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Description of key information

Combined 28-Day Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of FRESKOMENTHE by Dietary Administration in Rats test dated on 2017 and performed according to the OECD Guideline No. 422

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Appearance: Colourless to yellow liquid
Batch: VE00478017
Purity/Composition: See Certificate of Analysis (Appendix 3)
Test item storage: At room temperature protected from light
Stable under storage conditions until: 09 May 2019 (expiry date)
Additional information
Test Facility test item number: 207284/C
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil.
Chemical name (IUPAC, synonym or trade name): 2-(1-Methylpropyl)Cyclohexanone
CAS number: 14765-30-1
Molecular formula: C10H18O
Volatile: Vapour pressure: 0.08 hPa at 20.0°C
Specific gravity / density:
0.9159 (20/20 ̊C)
0.9139 (20/4 ̊C)
0.9129 (25/25 ̊C)

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis was provided to the Test Facility and is presented in Appendix 3.

Reserve Samples
For each batch (lot) of Test Item, a reserve sample (approximately 0.6 grams) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item(s) were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item was discarded. 
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat: Crl:WI(Han) (outbred, SPF-Quality). Untreated, nulliparous, non-pregnant females and untreated
males will be used at the initiation of the study.
Sex:
male/female
Details on test animals and environmental conditions:
This species and strain of rat has been recognized as appropriate for general and reproduction toxic
ity studies. Charles River Den Bosch has general and reproduction/developmental historical data in
this species from the same strain and source. This animal model has been proven to be susceptible to
the effects of reproductive toxicants.
48 females and 40 males.
Acclimatisation: At least 5 days prior to start of pretest (females) or treatment (males).
Environmental controls for the animal room are 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. The light/dark cycle may
be interrupted for study related activities. Any variations to these conditions will be evaluated and
maintained in the raw data.
Route of administration:
oral: feed
Details on route of administration:
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The test item will be mixed without the use of a vehicle, directly with the required amount of powder feed. No correction will be made for the purity/composition of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses will be conducted on a single occasion during the treatment phase on samples as specified below, according to a validated method (Test Facility Study No. 507072):
The accuracy of diet preparations is considered acceptable if the mean measured concentrations are 80-120% of the target concentration. Homogeneity is demonstrated if the coefficient of variation is ≤10%.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15000ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for 1 day at room temperature (Test Facility Study No.507072 (method development and validation study)).
In addition, random back-up diet samples will be taken from diets used for analytical sampling and stored at ≤-15ºC for possible future analysis. Any remaining samples at finalization of the study report will be discarded.
If the analytical determinations are not performed on the day of preparation, the samples will be stored at ≤-15ºC.
Duration of treatment / exposure:
The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
Frequency of treatment:
The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
Dose / conc.:
650 ppm
Remarks:
48 mg/kg bw/day in males and 88 mg/kg bw/day in females.
Dose / conc.:
2 000 ppm
Remarks:
151 mg/kg bw/day in males and 226 mg/kg bw/day in females.
Dose / conc.:
6 000 ppm
Remarks:
377 mg/kg bw/day in males and 508 mg/kg bw/day in females.
No. of animals per sex per dose:
48 females and 40 males.
Control animals:
yes, concurrent no treatment
Details on study design:
Method Oral, by inclusion in the diet.
Frequency Ad libitum.
Dietary Inclusion Levels The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.
Exposure period Males will receive test diet for a minimum of 28 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period.
Females will receive test diet for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least thirteen days after delivery, up to and including the day before scheduled necropsy.
Positive control:
NA
Observations and examinations performed and frequency:
All animals will be deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water will be available.
All animals surviving to the end of the observation period and all moribund animals will be deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded.
Necropsy will be conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which deliver PND 14-16.
Females which fail to deliver Post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss Within 24 hours of litter loss.
Spontaneous deaths4 As soon as possible after death and always within 24 hours.
Euthanized in extremis6 When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.
The numbers of former implantation sites will be recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
For females which fail to deliver a complete nest, uterine contents (i.e. any fetuses, placenta and implantation sites) will be fixed (if applicable), but will not be examined histopathologically in first instance.
Samples of the tissues and organs specified on the next page will be collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Sacrifice and pathology:
Pups, younger than 7 days will be euthanized by decapitation.
All remaining pups (PND 7-15) will be sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater,
The Netherlands) by intraperitoneal (ip) injection.
Pups found dead during the weekend will be fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) if not necropsied on the same day.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) will be collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter will be collected into one serum tube. For further details, see section 3.10: Blood sampling for thyroid hormone analysis.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples will be collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) will be drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood will be collected into serum tubes.
Statistics:
The following statistical methods will be used to analyse the data:
• If the variables can be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one
t-test) based on a pooled variance estimate will be applied for the
comparison of the treated groups and the control groups for each sex.
• The Steel-test (Ref. 3; many-to-one rank test) will be applied if the data cannot be assumed to follow
a normal distribution.
• The Fisher Exact-test (Ref. 4) will be applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) will be applied to motor activity data to
determine intergroup differences. In case intergroup differences are seen, the Wilcoxon test (Ref. 6)
will be applied to compare the treated groups to the control group.
All tests will be two-sided and in all cases p < 0.05 will be accepted as the lowest level of significance.
Group means will be calculated for continuous data and medians will be calculated for discrete data
(scores) in the summary tables. Test statistics will be calculated on the basis of exact values for
means and pooled variances. Individual values, means and standard deviations may be rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter,
yet display different test statistics values.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs of toxicity were noted at 2000 ppm in most females (piloerection, starting in Week 3 of treatment) and at 6000 ppm in both sexes (piloerection in all animals, and hunched posture in one male and most females, mostly from Week 3 of treatment onwards). During the weekly arena observations, no additional treatment-related clinical signs were noted.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male treated at 2000 ppm (no. 25) was found dead after blood sampling on the scheduled day of necropsy. This animal showed no abnormalities in clinical appearance or body weight development. Dark red fluid was seen in the thoracic cavity at necropsy. No cause of death could be established from the slides examined microscopically. Based on the single occurrence, time of death and presence in an intermediate dose group, this unscheduled death was considered as unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, body weight gain was statistically significantly decreased at 6000 ppm from initiation of treatment onwards. Most males lost weight (up to 5% of the initial weight) in treatment Week 1 and about half of them lost some weight in Week 4. The weight loss in Week 4 was likely to be related to the overnight fasting prior to weighing (mistakenly, all males were fasted one day too early, see deviation in Appendix 8). Mean body weights of 6000 ppm males were statistically significantly decreased from Day 8 of the pre-mating period onwards (relative difference from controls: 14% after four weeks of treatment).
In females at 6000 ppm, body weight gain was decreased, generally statistically significantly, throughout the pre-mating, post-coitum and lactation period. Weight loss occurred in treatment Week 1 in most females (up to -10% of the initial body weight) and between Days 1-13 of lactation in 4/9 females (-2 or -5%). Mean body weights of 6000 ppm females were statistically significantly decreased throughout post-coitum and lactation (relative differences from control: about 10 and 20%, respectively). During the pre-mating period, mean body weights of 6000 ppm females did not differ significantly from those of controls (likely due to their slightly higher weight at initiation of treatment).
No treatment-related changes in body weights or body weight gain were noted in rats treated at 650 or 2000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated at 6000 ppm consumed less food than controls throughout the treatment period, most markedly (about 60-70%) during the first few days of treatment. Their overall mean food consumption was reduced by about 35 and 25% in the pre-mating and mating period, respectively. Males treated at 650 or 2000 ppm showed reduced food consumption (about 15-20%) during the first few treatment days. Food consumption after allowance for body weight showed a similar pattern (during the mating period, the differences from controls at 6000 ppm had become smaller due to the reduced body weights at this dose level).
Females at 6000 ppm consumed less food than controls throughout the pre-mating period (on average about 30%, about 70% during the first two treatment days), post-coitum (about 40%) and lactation (about 50%). To a lesser extent, food consumption was also reduced in females at 2000 ppm during the first few days of treatment (about 30%) and throughout post-coitum (about 20%) and lactation (about 15%). Food consumption after allowance for body weight showed a similar pattern.
Mean food intake of all groups on Days 21-22 and 22-23 showed a downward trend compared to the previous days. This was attributed to a reduced food intake due to littering of females across these groups and periods.
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Females treated at 6000 ppm showed statistically significantly reduced values for the number of reticulocytes and red blood cell distribution width (RDW). The relative differences from control values were 31 and 10%, respectively.
The statistically significantly higher mean corpuscular volume (MCV) noted in females at 2000 ppm was considered unrelated to treatment due to the lack of a dose-related response.
No treatment-related changes in hematology parameters were noted in male rats.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test-item related lower thymus weights (absolute and relative to bodyweights) and higher liver weights (relative to bodyweights) were noted in 6000 ppm males and females.
There was a test item-related effect on the final body weight. Males of the 6000 ppm group showed a 15% decrease in final body weight and females of the 6000 ppm group a 20% decrease. When expressed relative to body weight, some mean organ weight differences (heart, brain, kidneys of both sexes, testes of males and adrenal glands of females) were statistically significant when compared to the control group at 6000 ppm, but were considered to be the result of the lower final body weight at that dose level.
Any other differences, including those that reached statistical significance were considered not to be Freskomenthe-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values (including pregnancy-related variability for ovaries and uterus).
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test item-related microscopic findings were noted in the kidneys of males starting at 2000 ppm, in the spleen, urinary bladder and bone marrow of females at 6000 ppm, and in the thymus of rats of both sexes at 6000 ppm.
The following changes were observed in the kidneys of male rats:
• Hyaline droplet accumulation was observed at increased incidence and/or severity at 2000 and 6000 ppm (up to moderate).
• Granular cast(s) were observed at 2000 and 6000 ppm (minimal).
• Tubular basophilia was observed at increased incidence and/or severity at 2000 and 6000 ppm (up to slight).

