the males and females during the pre-mating and mating periods and during the administration period after the end of mating period were calculated based on the most recent, individually measured body weights. Furthermore, the amounts of solutions to be administered to the females during the gestation period and after delivery were calculated based on individual body weights measured on Days 0 (mating confirmation date), 7, 14, and 20 and the date of delivery completion (Day 0 of nursing period). Solutions were administered once a day, collected while being stirred with a magnetic stirrer.

- Method of formulation:
Administration solutions of 10, 40, and 160 mg/mL doses were prepared by mixing the investigational substance thoroughly in corn oil (preparation examples, 50 mg/kg bw/day group: dilute 5.0 g of the investigational substance with corn oil to create 500 mL solution using a measuring cylinder; 200 mg/kg bw/day group: dilute 20.0 g of the investigational substance with corn oil to create 500 mL solution using a measuring cylinder; 800 mg/kg bw/day group: dilute 80.0 g of the investigational substance with corn oil to create 500 mL solution using a measuring cylinder).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 11 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was housed individually with nesting materials

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the administration solutions were performed based on the analytical methods established by the “Validation of 2-Heptan-1-ylcyclopentan-1-one Concentration Measurement Methods and the Examination of its Stability in the Medium [Study No.: D642 ( 314–051 )]”. The stability of the investigational substance was verified by measuring its concentration in the samples obtained from 10 and 160 mg/mL administration solutions that were stored in low temperatures for 8 days right after preparation and then left for 1 more day at room temperature. As a result, it was verified that the investigational substance is stable since the ratio of concentration of the substance right after preparation to mean concentration (retention rate) meets the criteria (90.0 to 110.0%) with relative standard deviation of 5.0% or less. Therefore, administration solutions were stored under light-shielded conditions in low temperatures and used within 8 days of preparation. Incidentally, storage temperatures inside the storage location of the administration solutions (prefabricated low-temperature storage ch. 66, allowable range: 1 to 9˚C) ranged from 2.5 to 7.2˚C.
Analysis of concentration / homogeneity was performed to all administration solutions at the time of the first and last preparation. According to the result, all of the administration solutions met the criteria for concentration / homogeneity compared to the configured concentration (10, 40, and 160 mg/mL) (ratio to configured concentration: 90.0 to 110.0%, relative standard deviation: 5.0% or less), and the investigational substance was not detected in the solution administered to the control group. Therefore, the administration solutions were verified to have been prepared properly.

Duration of treatment / exposure:

Duration of treatment / exposure
Duration of administration to males was 42 consecutive days, including 14 days before mating, 14 days of mating period, and 14 days after the end of mating period. Duration of administration to the females in the mating group was 14 days before mating, the mating period (maximum of 11 days until successful mating), and from the gestation period to Day 4 of nursing period (with delivery completion date as Day 0) for females with confirmed mating (42 to 52 days). Administration to the females that did not deliver after mating confirmation was continued until the day before the autopsy on Day 25 of gestation (Day 24 of gestation). Duration of administration to the females in the non-mating group was consecutive 42 days. Administration to the males and females in the recovery testing groups was suspended for 14 days from the day after the final administration (Day 0 of recovery).

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12/sex in mating groups
10/sex in non-mating groups (control and 800 mg/kg bw/day); 5 of the latter in the recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: In the single-dose study of 2-Heptan-1-ylcyclopentan-1-one [study no.: D643 ( 314–052 )], there were no deaths of animals from administration of 2,000 mg/kg bw. Furthermore, a preliminary 2-week administration study [study no.: D646 ( 314–055 )] was conducted with 60, 200, 600, and 1,000 mg/kg bw/day dose levels. According the results, there were no deaths of male or female animals, and no changes attributed to administration of the investigational substance were observed in the general conditions, body weight, amount of feed intake, hematological tests, and autopsy findings. In urinalysis, squamous cell positive (1+ to 2+) was observed in 3 out of 6 cases of males in the 1,000 mg/ kg bw/day group. In blood coagulation test, prolongation of the activated partial thromboplastin time was observed in the males in 600 and 1,000 mg/kg bw/day groups. In blood biochemistry test, high values of total cholesterol and phospholipids in the males and females in 600 and 1,000 mg/kg bw/day groups and high values of γ-GTP in the males and females in the 1,000 mg/kg bw/day group were observed. Furthermore, the males in the 1,000 mg/kg bw/day group exhibited low values of neutral fat. According to the measurement of organ weights at autopsies, high liver weight values were observed in the males and females in 600 and 1,000 mg/kg bw/day groups. Moreover, high kidney weight values were observed in the males in 600 and 1,000 mg/kg bw/day groups.
Duration of administration in the main study was extended from the duration in the preliminary study, and the main study included pregnant females as subjects of administration. However, 800 mg/kg bw/day was adopted as the dose for the high-dosage group given that the changes observed in the preliminary study were minor. Doses for lower-dosage groups were set at 200 and 50 mg/kg/day through divisions by a common ratio of 4.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: 3 times daily, once a day during the recovery period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: included in FOB

BODY WEIGHT:
- Time schedule for examinations: Body weights of males and the females in the non-mating groups were measured on Days 0 (date of the start of administration), 7, 14, 21, 28, 35, 41, and 42 (date of autopsy or Day 0 of recovery) of administration, and the amount of increase in body weight from Day 0 to 41 was calculated. Body weights of the males and females in the recovery testing groups measured after the end of administration period and on Days 0, 7, 13, and 14 (autopsy day) of recovery, and the increase in body
weight from Day 0 to 13 of recovery was calculated.
Body weights of the females in the mating groups were measured on Days 0 (date of the start of administration), 7, and 14 of administration, and the increase in body weight from Day 0 to 14 of administration was calculated. Furthermore, body weights of females that mated were measured on Days 0, 7, 14, and 20 of gestation as well as on Days 0, 4, and 5 (autopsy day) of nursing period after delivery, and the increase in body weight during each period, from Day 0 to 20 of gestation and from Day 0 to 4 of nursing period, was calculated. Body weights of females whose offspring all died were measured also at
the time of the discovery of offspring deaths. Body weights of females without any natural deliveries were measured also on autopsy day on Day 25 of gestation.

FOOD CONSUMPTION:
- Feed weights for males and the females in the non-mating groups were measured on Days 0 (date of the start of administration), 7, 14, 21, 28, 35, and 41 (day before autopsy) of administration to find the feed intake from one measurement day to the next, and the mean daily feed intake was calculated. Furthermore, the cumulative feed intake between Day 0 and 14 and between Day 21 and 41 of administration for males and between Day 0 and 41 of administration for the females in the non-mating group was calculated. Feed weights for the males and females in the recovery testing groups were measured after
the end of administration period, on Days 0, 7, and 13 (day before autopsy) of recovery to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed intake from Day 0 to 13 of recovery were calculated.
Feed weights given for the females in the mating groups were measured on Days 0 (date of the start of administration), 7, and 14 of administration to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed intake from Day 0 to Day 14 of administration were calculated. In addition, feed weights for mated females were measured on Days 0, 7, 14, 18 and 20 of gestation and Days 0 and 4 of nursing period after delivery to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed intake from Day 0 to 20 of gestation were calculated.

FOOD EFFICIENCY: no

WATER CONSUMPTION : No.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: no information
- Animals fasted: No.
- How many animals: 5 mated and 5 non-mated/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: no information
- Animals fasted: No.
- How many animals: 5 mated and 5 non-mated/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: Yes, 5/sex/group (non-mating groups) at the end of the administration period and at the end of the recovery period (pH, occult blood, ketone bodies, glucose, protein, bilirubin, and urobilinogen , amounts of urine and color tones, urine electrolyte concentration (sodium, potassium, and chloride), osmotic pressure, microscopic examinations were conducted using the sediments (erythrocytes, leukocytes, squamous cells, transitional epithelial cells, renal tubular epithelial cells, casts, fat globules, mucous threads, and crystals were investigated).

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
(detailed observation of symptoms, pupillary reflex, approaching reaction, tactile response, auditory response, grip strengths of the fore and hind limbs, locomotor activity) The FOB was performed on all surviving animals. Of the observation items, detailed observation of symptoms was conducted once before grouping and once every week during administration and recovery periods, except for mated females and females that delivered offspring, to whom observations were conducted on Days 7 and 14 and on Day 4 of nursing period, respectively.
Observation of kinesthetic response to various stimuli and measurement of grip strength and locomotor activity were performed on 5 animals from each group, on Day 41 of administration to males and the females in the non-mating group and on Day 4 of nursing period to post-delivery females (in the order of the dates of delivery completion. The same were performed on the males and females in the recovery testing group on Day 41 of administration and Day 13 of recovery. Detailed observation of symptoms was performed on all animals before grouping. Observations during the administration period were started at 30 minutes after administration.

Oestrous cyclicity (parental animals):

yes

Sperm parameters (parental animals):

Parameters examined in male parental generations:
The spermatogenic cycles (stage VII or VIII) were examined using the PAS response samples in all the males in the control and 800 mg/kg bw/day groups (including recovery testing groups) and the male that resulted in gestation failure, found in the 50 mg/kg bw/day group.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
Autopsies were performed on males and the females in the non-mating groups on Day 42 of administration, the post-delivery females on Day 5 of nursing period, the females that did not deliver on Day 25 of gestation, the males and females in the recover testing groups on Day 14 of recovery, and the female whose offspring all died on the day of discovery of deaths (Day 4 of nursing period).

GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
-organ weights of Epididymides, Kidneys, Liver and Testes
-histopathology was performed according to Guiodelines; liver and kidneys of all groups were examined

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificd at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic); mortality/viability, clinical signs, body weight, sex.

GROSS NECROPSY
- Gross necropsy consisted of external examinations

Statistics:

The following statistical methods were used to analyse the data:
Bartlett’s test for equal variance was first performed. In the case of equal variance, statistical significance between the control and each administration group were tested using Dunnet’s multiple comparison test. In the case of unequal variance based on Bartlett’s test, statistical significance between the control and each administration group was tested using Steel’s test.
For birthrate, mating rate, and conception rate, x2 test was adopted.
The rate of incidence of abnormal sexual cycles, test items in detailed observation of symptoms and observation of kinaesthetic response to various stimuli other than standing, defecation, and urination, sex ratio per group, and rate of incidence of histopathological findings were tested using Fisher’s exact test.

Bartlett’s test for equal variance was conducted with 5% level of significance, and the other tests were conducted with two-tailed significance levels at 5% and 1%.

Reproductive indices:

Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups before planned necropsy / number of pups born alive) x 100
Implantation rate = (number of implantation sites / number of pregnancy corpora lutea) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Sialorrhea was sporadically observed, in 5/12, 4/12, and 3/10 cases from the males in the 800 mg/kg bw/ day group, females in the mating groups, and females in the non-mating groups, respectively. In addition, no changes in the general conditions were observed in either males or females during the recovery period. Moreover, unclean fur coat condition (around the anus / reproductive organ) was observed during the nursing period in a female in the 800 mg/kg bw/day, whose offspring all died.
There were no abnormal findings in in-cage observation items (observed lower or higher numbers of some postures were judged to be casual due to the lack of similar behaviors on other observation dates). In observations outside the cage, mild sialorrhea was observed in 3 to 5 cases in the 800 mg/kg bw/ day group since Day 16 of administration, with significantly high number of occurrences exhibited on Day 41. Although 1 case of mild sialorrhea was also observed in the 200 mg/kg bw/day on Day 23 of administration, this was judged to be a casual change given the frequency of its occurrence. There was no difference in the number of occurrences of other findings noted during observations outside the cage between the control and the groups treated with the investigational substance. In the open-field observations, the number of times the animals stood with support was significantly low before grouping in animals later assigned to the 50 mg/kg bw/day group. However, this change occurred before administration, and it was not related to administration of the investigational substance. In addition, the same group exhibited significantly low frequency of defecation on Day 41 of administration, although this was not judged to be the effect of administration of the investigational substance since this change was not observed on other measurement dates and did not relate to the dosage. There was no significant difference in the number of occurrences of other findings during the open-field observations between the control and the groups treated with the investigational substance.
In the females in the non-mating groups, there was no difference in the number of occurrences of in-cage observation items between the control and the 800 mg/kg bw/day groups. In observations outside the cage, the 800 mg/kg bw/day group exhibited significantly high frequency of “extremely easy” and significantly low frequency of “easy” as the ease of extracting the animal from the cage on Day 24 of administration, although this was not related to administration of the investigational drug since the frequency of either evaluation was similar on other observation dates. Moreover, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrences of other findings noted in observations outside the cage. In open-field observations, the frequency of standing with support was significantly high in the 800 mg/kg bw/day group on Day 8 of administration, though this was not judged to be the effect of administration of the investigational substance as this change was not observed on other measurement dates. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrence of other findings noted in open-field observations. There were no abnormal findings in any of the observation items in observations conducted during the recovery period.
In the females in the mating groups, the number of occurrences of “sitting by lowering the hip” was significantly high and the number of occurrences of “standing and sitting while paying attention to the observer” was significantly low in the 50 mg/kg bw/day group on Day 4 of nursing period, though these changes were judged to be casual due to the lack of similar behaviors on other observation dates. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in in-cage observations. In observations outside the cage, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration, and the number of occurrences on Day 4 of nursing period was significantly high. Incidentally, 1 case in the 800 mg/kg bw/day group exhibited severe sialorrhea on Day 14 of gestation. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in observations outside the cage. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of findings noted in open-field observations.

Mortality:

no mortality observed

Description (incidence):

No deaths or moribund conditions were observed in either male or female animals throughout the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences between the control and the groups treated with the investigational substance were found in males and the females in the non-mating groups on any of the measurement dates during administration and recovery periods. Although the body weight increase value higher than the control group observed in the females in the 50 mg/kg bw/day mating group during the gestation period was statistically significant, no statistically significant differences between the control group and the 200 and 800 mg/kg bw/day groups were found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, no statistically significant differences in feed intake on measurement dates during the administration period or in the cumulative feed intake between the control and the groups treated with the investigational drugs were found. During the recovery period, the mean daily intake from Day 0 to 7 of recovery was significantly higher in the 800 mg/kg bw/day group. However, this change was judged to be a casual occurrence since the difference was small and no difference in the cumulative feed intake was observed during the recovery period.
In the females in the non-mating groups, no statistically significant differences in feed intake on measurement dates during the administration period or in the cumulative food intake between the control and the 800 mg/kg bw/day groups were found. During the recovery period, the mean daily intake from Day 7 to 13 of recovery was significantly higher in the 800 mg/kg group. However, this change was judged to be a casual occurrence since the difference was small and no difference in the cumulative feed intake was observed during the recovery period. In the females in the mating groups, the mean daily feed intake on Day 0 to 7 and Day 7 to 14 of gestation and Day 0 to 4 of nursing period was significantly high in the 50 mg/kg bw/day group, with also significantly high cumulative feed intake during the gestation period. No significant differences were found between the control and the 200 and 800 mg/kg bw/day groups.

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 males, although the platelet count in the 800 mg/kg bw/day group was significantly higher than the control group at the end of administration period, this change was minor and was not judged to be the effect of administration of the investigational substance. At the end of recovery period, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance.
In the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the 800 mg/kg bw/day administration group at the end of administration period. Although the ratio of eosinophils was significantly lower in the 800 mg/kg bw/day group than the control group at the end of recovery period, this change was minor and judged to be a casual occurrence. In the females in the mating group, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance. No statistically significant differences were found between the control and the groups treated with the investigational substance in males and females or in females in the mating groups at the end of administration period and at the end of recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, the total protein, phospholipids, total bilirubin, and γ-GTP were significantly higher in the 800 mg/kg bw/day group than the control group at the end of administration period. At the end of recovery period, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance.
In the females in the non-mating groups, blood glucose, BUN, total bilirubin, and sodium were significantly lower in the 800 mg/kg bw/day group than the control group at the end of administration period. At the end of recovery period, ALP was significantly lower in the 800 mg/kg bw/day group than the control group. In the females in the mating groups, phospholipids, total cholesterol, and γ-GTP were significantly higher in the 800 mg/kg bw/day group than the control group.
In males, α2-globulin concentration was significantly higher and γ-globulin ratio and concentration significantly lower in the 800 mg/kg bw/day group than the control group at the end of administration period.
At the end of recovery period, α1-globulin concentration was significantly higher in the 800 mg/kg bw/day group than the control group.
In the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance at the end of administration period and at the end of recovery period. In the females in the mating groups, α1-globulin concentration was significantly higher in the 800 mg/kg bw/day group. In addition, the albumin ratio was significantly low in the 200 mg/kg bw/day group, though this change was casual with no relations to the dose.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males, the 800 mg/kg bw/day group exhibited a tendency of high urine volumes on the week of the end of administration compared to the control group. Furthermore, in urinary sediment tests, renal tubular epithelial cells (1+) was found 3/5 cases and 3/5 cases in the 200 and 800 mg/kg bw/day groups, respectively, and renal tubular epithelial cells (2+) was found in 2/5 cases in the 800 mg/kg bw/day group. At the end of recovery period, the concentration and total discharge volume of urine chloride were significantly lower in the 800 mg/kg bw/day group than the control group.
In the females in the non-mating groups, urine volumes were significantly higher and the urine osmotic pressure and urine sodium, urine potassium, and urine chloride concentrations were significantly lower in the 800 mg/kg bw/day group than the control on the week of the end of administration. Furthermore, in urinary sediment tests, renal tubular epithelial cells (+1) was observed in 2/5 cases in the 800 mg/kg bw/ day group. At the end of recovery period, the total urine sodium discharge volume was significantly higher in the 800 mg/kg bw/daygroup than the control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the observations of kinaesthetic response to various stimuli, there were no changes attributed to administration of the investigational substance in either males or females.
No statistically significant differences in the fore-limb and hind-limb grip strengths were found between the control and the groups treated with the investigational substance in males and females or in females in the mating groups at the end of administration period and at the end of recovery period.
In males, the amount of activity from 50 to 60 minutes after the start of measurement at the end of administration period was statistically low and the measured values from 30 to 40 minutes after the start of measurement at the end of recovery period was statistically high in the 800 mg/kg bw/day group compared to the control. However, these changes were not judged to be related to administration of the investigational substance, since no effects related to acceleration or suppression of activity were found during the observations of general conditions or the detailed observations of symptoms.
In the females in the non-mating groups and the females in the mating groups, there was no difference between the control and the groups treated with the investigational substance in the amount of locomotor activity at the end of administration period and the end of recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

