2-(2-(4-methyl-3-cyclohexen-1-yl)propyl)cyclopentanone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 2014 to 13 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

There were no deviations from standard operating procedures that affected the integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity stzdy in rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Molecular formula:

C14H24O1

IUPAC Name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Constituent 2

Reference substance name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Constituent 3

Reference substance name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Constituent 4

Reference substance name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Test material form:

liquid

Specific details on test material used for the study:

Substance name (as stated in the report): NECTARYL
Description: Clear colourless liquid (determined at WIL Research Europe B.V.)
Batch: VE00318744
Stable under storage conditions until: 14 March 2015 (expiry date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality)

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany
- Total number of animals: 30 males, 30 females (females were nulliparous and non-pregnant).
- Age at start of treatment: Approximately 6 weeks
- Identification: Earmark and tattoo
- Randomization: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
- Acclimatization period: At least 5 days before the start of treatment under laboratory conditions
- Health inspection: Upon receipt of the animals

- Animal husbandry conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study
- Accommodation: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage- enrichment or bedding material.
- Diet: Free access to prepared diets. During the acclimatization and recovery period, animals had free access to standard pelleted rodent diet (SM R/M- Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours
- Water: Free access to tap water except during motor activity measurements

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Oral, by inclusion in the diet (ad libitum)

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- Dietary Inclusion Levels. The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test substance intake was estimated based on the body weight and food consumption value

Analytical verification of doses or concentrations:

yes

Remarks:

once during the study

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the pretreatment and/or treatment phase, according to a validated method.
Samples of diet preparations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of diet preparations was considered acceptable if the mean measured concentrations are 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

- Duration of treatment: At least 28 days. Animals received test diet up to the day prior to necropsy.
- Duration of recovery: At least 14 days.

Frequency of treatment:

Ad libitum in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Group 1 main (5 males, 5 females)

Doses / concentrations 2

Dose / conc.:

0 ppm

Remarks:

Group 1 recovery (5 males, 5 females)

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

Group 2 main (5 males, 5 females)

Doses / concentrations 4

Dose / conc.:

3 000 ppm

Remarks:

Group 3 main (5 males, 5 females)

Doses / concentrations 5

Dose / conc.:

10 000 ppm

Remarks:

Group 4 main (5 males, 5 females)

Doses / concentrations 6

Dose / conc.:

10 000 ppm

Remarks:

Group 4 recovery (5 males, 5 females)

No. of animals per sex per dose:

5 males and 5 females per group / 1 dose level per group
Additional 5 males and 5 females in control group (group 1) and high dose group (group 4) to allow for a recovery period of 14 days after end of treatment period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose levels: Based on the results of a 10-day range finding study, the dose levels for this 28-day oral gavage study were selected to be 0, 1000, 3000 and 10.000 ppm.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

Functional Observations The following tests were performed:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R) and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent): all Recovery Group 1 and 4 animals in Week 4.
- fore- and hind-limb grip strength (GRIP) were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands (Score 0 = normal/present, score 1 = abnormal/absent): all Recovery Group 1 and 4 animals and all Main Group 2 and 3 animals* in Week 4, and all Recovery Group 1 and 4 animals* during the last week of the recovery period.
- motor activity test (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA)): all Recovery Group 1 and 4 and Main Group 2 and 3 animals in Week 4. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
* Since a treatment-related effect on grip strength was suspected in Week 4.

Body weights: Weekly.

Food consumption: Weekly.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

Pathology:
- Necropsy: On the scheduled day of necropsy, animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands)
- Organ weights: The organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy.
- Histotechnology: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
- Histopathology: The following slides were examined by a pathologist:
- All tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- Kidney and thyroid gland of all male animals of Groups 2 and 3 and recovery group male animals, based on (possible) treatment-related changes in these organs,
- All gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross
observations with microscopic findings.
Histopathology was subjected to a peer review.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or abnormalities during weekly arena observations were noted during the observation period.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain of males and females at 3000 and 10,000 ppm was reduced throughout treatment (and a statistically significant reduction in body weight was recorded for females at 10,000 ppm from Week 3 onwards, and at 3000 ppm in Week 4 (Day 22), and for males at 10,000 ppm in Week 2), achieving a level of statistical significance on most occasions.