The following changes were observed in females at 6000 ppm:
• Spleen: an increased incidence and severity (up to slight) of yellow-brown pigmentation (consistent with hemosiderin).
• Urinary bladder: minimal hypertrophy of the urothelium, characterized by a uniform, diffuse thickening of the urothelium without an apparent increase in cell numbers.
• Bone marrow: an increased incidence and/or severity of adipocytes (up to slight), and decreased cellularity of hematopoietic cells (minimal).

In the thymus, lymphoid atrophy was noted in 6000 ppm males (minimal) and females (minimal or slight).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
2 000 ppm
System:
other: Body weight
Conclusions:
Parental NOAEL:
650 ppm1, based on renal toxicity in males starting at 2000 ppm (hyaline droplet accumulation, accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia).
Note: This type of renal toxicity is specific for male rats and generally considered not to be relevant for human risk assessment. When this renal toxicity is excluded from the NOAEL derivation, the NOAEL under the conditions of this study was 2000 ppm, based on clinical signs of toxicity (piloerection and/or hunched posture) and reduced body weight gain and food consumption at 6000 ppm in both sexes.
Executive summary:

Parental NOAEL: 2000 ppm ( 151 mg/kg bw/day in males and 226 mg/kg bw/day in females).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
151 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good
System:
other: Clinical signs

Additional information

Justification for classification or non-classification