As findings that were potentially related to administration of the investigational substance, hepatocellular hypertrophy in the liver was observed in 4/7 cases of males in the 800 mg/kg bw/daygroup at the end of administration period. Moreover, renal hyaline / eosinophilic globules were found as mild symptoms in 6/7, 10/11, 11/12, and 2/7 cases and as moderate symptoms in 0/7, 0/11, 1/12, and 5/7 cases in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, and a statistically significant dose-dependent aggravation of the degrees of symptoms was found.
There were no findings potentially related administration of the investigational substance in the females in the mating and non-mating groups and the female that failed gestation in the 50 mg/kg bw/day group.
In the female that did not deliver offspring in the 200 mg/kg bw/day group, endometrial hyperplasia and goblet cell metaplasia in vaginal epithelium were found.
In the dam whose offspring all died in the 800 mg/kg bw/day group, thymus atrophy, necrosis of duodenal villi, basophilic tubules, acidophilic changes, and vacuolation of the kidneys, bladder edema, and intraperitoneal hyperpigmentation of the Harderian gland were found.
In the observations of spermatogenic cycles, the number of spermatogonia, number of preleptotene spermatocytes, number of pachytene spermatocytes, and number of spermatids per number of Sertoli cells were all similar in the 800 mg/kg bw/daygroup to the control group at the end of administration period and at the end of recovery period, and no effects of administration of the investigational substance were observed. Moreover, no changes in the spermatogenic cycles were found in the male that failed gestation in the 50 mg/kg bw/day group.
All the other findings observed in the males and females at the end of administration period and at the end of recovery period were believed to be naturally occurring changes given the number of occurrences and severity of the conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The average sexual cycles were 4.0, 4.4, 4.0, and 4.2 days in the control, 50, 200, and 800 mg/kg bw/day, respectively, and while a statistically significant prolongation of cycles was found in the 50 mg/kg bw/day group compared to the control group, this was likely due to 1 incidental case with the 6-day sexual cycle and not due to the effects of administration of the investigational substance since tendencies of prolonged cycles were not observed in the 200 and 800 mg/kg bw/day groups. While abnormal sexual cycles were found in 0/12, 1/12, 1/12, and 0/12 cases in the animals in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, no statistically significant differences in the rates of incidence of abnormal sexual cycles were found. Incidentally, the animal in the 200 mg/kg bw/day group that exhibited an abnormal sexual cycle was likely experiencing a false pregnancy since its anestrous period had been continuing for 15 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the observations of spermatogenic cycles, the number of spermatogonia, number of preleptotene spermatocytes, number of pachytene spermatocytes, and number of spermatids per number of Sertoli cells were all similar in the 800 mg/kg bw/day group to the control group at the end of administration period and at the end of recovery period, and no effects of administration of the investigational substance were observed. Moreover, no changes in the spermatogenic cycles were found in the male that failed gestation in the 50 mg/kg bw/day group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating was successful in all cases, with a mating rate of 100%. Conception was unsuccessful in 1/12 couples in the 50 mg/kg bw/day group, with a conception rate of 91.7%. Conception was successful in all the other groups, with a conception rate of 100%.
In the observations of sexual cycles, the average sexual cycles were 4.0, 4.4, 4.0, and 4.2 days in the control, 50, 200, and 800 mg/kg bw/day, respectively, and while a statistically significant prolongation of cycles was found in the 50 mg/kg bw/day group compared to the control group, this was likely due to 1 incidental case with the 6-day sexual cycle and not due to the effects of administration of the investigational substance since tendencies of prolonged cycles were not observed in the 200 and 800 mg/kg bw/day groups. While abnormal sexual cycles were found in 0/12, 1/12, 1/12, and 0/12 cases in the animals in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, no statistically significant differences in the rates of incidence of abnormal sexual cycles were found. Incidentally, the animal in the 200 mg/kg bw/day group that exhibited an abnormal sexual cycle was likely experiencing a false pregnancy since its anestrous period had been continuing for 15 days.

The effects seen on liver are considered substance related, as the substance is a fatty chain which will be metabolised in the liver. The observed activation of the liver is considered an adaptive effect related to the increase in fatty acid burden in general and not specifically to this substance and therefore considered to be not adverse in nature.
The effects observed in the renal tubular cells of high dose males are not accompanied by other effects on the kidney and is likely associated with alpha hydrocarbon nephropathy. the observed increase in urinary volume did not show a dose relationship, and did not show a statistical significantly increase in the high dose group. As alpha hydrocarbon nephropathy is specific for male rats and not relevant for human risk assessment, this observation will not be considered for the NOAEL. in summary, the observed minor effects on liver and kidneys are considered either adaptive, not related to any dysfunction of these organs or species specific. therefore it is concluded that no adverse effects were observed up to and including 800 mg/kg bw/day, the highest dose tested.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

In conclusion, treatment with the substance, dosed via oral gavage at dose levels 50, 200 or 800 mg/kg bw/day resulted in a NOAEL for males and females of 800 mg/kg bw/day based on absence of adverse effects at >= 800 mg/kg bw/day. The NOAEL for reproductive toxicity: fertility and developmental toxicity is >= 800 mg/kg bw/day, since no adverse effects were observed in this OECDTG 422 test.

Executive summary:

The present summary on reproductive toxicity (fertility and developmental toxicity) the repeated dose toxicity is included.

Introduction and methods: For Fleuramone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD TG 422 was performed with rat, with dose groups 0, 50, 200 and 800 mg/kg bw/day. The control and 800 mg/kg bw/day groups also included non-mating and recovery groups. The following repeated dose toxicity parameters were recorded: mortality, clinical signs body weight and food consumption, haematology parameters and organ weights of liver, kidney, testis and epididymides. These organs were also microscopically evaluated. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analytics.

Results: Clinically: No deaths or moribund conditions were observed in either male or female animals, and there were no effects of administration of the investigational substance on body weight, amount of food intake, haematological tests, and blood coagulation tests. In terms of detailed observations based on observations of general conditions and FOB, sialorrhea (excessive salivation) was observed in males and females in the 800 mg/kg bw/day group although this change was mild and temporary, and it was not judged to be the result of toxicity from the administration of the investigational substance.

In urinalysis, males and females in the 800 mg/kg bw/day group exhibited increase or tendency of increase in the amount of urine at the end of the administration period with tubular epithelial cells found in the urinary sediments and considered to be related to the substance. In addition, tubular epithelial cells in the urinary sediments were also found in males in the 200 mg/kg bw/day group, although this was not judged to be caused definitely by the administration of the substance due to the lack of other changes.

Haematology:No effects were seen.

Blood biochemistry: Blood biochemistry tests of males and females in the 800 mg/kg bw/day group suggested effects on increased liver function: increase in phospholipids (23; 50%); in cholesterol (30; 44%) increase in gamma GTP (50; 200%) in males and females, respectively. The protein alpa2-globuline was increased in males only up to 22%. At 200 mg/kg bw these effects were not seen (or only to a very slight degree).

Organ liver effects: Males and females in the 800 mg/kg bw/day group exhibited relative liver weights increases of 37 and 25%, in males and females, respectively. In the 200 mg/kg bw group in males this increase was 8%. In histopathological examinations, hypertrophy of hepatocytes was observed in the males. The effects seen on liver are considered substance related, as the substance has an alkyl chain which will be metabolised as a fatty acid. The effect is therefore considered an adaptive effect and not considered to be not adverse in nature.

Organ kidney effects: The organ weights of the kidneys were increased to 20 and 14%, in males and females, respectively. In the renal tubular cells of high dose males eosinophilic droplets were seen. In combination with the higher alpha2-globulin increase in males these are considered related to the alpha-hydrocarbon nephropathy, being a male rat specific phenomenon. The tubular epithelial cells in the urine are also anticipated to be related to this effect. The observed increase in urinary volume did not show a dose relationship, and did not show a statistical significant increase in the high dose group.

Other organ effects including reproductive toxicity organs: No other effects were observed.

Reproductive toxicity-Fertility: In reproductive capacity tests, there were no effects of administration of the investigational substance on average sexual cycles, rate of incidence of abnormal sexual cycles, coitus ability, and fertility. Abnormal delivery conditions were not observed in any of the administration groups at observations during deliveries. No effects of administration of the investigational substance on the gestation period, corpus luteum count, number of implantation sites, implantation rate and rate of deliveries were observed in the dams.

Reproductive toxicity-Developmental toxicity: In terms of the offspring, there were no effects of administration of the investigational substance on the number of offspring, birth rate, sex ratio, body weight on Day 0 and Day 4 of nursing period, number of surviving offspring, and rate of surviving offspring after nursing period, and no abnormalities related to administration of the investigational substance were observed in external inspections of the new borns or autopsies on Day 4 of the nursing period. Based on the absence of adverse effects on reproductive and developmental parameters, the NOAEL for reproductive toxicity and for developmental toxicity is >=800 mg/kg bw/day.