During the recovery period, body weight and body weight gain remained statistically significantly lower for females at 10,000 ppm. However, weight gain compared to Day 1 of the recovery period was similar to control levels (but statistically significantly lower when compared to day 1 of the treatment phase). Therefore, it was considered that no treatment-related effect on weight gain was present during the recovery phase.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was considered to have been unaffected by treatment.
The higher relative food intake of males at 10,000 ppm over Weeks 2-3 and of females at 10,000 ppm over Weeks 3-4 of the treatment period was due to the lower body weight, and not due to a primary effect on food intake.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in haematology parameters occurred.

The changes in red blood cell parameters (higher red blood cell distribution width, and lower haemoglobin and haematocrit level in males at 10,000 ppm, and lower mean corpuscular haemoglobin concentration in females at 3000 and 10,000 ppm) at the end of treatment were considered not to be related to treatment. These changes were very slight in nature (i.e. means remained within the range considered normal for rats of this age and strain), occurred in the absence of a clear dose-related trend, did not result in a treatment-related change in red blood cell counts, occurred in the absence of any supportive histopathological changes, and were absent at the end of the recovery period.
Other statistically significant changes in haematology parameters were also considered to be unrelated to treatment as these occurred in the absence of a dose-related trend, were absent at the end of the treatment period, and remained within the range considered normal for rats of this age and strain.

The lower prothrombin time in males at 10,000 ppm at the end of treatment was considered to be of no toxicological relevance since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity, and since this change had recovered to levels similar to those seen in control animals.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters were considered to be related to treatment:
- Lower total protein level in females at 10,000 ppm,
- Higher creatinine level in males at 10,000 ppm,
- Higher cholesterol level in males and females at 10,000 ppm (not statistically significant for
females),
- Higher potassium level in males at 3000 and 10,000 ppm.

At the end of the recovery period, these changes had recovered to levels similar to those seen in control animals.
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend, were absent at the end of the treatment period and/or remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The statistically significantly higher fore- and hindlimb grip strength of males at 1000 and 3000 ppm and of females at 1000 ppm at the end of treatment was considered unrelated to treatment since no dose-related trend was apparent.
Motor activity ambulation counts of females at 10,000 ppm appeared lower than control counts at the end of treatment. Given that the control mean was considered slightly high based on data expected from rats of the same age and strain, and the mean at 10,000 ppm was within this range considered normal, it was considered that there was no treatment-related effect on motor activity. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment at the end of the treatment period:
- Higher absolute and relative liver weights in males at 3000 and 10,000 ppm and females at 10,000 ppm (not statistically significant for absolute liver weights of males at 3000 ppm). At 10,000 ppm, relative liver weights were increased with approximately 45% and 33% for males and females, respectively. At 3000 ppm, relative liver weights were increased with approximately 17%.
- Higher relative thyroid weights in males at 10,000 ppm; relative thyroid weights were increased with approximately 20%). It was considered not adverse in nature (see histopathology).
- Higher absolute and relative kidney weights in males at 3000 and 10,000 ppm (not statistically significant for absolute kidney weights of males); relative kidney weights were increased with approximately 12% and 13% at 3000 and 10,000 ppm, respectively.