Summary of the effects and the derivation of the NOAEL in the OECD TG 422. The observed effects on liver are considered to be adaptive in nature. The 800 mg/kg bw is considered to be the close maximum tolerable dose considering the metabolic capacity of the liver as the substance is readily absorbed and metabolised. The absence of liver effect after the recovery period supports this. The effects on the kidneys are considered adaptive too, not related to any dysfunction of this organs or are considered to be specific to male rats.

Systemic effects: Therefore it is concluded that no adverse systemic effects were observed >= 800 mg/kg bw/day

Reproductive toxicity, fertility and developmental: Based on the absence of adverse effects at 800 mg/kg bw/day, the NOAEL for fertility and developmental toxicity is >= 800 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

One repeated dose toxicity study with reproduction/developmental toxicity screening study is available, conducted according to OECD guideline 422 in compliance with GLP, resulting in adequate information for the endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The executive summary below is a copy from the repeated dose toxicity summary.

Introduction and methods: For Fleuramone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD TG 422 was performed with rat, with dose groups 0, 50, 200 and 800 mg/kg bw/day. The control and 800 mg/kg bw/day groups also included non-mating and recovery groups. The following repeated dose toxicity parameters were recorded: mortality, clinical signs body weight and food consumption, haematology parameters and organ weights of liver, kidney, testis and epididymides. These organs were also microscopically evaluated. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analytics.

Results: Clinically: No deaths or moribund conditions were observed in either male or female animals, and there were no effects of administration of the investigational substance on body weight, amount of food intake, haematological tests, and blood coagulation tests. In terms of detailed observations based on observations of general conditions and FOB, sialorrhea (excessive salivation) was observed in males and females in the 800 mg/kg bw/day group although this change was mild and temporary, and it was not judged to be the result of toxicity from the administration of the investigational substance.

In urinalysis, males and females in the 800 mg/kg bw/day group exhibited increase or tendency of increase in the amount of urine at the end of the administration period with tubular epithelial cells found in the urinary sediments and considered to be related to the substance. In addition, tubular epithelial cells in the urinary sediments were also found in males in the 200 mg/kg bw/day group, although this was not judged to be caused definitely by the administration of the substance due to the lack of other changes.

Haematology:No effects were seen.

Blood biochemistry: Blood biochemistry parameters of males and females in the 800 mg/kg bw/day group show effects on increased liver function: increase in phospholipids (23; 50%); in cholesterol (30; 44%) increase in gamma GTP (50; 200%) in males and females, respectively. The protein alpa2-globuline was only increased in males up to 22%. At 200 mg/kg bw these effects were not seen (or only to a very slight degree).

Organ liver effects: Males and females in the 800 mg/kg bw/day group exhibited relative liver weights increases of 37 and 25%, in males and females, respectively. In the 200 mg/kg bw group in males this increase was 8%. In histopathological examinations, hypertrophy of hepatocytes was observed in the males. The effects seen on liver are considered substance related, as the substance has an alkyl chain which will be metabolised as a fatty acid. The effect is therefore considered an adaptive effect and not considered adverse in nature.

Organ kidney effects: The organ weights of the kidneys were increased to 20 and 14%, in males and females, respectively. In the renal tubular cells of high dose males eosinophilic droplets were seen. In combination with the higher alpha2-globulin increase in males these are considered related to the alpha-hydrocarbon nephropathy, being a male rat specific phenomenon. The tubular epithelial cells in the urine are also anticipated to be related to this effect too. The observed increase in urinary volume did not show a dose relationship, and did not show a statistical significant increase in the high dose group.

Other organ effects including reproductive toxicity organs: No other effects were observed.

Reproductive toxicity-Fertility: In reproductive capacity tests, there were no effects of administration of the investigational substance on average sexual cycles, rate of incidence of abnormal sexual cycles, coitus ability, and fertility. Abnormal delivery conditions were not observed in any of the administration groups at observations during deliveries. No effects of administration of the investigational substance on the gestation period, corpus luteum count, number of implantation sites, implantation rate and rate of deliveries were observed in the dams.

Reproductive toxicity-Developmental toxicity: In terms of the offspring, there were no effects of administration of the investigational substance on the number of offspring, birth rate, sex ratio, body weight on Day 0 and Day 4 of nursing period, number of surviving offspring, and rate of surviving offspring after nursing period, and no abnormalities related to administration of the investigational substance were observed in external inspections of the new borns or autopsies on Day 4 of the nursing period. Based on the absence of adverse effects on reproductive and developmental parameters, the NOAEL for reproductive toxicity and for developmental toxicity is >=800 mg/kg bw/day.

Summary of the effects and the derivation of the NOAEL in the OECD TG 422. The observed effects on liver are considered to be adaptive in nature. The 800 mg/kg bw is considered to be the close maximum tolerable dose considering the metabolic capacity of the liver as the substance is readily absorbed and metabolised. The absence of liver effect after the recovery period supports this. The effects on the kidneys are considered adaptive too, not related to any dysfunction of this organs or are considered to be specific to male rats.

Systemic effects: Therefore it is concluded that no adverse systemic effects were observed >= 800 mg/kg bw/day

Reproductive toxicity, fertility and developmental: Based on the absence of adverse effects at 800 mg/kg bw/day, the NOAEL for fertility and developmental toxicity is >= 800 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

NOAEL = 800 mg/kg bw/day, the highest dose tested 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 dec 2011 - 27 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods of Testing New Chemical Substances (PFSB Notification No. 0331-7 on March 31, 2011; MIB Notification No. 5 on March 29, 2011; EPPB Notification No. 110331009)

Version / remarks:

March 2011

Deviations:

no

Principles of method if other than guideline:

An additional group of 10/sex was not mated in the control and 800 mg/kg bw/day groups Of these after completing the 42-day repeat dose administration, 5 cases each of the males and non-mating females in the control and 800 mg/kg bw/day groups were assigned to the recovery-study group to examine recovery from the toxicities through 14 days of drug administration.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)[SPF]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center of Charles River Laboratories Japan, Inc.,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old
- Weight at study initiation: males: 323 to 381 g; females: 204 to 248 g
- Fasting period before study: no
- Housing: The animals were placed individually in rearing cages covered in the front and bottom with stainless nets and reared with washable rearing machines. However, successfully mated female animals were reared from Day 18 of gestation to Day 4 of nursing period in Ekonkeji with nesting materials.
- Diet: The animals were fed freely with radiation-sterilized solid feeds (CRF-1, lot no.: 110106, Oriental Yeast).
- Water: Tap-water (Iwata City Waterworks) was taken freely as the drinking water by the animals through automatic water nozzles or the water bottles.
- Acclimation period: 8 days (after a 5 day quarantine period)

DETAILS OF FOOD AND WATER QUALITY: Analysis of contaminations in the feeds was conducted by Eurofin Scientific, verifying that they were within the acceptable range drafted by Japan Experimental Animal Feed Association, and the analysis results were retained by BSRC. Inspection of water quality of the tap water based on Water Supply Act was conducted in October 2011 by EcoPro Research Company Limited, and it was verified that the analysis results meet the water quality standards for drinking water (MHLW Ordinance No. 101 on May 30, 2003). Moreover, bacteriological tests (general bacteria and coliform tests) were conducted by BSRC in November and December 2011 and January 2012, verifying that bacteria are not detected in the water. The test results (certificate of water quality test: No. 113689-3, bacterial tests: No. GT11-11, GT11-12, and GT12-01) were retained by BSRC. Contaminations that could have affected the study were not present in the nesting materials, feeds, and water used. Therefore, there were no changes in environmental factors with any potential impacts on the reliability of the data during the rearing period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 to 24.4
- Humidity (%): 41.0 to 60.5
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 November 2011 To: 27 December 2011

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The volume of administration, 0.5 mL per 100 g of body weight was adopted, and the amounts of solutions to be administered to the males and females during the pre-mating and mating periods and during the administration period after the end of mating period were calculated based on the most recent, individually measured body weights. Furthermore, the amounts of solutions to be administered to the females during the gestation period and after delivery were calculated based on individual body weights measured on Days 0 (mating confirmation date), 7, 14, and 20 and the date of delivery completion (Day 0 of nursing period). Solutions were administered once a day, collected while being stirred with a magnetic stirrer.

- Method of formulation:
Administration solutions of 10, 40, and 160 mg/mL doses were prepared by mixing the investigational substance thoroughly in corn oil (preparation examples, 50 mg/kg bw/day group: dilute 5.0 g of the in vestigational substance with corn oil to create 500 mL solution using a measuring cylinder; 200 mg/kg bw/ day group: dilute 20.0 g of the investigational substance with corn oil to create 500 mL solution using a measuring cylinder; 800 mg/kg bw/day group: dilute 80.0 g of the investigational substance with corn oil to create 500 mL solution using a measuring cylinder).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the administration solutions were performed based on the analytical methods established by the “Validation of 2-Heptan-1-ylcyclopentan-1-one Concentration Measurement Methods and the Examination of its Stability in the Medium [Study No.: D642 ( 314–051 )]”. The stability of the investigational substance was verified by measuring its concentration in the samples obtained from 10 and 160 mg/mL administration solutions that were stored in low temperatures for 8 days right after preparation and then left for 1 more day at room temperature. As a result, it was verified that the investigational substance is stable since the ratio of concentration of the substance right after preparation to mean concentration (retention rate) meets the criteria (90.0 to 110.0%) with relative standard deviation of 5.0% or less. Therefore, administration solutions were stored under light-shielded conditions in low temperatures and used within 8 days of preparation. Incidentally, storage temperatures inside the storage location of the administration solutions (prefabricated low-temperature storage ch. 66, allowable range: 1 to 9˚C) ranged from 2.5 to 7.2˚C.
Analysis of concentration / homogeneity was performed to all administration solutions at the time of the first and last preparation. According to the result, all of the administration solutions met the criteria for concentration / homogeneity compared to the configured concentration (10, 40, and 160 mg/mL) (ratio to configured concentration: 90.0 to 110.0%, relative standard deviation: 5.0% or less), and the investigational substance was not detected in the solution administered to the control group. Therefore, the administration solutions were verified to have been prepared properly.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): females were housed individually with nesting materials

Duration of treatment / exposure:

Duration of treatment / exposure
Duration of administration to males was 42 consecutive days, including 14 days before mating, 14 days of mating period, and 14 days after the end of mating period. Duration of administration to the females in the mating group was 14 days before mating, the mating period (maximum of 11 days until successful mating), and from the gestation period to Day 4 of nursing period (with delivery completion date as Day 0) for females with confirmed mating (42 to 52 days). Administration to the females that did not deliver after mating confirmation was continued until the day before the autopsy on Day 25 of gestation (Day 24 of gestation). Duration of administration to the females in the non-mating group was consecutive 42 days. Administration to the males and females in the recovery testing groups was suspended for 14 days from the day after the final administration (Day 0 of recovery).

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12/sex in mating groups
10/sex in non-mating groups (control and 800 mg/kg bw/day); 5 of the latter in the recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: IIn the single-dose study of 2-Heptan-1-ylcyclopentan-1-one [study no.: D643 ( 314–052 )], there were no deaths of animals from administration of 2,000 mg/kg bw. Furthermore, a preliminary 2-week administration study [study no.: D646 ( 314–055 )] was conducted with 60, 200, 600, and 1,000 mg/kg bw/day dose levels. According the results, there were no deaths of male or female animals, and no changes attributed to administration of the investigational substance were observed in the general conditions, body weight, amount of feed intake, hematological tests, and autopsy findings. In urinalysis, squamous cell positive (1+ to 2+) was observed in 3 out of 6 cases of males in the 1,000 mg/ kg bw/day group. In blood coagulation test, prolongation of the activated partial thromboplastin time was observed in the males in 600 and 1,000 mg/kg bw/day groups. In blood biochemistry test, high values of total cholesterol and phospholipids in the males and females in 600
and 1,000 mg/kg bw/day groups and high values of γ-GTP in the males and females in the 1,000 mg/ kg bw/day group were observed. Furthermore, the males in the 1,000 mg/kg bw/day group exhibited low values of neutral fat. According to the measurement of organ weights at autopsies, high liver weight values were observed in the males and females in 600 and 1,000 mg/kg bw/day groups. Moreover, high kidney weight values were observed in the males in 600 and 1,000 mg/kg bw/day groups. Duration of administration in the main study was extended from the duration in the preliminary study, and the main study included pregnant females as subjects of administration. However, 800 mg/kg bw/day was adopted as the dose for the high-dosage group given that the changes observed in the preliminary study were minor. Doses for lower-dosage groups were set at 200 and 50 mg/kg/day through divisions by a common ratio of 4.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: 3 times daily, once a day during the recovery period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: included in FOB

BODY WEIGHT:
- Time schedule for examinations: Body weights of males and the females in the non-mating groups were measured on Days 0 (date of the start of administration), 7, 14, 21, 28, 35, 41, and 42 (date of autopsy or Day 0 of recovery) of administration, and the amount of increase in body weight from Day 0 to 41 was calculated. Body weights of the males and females in the recovery testing groups measured after the end of administration period and on Days 0, 7, 13, and 14 (autopsy day) of recovery, and the increase in body weight from Day 0 to 13 of recovery was calculated. Body weights of the females in the mating groups were measured on Days 0 (date of the start of administration), 7, and 14 of administration, and the increase in body weight from Day 0 to 14 of administration was calculated. Furthermore, body weights of females that mated were measured on Days 0, 7, 14, and 20 of gestation as well as on Days 0, 4, and 5 (autopsy day) of nursing period after delivery, and the increase in body weight during each period, from Day 0 to 20 of gestation and from Day 0 to 4 of nursing period, was calculated. Body weights of females whose offspring all died were measured also at the time of the discovery of offspring deaths. Body weights of females without any natural deliveries were measured also on autopsy day on Day 25 of gestation.

FOOD CONSUMPTION:
- Feed weights for males and the females in the non-mating groups were measured on Days 0 (date of the start of administration), 7, 14, 21, 28, 35, and 41 (day before autopsy) of administration to find the feed intake from one measurement day to the next, and the mean daily feed intake was calculated. Furthermore, the cumulative feed intake between Day 0 and 14 and between Day 21 and 41 of administration for males and between Day 0 and 41 of administration for the females in the non-mating group was calculated. Feed weights for the males and females in the recovery testing groups were measured after the end of administration period, on Days 0, 7, and 13 (day before autopsy) of recovery to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed in take from Day 0 to 13 of recovery were calculated. Feed weights given for the females in the mating groups were measured on Days 0 (date of the start of administration), 7, and 14 of administration to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed intake from Day 0 to Day 14 of administration were calculated. In addition, feed weights for mated females were measured on Days 0, 7, 14, 18 and 20 of gestation and Days 0 and 4 of nursing period after delivery to find the feed intake from one measurement day to the next, and the mean daily feed intake and the cumulative feed intake from Day 0 to 20 of gestation were calculated.

FOOD EFFICIENCY: no

WATER CONSUMPTION : No.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: no information
- Animals fasted: No.
- How many animals: 5 mated and 5 non-mated/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: no information
- Animals fasted: No.
- How many animals: 5 mated and 5 non-mated/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: Yes, 5/sex/group (non-mating groups) at the end of the administration period and at the end of the recovery period (pH, occult blood, ketone bodies, glucose, protein, bilirubin, and urobilinogen , amounts of urine and color tones, urine electrolyte concentration (sodium, potassium, and chloride), osmotic pressure, microscopic examinations were conducted using the sediments (erythrocytes, leukocytes, squamous cells, transitional epithelial cells, renal tubular epithelial cells, casts, fat globules, mucous threads, and crystals were investigated)

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
(detailed observation of symptoms, pupillary reflex, approaching reaction, tactile response, auditory response, grip strengths of the fore and hind limbs, locomotor activity)
The FOB was performed on all surviving animals. Of the observation items, detailed observation of symptoms was conducted once before grouping and once every week during administration and recovery periods, except for mated females and females that delivered offspring, to whom observations were conducted on Days 7 and 14 and on Day 4 of nursing period, respectively.
Observation of kinesthetic response to various stimuli and measurement of grip strength and locomotor activity were performed on 5 animals from each group, on Day 41 of administration to males and the females in the non-mating group and on Day 4 of nursing period to post-delivery females (in the order of the dates of delivery completion. The same were performed on the males and females in the recovery testing group on Day 41 of administration and Day 13 of recovery.
Detailed observation of symptoms was performed on all animals before grouping. Observations during the administration period were started at 30 minutes after administration.

SACRIFICE
Autopsies were performed on males and the females in the non-mating groups on Day 42 of administration, the post-delivery females on Day 5 of nursing period, the females that did not deliver on Day 25 of gestation, the males and females in the recover testing groups on Day 14 of recovery, and the female whose offspring all died on the day of discovery of deaths (Day 4 of nursing period).

GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
-histopathology was performed according to Guidelines; liver and kidneys of all groups were examined.
-organ weights: Epididymides, Kidneys, Liver and Testes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All offspring killed or dying prior to scheduled termination, and of those killed at the end of the study were subjected to detailed necropsy.

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data:
1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For comparisons involving two groups only t-tests were used, for all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For comparisons involving two groups only, Wilcoxon’s rank sum tests (Wilcoxon 1945) were used. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length estimates an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.