At the end of the recovery period, absolute and relative liver weights remained higher for males at 10,000 ppm (not statistically significant for absolute weights of males).
The statistically significant higher relative liver weight of females at 10,000 ppm at the end of the recovery period was attributed to the lower terminal body weights, and absolute liver weights of these females were lower instead of higher than controls. It was therefore considered that liver weights of females at the end of the recovery period were unaffected.
Other statistically significant changes in absolute and relative organ weights were considered unrelated to treatment as these occurred in the absence of a dose-related trend, were attributed to the lower terminal body weights and/or remained within the range considered normal for rats of this age and strain.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates.
These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings consisted of:
-Thyroid gland: an increased incidence in minimal follicular cell hypertrophy/hyperplasia in males treated at 10,000 ppm. This finding was still present at the end of the recovery period. Given the slight severity of this finding, this was considered not to be related to the slightly higher relative thyroid weights recorded for males at 10,000 ppm at the end of treatment, as a more pronounced histopathological lesion would be expected to occur with a thyroid weight change. As such, the follicular cell hypertrophy/hyperplasia and higher thyroid weight were considered not adverse in nature.
- Kidneys: a dose-dependent increased incidence and severity in hyaline droplet accumulation in all treated males.
At the end of the recovery period, these lesions showed no recovery (thyroid) or partial recovery (kidneys).
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mean test article intake over the study period was as follows:

Group Nominal dietary Average intake [mg test substance/kg body weight]
inclusion level (range indicated within brackets)
[ppm] males females

2 1000 93 (84-105) 89 (85-96)
3 3000 285 (260-313) 274 (265-282)
4 10,000 981 (938-1062) 901 (889-938)

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: (corresponding to 285 and 274 mg NECTARYL/kg body weight/day, for males and females respectively)

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Adverse Effect Level (NOAEL) for NECTARYL of 3000 ppm was established (corresponding to 285 and 274 mg NECTARYL/kg body weight/day, for males and females respectively) in this dietary 28d repeated dose toxicity study.

Executive summary:

The Repeated dose 28-day oral toxicity study with NECTARYL by dietary administration in the rat, followed by a 14-day recovery period was performed in 2014 according to the OECD Guideline No. 407.

Based on the results of a 10-day range finding study (Project 505867; See APPENDIX 5), the dose levels for this 28-day oral gavage study were selected to be 0, 1000, 3000 and 10.000 ppm.

The test substance, was administered daily for 28 days by dietary administration to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.

Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4 and grip strength also at the end of the recovery period; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. Dietary analyses confirmed that diets were prepared accurately and homogenously.

At the end of treatment, liver weights were increased with approximately 45% and 33% for males and females, respectively, and with approximately 17% at 3000 ppm in males (relative to body weight). Liver weights remained higher at the end of the recovery period for males at 10,000 ppm. Several clinical biochemistry changes were recorded that may be related to altered liver function including lower total protein level in females at 10,000 ppm, and higher cholesterol level in males and females at 10,000 ppm. Although there was no microscopic evidence of any degenerative changes suggestive of hepatocytotoxicity, the magnitude of change in liver weight at 10,000 ppm was considered adverse in nature.

Other changes that were considered to be related to treatment (lower body weight/body weight gain of both sexes at 3000 and 10,000 ppm, and clinical biochemistry changes consisting of higher creatinine and potassium level in males at 10,000 ppm (and for potassium level also at 3000 ppm), lower total protein level in females at 10,000 ppm and higher cholesterol level in males and females at 10,000 ppm) were considered not adverse in nature.

The histopathological changes in male thyroid (increased incidence in minimal follicular cell hypertrophy/hyperplasia) and kidneys (increased incidence and severity in hyaline droplet accumulation) at 10000 ppm at the end of the treatment and recovery period were not considered to be adverse. This was based on the mild (increased) severity, the absence of accompanying degenerative changes and the fact that both findings can be observed as background finding at low incidence and severity. Moreover, it is known that hyaline droplet accumulation in the rat kidney, representing alpha2µglobulin, is not relevant for humans. Weight changes of these organs (which were reversible within 14-days) were considered unrelated to the accompanying histopathological change in these organs, since a more pronounced histopathological change would be expected to occur with concurrent organ weight changes. As such, these organ weight changes were considered not adverse in nature.

Adverse Effect Level (NOAEL) for NECTARYL of 3000 ppm was established (corresponding to 285 and 274 mg NECTARYL/kg body weight/day, for males and females respectively).