Indices:

REPRODUCTIVE INDICES:
Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100

OFFSPRING VIABILITY INDICES
Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100
Percentage of males : Number of males in litter/ Total number of offspring in litter x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sialorrhea was sporadically observed in 4/12, and 3/10 cases from the females in the mating groups, and females in the non-mating groups, respectively. In addition, no changes in the general conditions were observed in females during the recovery period. Moreover, unclean fur coat condition (around the anus / reproductive organ) was observed during the nursing period in a female in the 800 mg/kg bw/day, whose offspring all died.
There were no abnormal findings in in-cage observation items (observed lower or higher numbers of some postures were judged to be casual due to the lack of similar behaviors on other observation dates). In observations outside the cage, mild sialorrhea was observed in 3 to 5 cases in the 800 mg/kg bw/ day group since Day 16 of administration, with significantly high number of occurrences exhibited on Day 41. Although 1 case of mild sialorrhea was also observed in the 200 mg/kg bw/day on Day 23 of administration, this was judged to be a casual change given the frequency of its occurrence. There was no difference in the number of occurrences of other findings noted during observations outside the cage between the control and the groups treated with the investigational substance. In the open-field observations, the number of times the animals stood with support was significantly low before grouping in animals later assigned to the 50 mg/kg bw/day group. However, this change occurred before administration, and it was not related to administration of the investigational substance. In addition, the same group exhibited significantly low frequency of defecation on Day 41 of administration, although this was not judged to be the effect of administration of the investigational substance since this change was not observed on other measurement dates and did not relate to the dosage. There was no significant difference in the number of occurrences of other findings during the open-field observations between the control and the groups treated with the investigational substance. In the females in the non-mating groups, there was no difference in the number of occurrences of in-cage observation items between the control and the 800 mg/kg bw/day groups. In observations outside the cage, the 800 mg/kg bw/day group exhibited significantly high frequency of “extremely easy” and significantly low frequency of “easy” as the ease of extracting the animal from the cage on Day 24 of administration, although this was not related to administration of the investigational drug since the frequency of either evaluation was similar on other observation dates. Moreover, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrences of other findings noted in observations outside the cage. In open-field observations, the frequency of standing with support was significantly high in the 800 mg/kg bw/day group on Day 8 of administration, though this was not judged to be the effect of administration of the investigational substance as this change was not observed on other measurement dates. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrence of other findings noted in open-field observations. There were no abnormal findings in any of the observation items in observations conducted during the recovery period. In the females in the mating groups, the number of occurrences of “sitting by lowering the hip” was significantly high and the number of occurrences of “standing and sitting while paying attention to the observer” was significantly low in the 50 mg/kg bw/day group on Day 4 of nursing period, though these changes were judged to be casual due to the lack of similar behaviors on other observation dates. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in in-cage observations. In observations outside the cage, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration, and the number of occurrences on Day 4 of nursing period was significantly high. Incidentally, 1 case in the 800 mg/kg bw/day group exhibited severe sialorrhea on Day 14 of gestation. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in observations outside the cage. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of findings noted in open-field observations.

Mortality:

no mortality observed

Description (incidence):

No deaths or moribund conditions were observed in either male or female animals throughout the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences between the control and the groups treated with the investigational substance were found in the females in the non-mating groups on any of the measurement dates during administration and recovery periods. Although the body weight increase value higher than the control group observed in the females in the 50 mg/kg bw/day mating group during the gestation period was statistically significant, no statistically significant differences between the control group and the 200 and 800 mg/kg bw/day groups were found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the females in the non-mating groups, no statistically significant differences in feed intake on measure ment dates during the administration period or in the cumulative food intake between the control and the 800 mg/kg bw/day groups were found. During the recovery period, the mean daily intake from Day 7 to 13 of recovery was significantly higher in the 800 mg/kg group. However, this change was judged to be a casual occurrence since the difference was small and no difference in the cumulative feed intake was observed during the recovery period. In the females in the mating groups, the mean daily feed intake on Day 0 to 7 and Day 7 to 14 of gestation and Day 0 to 4 of nursing period was significantly high in the 50 mg/kg bw/day group, with also significantly high cumulative feed intake during the gestation period. No significant differences were found between the control and the 200 and 800 mg/kg bw/day groups.

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 the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the 800 mg/kg bw/day administration group at the end of administration period. Although the ratio of eosinophils was significantly lower in the 800 mg/kg bw/day group than the control group at the end of recovery period, this change was minor and judged to be a casual occurrence. In the females in the mating group, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance. No statistically significant differences were found between the control and the groups treated with the investigational substance in males and females or in females in the mating groups at the end of administration period and at the end of recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the females in the non-mating groups, blood glucose, BUN, total bilirubin, and sodium were significantly lower in the 800 mg/kg bw/day group than the control group at the end of administration period. At the end of recovery period, ALP was significantly lower in the 800 mg/kg bw/day group than the control group. In the females in the mating groups, phospholipids, total cholesterol, and γ-GTP were significantly higher in the 800 mg/kg bw/day group than the control group.
In the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance at the end of administration period and at the end of recovery period. In the females in the mating groups, α1-globulin concentration was significantly higher in the 800 mg/kg bw/day group. In addition, the albumin ratio was significantly low in the 200 mg/kg bw/day group, though this change was casual with no relations to the dose.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In the females in the non-mating groups, urine volumes were significantly higher and the urine osmotic pressure and urine sodium, urine potassium, and urine chloride concentrations were significantly lower in the 800 mg/kg bw/day group than the control on the week of the end of administration. Furthermore, in urinary sediment tests, renal tubular epithelial cells (+1) was observed in 2/5 cases in the 800 mg/kg bw/ day group. At the end of recovery period, the total urine sodium discharge volume was significantly higher in the 800 mg/kg bw/daygroup than the control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the females in the non-mating groups, there was no difference in the number of occurrences of in-cage observation items between the control and the 800 mg/kg bw/day groups. In observations outside the cage, the 800 mg/kg bw/day group exhibited significantly high frequency of “extremely easy” and significantly low frequency of “easy” as the ease of extracting the animal from the cage on Day 24 of administration, although this was not related to administration of the investigational drug since the frequency of either evaluation was similar on other observation dates. Moreover, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrences of other findings noted in observations outside the cage. In open-field observations, the frequency of standing with support was significantly high in the 800 mg/kg bw/day group on Day 8 of administration, though this was not judged to be the effect of administration of the investigational substance as this change was not observed on other measurement dates. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrence of other findings noted in open-field observations. There were no abnormal findings in any of the observation items in observations conducted during the recovery period.
In the females in the mating groups, the number of occurrences of “sitting by lowering the hip” was significantly high and the number of occurrences of “standing and sitting while paying attention to the observer” was significantly low in the 50 mg/kg bw/day group on Day 4 of nursing period, though these changes were judged to be casual due to the lack of similar behaviors on other observation dates. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in in-cage observations. In observations outside the cage, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration, and the number of occurrences on Day 4 of nursing period was significantly high. Incidentally, 1 case in the 800 mg/kg bw/day group exhibited severe sialorrhea on Day 14 of gestation. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in observations outside the cage. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of findings noted in open-field observations In the observations of kinaesthetic response to various stimuli, there were no changes attributed to a dministration of the investigational substance in females.
No statistically significant differences in the fore-limb and hind-limb grip strengths were found between the control and the groups treated with the investigational substance in females or in females in the mating groups at the end of administration period and at the end of recovery period.
In the females in the non-mating groups and the females in the mating groups, there was no difference between the control and the groups treated with the investigational substance in the amount of locomotor activity at the end of administration period and the end of recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the females in the non-mating groups, the absolute and relative liver weights and the relative kidney weights were significantly high in the 800 mg/kg bw/day group at the end of administration period. The absolute heart weights were significantly low and the relative pituitary gland and liver weights were significantly high in the 800 mg/kg group at the end of recovery period, though these changes were minor. In the females in the mating group, the absolute and relative liver, kidney, and ovary weights were significantly high in the 800 mg/kg bw/day group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the females in the non-mating groups, black spots in the stomach, accentuated lobular patterns and red spots in the liver, renal cysts and scarring, and white spots in the adrenal glands were found at the end of administration period. At the end of recovery period, hepatodiaphragmatic nodules and renal scarring were found.
In the females in the mating groups, gastric nodules and white spots, diverticula of small intestine, brown spots and white spots in the liver, ascites, renal cysts and scarring, ovarian cysts, fallopian cysts, and vaginal nodules were found. Any of these findings were either found in the control group as well or occurred as singular instances, and were not related to administration of the investigational substance. In addition, 2 cases of females that did not deliver offspring in the 200 mg/kg bw/day were both found to have enlarged vaginal cavity. The dam in the 800 mg/kg bw/day group whose offspring all died was found to have thymus atrophy, dilated proventricular cavity, and enlarged and pale kidneys, and it was emaciated. There were no abnormal findings in the female that failed gestation in the 50 mg/kg bw/day group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no findings potentially related administration of the investigational substance in the females in the mating and non-mating groups and the female that failed gestation in the 50 mg/kg bw/day group. In the female that did not deliver offspring in the 200 mg/kg bw/day group, endometrial hyperplasia and goblet cell metaplasia in vaginal epithelium were found.
In the dam whose offspring all died in the 800 mg/kg bw/day group, thymus atrophy, necrosis of duodenal villi, basophilic tubules, acidophilic changes, and vacuolation of the kidneys, bladder edema, and intraperitoneal hyperpigmentation of the Harderian gland were found.
All the other findings observed in the females at the end of administration period and at the end of recovery period were believed to be naturally occurring changes given the number of occurrences and severity of the conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, 2 cases were dissected without delivery confirmation by Day 25 of gestation, although both cases were found in the autopsies to be pregnant with some fetal resorption. Therefore, the birthrate in the 200 mg/kg bw/day group was 83.3%. No abnormalities in the delivery conditions were observed in the other groups, with a birthrate of 100%. the duration of gestation was almost the same across the groups.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, 2 cases were dissected without delivery confirmation by Day 25 of gestation, although both cases were found in the autopsies to be pregnant with some fetal resorption. Therefore, the birthrate in the 200 mg/kg bw/day group was 83.3%. No abnormalities in the delivery conditions were observed in the other groups, with a birthrate of 100%. the duration of gestation was almost the same across the groups.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

In the 800 mg/kg bw/day group, there was 1 case of dam whose offspring all died on Day of nursing period. During the nursing period, 1, 0, 1, and 1 case of cannibalized offspring and 5, 5, 8, and 17 cases of dead offspring were observed in the control, 50, 200, and 800 mg/kg groups, respectively, although there was no inter-group difference in the mean number of occurrences. There was no inter-group differences in the rate of offspring survival on Day 0 of nursing period and the rate of survival of nursed offspring on Day 4 of nursing period.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The duration of gestation was almost the same across the groups.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Details on maternal toxic effects:


CLINICAL SIGNS:
Sialorrhea was sporadically observed in 4/12, and 3/10 cases from females in the mating groups, and females in the non-mating groups, respectively. In addition, no changes in the general conditions were observed in females during the recovery
period. Moreover, unclean fur coat condition (around the anus / reproductive organ) was observed during the nursing period in a female in the 800 mg/kg bw/day, whose offspring all died.

BODY WEIGHT:
No statistically significant differences between the control and the groups treated with the investigational substance were found in the females in the non-mating groups on any of the measurement dates during administration and recovery periods.
Although the body weight increase value higher than the control group observed in the females in the 50 mg/kg bw/day mating group during the gestation period was statistically significant, no statistically significant differences between the control group and the 200 and 800 mg/kg bw/day groups were found.

FOOD CONSUMPTION:
In the females in the non-mating groups, no statistically significant differences in feed intake on measure ment dates during the administration period or in the cumulative food intake between the control and the 800 mg/kg bw/day groups were found. During the recovery period, the mean daily intake from Day 7 to 13 of recovery was significantly higher in the 800 mg/kg group. However, this change was judged to be a casual occurrence since the difference was small and no difference in the cumulative feed intake was observed during the recovery period.
In the females in the mating groups, the mean daily feed intake on Day 0 to 7 and Day 7 to 14 of gestation and Day 0 to 4 of nursing period was significantly high in the 50 mg/kg bw/day group, with also significantly high cumulative feed intake during the gestation period. No significant differences were found between the control and the 200 and 800 mg/kg bw/day groups.

HAEMATOLOGICAL FINDINGS:
In the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the 800 mg/kg bw/day administration group at the end of administration period. Although the ratio of eosinophils was significantly lower in the 800 mg/kg bw/day group than the control group at the end of recovery period, this change was minor and judged to be a casual occurrence.
In the females in the mating group, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance. No statistically significant differences were found between the control and the groups treated with the investigational substance in males and females or in females in the mating groups at the end of administration period and at the end of recovery period.

CLINICAL BIOCHEMISTRY FINDINGS:
In the females in the non-mating groups, blood glucose, BUN, total bilirubin, and sodium were significantly lower in the 800 mg/kg bw/day group than the control group at the end of administration period. At the end of recovery period, ALP was significantly lower in the 800 mg/kg bw/day group than the control group.
In the females in the mating groups, phospholipids, total cholesterol, and γ-GTP were significantly higher in the 800 mg/kg bw/day group than the control group.
In the females in the non-mating groups, there was no statistically significant difference in any of the test items between the control and the groups treated with the investigational substance at the end of administration period and at the end of recovery period.
In the females in the mating groups, α1-globulin concentration was significantly higher in the 800 mg/kg bw/day group. In addition, the albumin ratio was significantly low in the 200 mg/kg bw/day group, though this change was casual with no relations to the dose.

URINALYSIS:
In the females in the non-mating groups, urine volumes were significantly higher and the urine osmotic pressure and urine sodium, urine potassium, and urine chloride concentrations were significantly lower in the 800 mg/kg bw/day group than the control on the week of the end of administration. Furthermore, in urinary sediment tests, renal tubular epithelial cells (+1) was observed in 2/5 cases in the 800 mg/kg bw/ day group. At the end of recovery period, the total urine sodium discharge volume was significantly higher in the 800 mg/kg bw/daygroup than the control group.

BEHAVIOUR (FUNCTIONAL FINDINGS):
In the females in the non-mating groups, there was no difference in the number of occurrences of in-cage observation items between the control and the 800 mg/kg bw/day groups. In observations outside the cage, the 800 mg/kg bw/day group exhibited significantly high frequency of “extremely easy” and significantly low frequency of “easy” as the ease of extracting the animal from the cage on Day 24 of administration, although this was not related to administration of the investigational drug since the frequency of either evaluation was similar on other observation dates. Moreover, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrences of other findings noted in observations outside the cage. In open-field observations, the frequency of standing with support was significantly high in the 800 mg/kg bw/day group on Day 8 of administration, though this was not judged to be the effect of administration of the investigational substance as this change was not observed on other measurement dates. There was no significant difference between the control and the 800 mg/kg bw/day group in the number of occurrence of other findings noted in open-field observations.
There were no abnormal findings in any of the observation items in observations conducted during the recovery period. In the females in the mating groups, the number of occurrences of “sitting by lowering the hip” was significantly high and the number of occurrences of “standing and sitting while paying attention to the observer” was significantly low in the 50 mg/kg bw/day group on Day 4 of nursing period, though these changes were judged to be casual due to the lack of similar behaviors on other observation dates. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in in-cage observations. In observations
outside the cage, mild sialorrhea was observed in 1 to 4 cases in the 800 mg/kg bw/day group since Day 8 of administration, and the number of occurrences on Day 4 of nursing period was significantly high. Incidentally, 1 case in the 800 mg/kg bw/day group exhibited severe sialorrhea on Day 14 of gestation. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of other findings noted in observations outside the cage. There was no significant difference between the control and the groups treated with the investigational substance in the number of occurrences of findings noted in open-field observations In the observations of kinaesthetic response to various stimuli, there were no changes attributed to administration of the investigational substance in females. No statistically significant differences in the fore-limb and hind-limb grip strengths were found between the
control and the groups treated with the investigational substance in females or in females in the mating groups at the end of administration period and at the end of recovery period.
In the females in the non-mating groups and the females in the mating groups, there was no difference between the control and the groups treated with the investigational substance in the amount of locomotor
activity at the end of administration period and the end of recovery period.

ORGAN WEIGHT:
In the females in the non-mating groups, the absolute and relative liver weights and the relative kidney weights were significantly high in the 800 mg/kg bw/day group at the end of administration period. The absolute heart weights were significantly low and the relative pituitary gland and liver weights were significantly high in the 800 mg/kg group at the end of recovery period, though these changes were minor.
In the females in the mating group, the absolute and relative liver, kidney, and ovary weights were significantly high in the 800 mg/kg bw/day group.

GROSS PATHOLOGY:
In the females in the non-mating groups, black spots in the stomach, accentuated lobular patterns and red spots in the liver, renal cysts and scarring, and white spots in the adrenal glands were found at the end of administration period. At the end of recovery period, hepatodiaphragmatic nodules and renal scarring were found.
In the females in the mating groups, gastric nodules and white spots, diverticula of small intestine, brown spots and white spots in the liver, ascites, renal cysts and scarring, ovarian cysts, fallopian cysts, and vaginal nodules were found.
Any of these findings were either found in the control group as well or occurred as singular instances, and were not related to administration of the investigational substance.
In addition, 2 cases of females that did not deliver offspring in the 200 mg/kg bw/day were both found to have enlarged vaginal cavity. The dam in the 800 mg/kg bw/day group whose offspring all died was found to have thymus atrophy, dilated proventricular cavity, and enlarged and pale kidneys, and it was emaciated. There were no abnormal findings in the male and female that failed gestation in the 50 mg/kg bw/day group.

HISTOPATHOLOGY:
There were no findings potentially related administration of the investigational substance in the females in the mating and non-mating groups and the female that failed gestation in the 50 mg/kg bw/day group.
In the female that did not deliver offspring in the 200 mg/kg bw/day group, endometrial hyperplasia and goblet cell metaplasia in vaginal epithelium were found.
In the dam whose offspring all died in the 800 mg/kg bw/day group, thymus atrophy, necrosis of duodenal villi, basophilic tubules, acidophilic changes, and vacuolation of the kidneys, bladder edema, and intraperitoneal hyperpigmentation of the Harderian gland were found.
All the other findings observed in the females at the end of administration period and at the end of recovery period were believed to be naturally occurring changes given the number of occurrences and severity of the conditions.



REPRODUCTIVE FUNCTION (ESTROUS CYCLE) AND REPRODUCTIVE PERFORMANCE:
Mating was successful in all cases, with a mating rate of 100%. Conception was unsuccessful in 1/12 couples in the 50 mg/kg bw/day group, with a conception rate of 91.7%. Conception was successful in all the other groups, with a conception rate of 100%. In the observations of sexual cycles, the average sexual cycles were 4.0, 4.4, 4.0, and 4.2 days in the control, 50, 200, and 800 mg/kg bw/day, respectively, and while a statistically significant prolongation of cycles was found in the 50 mg/kg bw/day group compared to the control group, this was likely due to 1 incidental case with the 6-day sexual cycle and not due to the effects of administration of the investigational substance since tendencies of prolonged cycles were not observed in the 200 and 800 mg/kg bw/day groups. While abnormal sexual cycles were found in 0/12, 1/12, 1/12, and 0/12 cases in the animals in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, no statistically significant differences in the rates of incidence of abnormal sexual cycles were found. Incidentally, the animal in the 200 mg/kg bw/day group that exhibited an abnormal sexual cycle was likely experiencing a false pregnancy since its anestrous period had been continuing for 15 days.
In the 200 mg/kg bw/day group, 2 cases were dissected without delivery confirmation by Day 25 of gestation, although both cases were found in the autopsies to be pregnant with some fetal resorption. Therefore, the birthrate in the 200 mg/kg bw/day group was 83.3%. No abnormalities in the delivery conditions were observed in the other groups, with a birthrate of 100%. In addition, the duration of gestation was almost the same across the groups In the 50 mg/kg bw/day group, the number of implantation sites was significantly higher than the control group, with a tendency of higher number of offspring. Incidentally, there were no differences in the number of implantation sites and number of offspring between the 200 and 800 mg/kg bw/day groups. There were no differences in the corpus luteum count, number of live-birth offspring on Day 0 of nursing period, and number of surviving offspring on Day 4 of nursing period between the control and the groups treated with the investigational substance, and no inter-group differences in the birthrate, implantation rate, delivery rate, and sex ratio were found. In the 800 mg/kg bw/day group, there was 1 case of dam whose offspring all died on Day of nursing period. During the nursing period, 1, 0, 1, and 1 case of cannibalized offspring and 5, 5, 8, and 17 cases of dead offspring were observed in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, although there was no inter-group difference in the mean number of occurrences. There were no inter-group differences in the rate of offspring survival on Day 0 of nursing period and the rate of survival of nursed offspring on Day 4 of nursing period.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

no maternal abnormalities were observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No differences in the body weights of newborns on Day 0 and of the offspring on Day of nursing period were found in either males or females between the control and the groups treated with the investigational substance.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No differences in the body weights of newborns on Day 0 and of the offspring on Day of nursing period were found in either males or females between the control and the groups treated with the investigational substance.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The birthrate in the 200 mg/kg bw/day group was 83.3%. No abnormalities in the delivery conditions were observed in the other groups, with a birthrate of 100%. In addition, the duration of gestation was almost the same across the groups.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, 2 cases were dissected without delivery confirmation by Day 25 of gestation, although both cases were found in the autopsies to be pregnant with some fetal resorption. Therefore, the birthrate in the 200 mg/kg bw/day group was 83.3%. No abnormalities in the delivery conditions were observed in the other groups, with a birthrate of 100%. In addition, the duration of gestation was almost the same across the groups. In the 50 mg/kg bw/day group, the number of implantation sites was significantly higher than the control group, with a tendency of higher number of offspring.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in the number of live-birth offspring on Day 0 of nursing period, and number of surviving offspring on Day 4 of nursing period between the control and the groups treated with the investigational substance, and no inter-group differences in the birthrate, implantation rate, delivery rate, and sex ratio were found.
In the 800 mg/kg bw/day group, there was 1 case of dam whose offspring all died on Day of nursing period. During the nursing period, 1, 0, 1, and 1 case of cannibalized offspring and 5, 5, 8, and 17 cases of dead offspring were observed in the control, 50, 200, and 800 mg/kg bw/day groups, respectively, although there was no inter-group difference in the mean number of occurrences. There no inter-group differences in the rate of offspring survival on Day 0 of nursing period and the rate of survival of nursed offspring on Day 4 of nursing period.

External malformations:

no effects observed

Description (incidence and severity):

In the external inspections of the newborns, no abnormalities were observed in any of the administration groups, including the control. In the observations of general conditions in the surviving offspring during the nursing period, animals with smaller frames were observed in 2 cases of males and 1 case of female in the 50 mg/kg bw/day group, 1 case of male in the 200 mg/kg bw/day group, and 1 case of female in the 800 mg/kg bw/day group, although these were likely naturally occurring differences given that they occurred in a small number of cases. There were no other changes in the general conditions of the surviving offspring. In the autopsies of the dead offspring during the nursing period, 1 case of visceral in a female in the 50 mg/kg bw/day group, 1 case of thymus remnants in the neck in a male in the 200 mg/kg bw/day group, and 1 case of lack of one kidney in a female in the 800 mg/kg bw/day group were found, although relations of these conditions to death could not be determined as they were all naturally occurring conditions. No abnormal findings were found in the other dead offspring. There were no findings attributed to the effects of the investigational substance in either males or females in the autopsies on Day 4 of nursing period. As findings, brown spots in the lungs, nodules, red spots, white spots, yellow spots, and yellowing of the livers, red spots and pyelectasis in the kidneys, and skin nodules were observed sporadically in males. In females, 1 case of pyelectasis was observed in the 800 mg/kg bw/day group.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

no adverse effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

no adverse effects observed up to and including 800 mg/kg bw/day, the highest dose tested

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, treatment with the substance, dosed via oral gavage at dose levels 50, 200 or 800 mg/kg bw/day resulted in a NOAEL for males and females of 800 mg/kg bw/day based on absence of adverse effects at 800 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity is at least 800 mg/kg bw/day, the highest dose tested..

Executive summary:

Introduction and methods: For Fleuramone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD TG 422 was performed with rat, with dose groups 0, 50, 200 and 800 mg/kg bw/day. The control and 800 mg/kg bw/day groups also included non-mating and recovery groups.The following repeated dose toxicity parameters were recorded: mortality, clinical signs body weight and food consumption, haematology parameters and organ weights of liver, kidney, testis and epididymides. These organs were also microscopically evaluated. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analytics.

Results: No deaths or moribund conditions were observed in either male or female animals, and there were no effects of administration of the investigational substance on body weight, amount of food intake, hematological tests, and blood coagulation tests. In terms of detailed observations based on observations of general conditions and FOB, sialorrhea was observed in females in the 800 mg/kg bw/day group although this change was mild and temporary, and it was not judged to be the result of toxicity from the administration of the investigational substance. In urinalysis, females in the 800 mg/kg bw/day group exhibited increase or tendency of increase in the amount of urine at the end of the administration period with tubular epithelial cells found in the urinary sediments, which was interpreted as the effect of the administration of the investigational substance. However, no clear evidence of effects on kidneys was found in histopathological tests, and this toxicity effect was mild. In pathological examinations, females in the 800 mg/kg bw/day group exhibited high liver weight values at the end of the administration period. Furthermore, blood biochemistry tests of females in the 800 mg/kg bw/day group suggested effects on the liver function. In addition, females in the 800 mg/kg bw/day group exhibited high kidney weight values, and although no findings related to weight changes were present in the histopathological examinations, some kind of effects from the administration of the investigational substance on the kidneys were suspected. At the end of the recovery period, no effects on liver or kidneys from the administration of the investigational substance were found, which suggested that recovery is achieved by suspending administration of the drug.

 

The effects seen on liver are considered substance related, as the substance is a fatty acid chain which will be metabolised in the liver. The observed activation of the liver is considered an adaptive effect related to the increase in fatty acid burden in general and not specifically to this substance and therefore considered to be not adverse in nature.

The observed increase in uruinary volume did not show a dose relationship and was not accompanied by histopathological changes in female kidneys. In summary, the observed minor effects on liver and kidneys are considered either adaptive and not related to any dysfunction of these organs. Therefore it is concuded that no adverse effects were observed up to and including 800 mg/kg bw/day, the highest dose tested.

Based on the absence of adverse effects at 800 mg/kg bw/day, the NOAEL for systemic effects/parental toxicity in this study is at least 800 mg/kg bw/day, the highest dose tested.

In reproductive capacity tests, there were no effects of administration of the investigational substance on average sexual cycles, rate of incidence of abnormal sexual cycles, coitus ability, and fertility. Abnormal delivery conditions were not observed in any of the administration groups at observations during deliveries. No effects of administration of the investigational substance on the gestation period, corpus luteum count, number of implantation sites, implantation rate and rate of deliveries were observed in the dams.

In terms of the offspring, there were no effects of administration of the investigational substance on the number of offspring, birthrate, sex ratio, body weight on Day 0 and Day 4 of nursing period, number of surviving offspring, and rate of surviving offspring after nursing period, and no abnormalities related to administration of the investigational substance were observed in external inspections of the newborns or autopsies on Day 4 of the nursing period. Based on the absence af adverse effects on reproductive and developmental parameters, the NOAEL for reproductive toxicity and for developmental toxicity is 800 mg/kg bw/day, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

One repeated dose toxicity study with reproduction/developmental toxicity screening study is available, conducted according to OECD guideline 422 in compliance with GLP, resulting in adequate information for the endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The executive summary for developmental toxicity is included in the repeated dose toxicity summary, which is copied into the fertility section and is not repeated in the present section.

Justification for classification or non-classification

The NOAEL of the substance is >=800 mg/kg bw/day for both reproduction toxicity and developmental toxicity, therefore, classification of is not warranted according to EU CLP ((EC) No. 1272/2008 and its updates).