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Key value for chemical safety assessment

Effects on fertility

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

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 422 study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Justification for study design:
OECD 422 guideline study conducted to GLP.
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 toxicity 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
Vehicle:
unchanged (no vehicle)
Details on 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.
Details on mating procedure:
Pretest Females will be housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating Animals will be housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating Males and females will be cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating Males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm).
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 15000 ppm 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.
Details on study schedule:
Source F0 Charles River Laboratories France, L'Arbresle Cedex, France or Charles River Deutschland, Sulzfeld, Germany. Details will be documented in raw data and report.
Age at start pretest Females: approximately 10-12 weeks.
Age at start F0-treatment Males: approximately 10-12 weeks.
Females: approximately 12-14 weeks.
Number of F0-animals 48 females and 40 males.
A total of 40 females will be selected at randomization before initiation of the pretest phase. Any selected female without at least two regular estrous cycles at the end of the pretest phase will be replaced by one of the 8 additional females having at least two regular estrous cycles. A total of 40 females with at least two regular estrous cycles will continue in the study. The supernumerary females will then be removed from the study,
and their estrous cycle results will be kept in the raw data but will not be reported.
Acclimatization F0 At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection F0 At least upon receipt of the animals.
Randomization F0 Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0 During pretest (females1) and treatment (males and females): by earmark and tattoo.
Mating procedures Following a minimum of 14 days of treatment for the males and females, they will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated.
A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male of proven fertility of the same group for a maximum of 7 days (if possible).
Parturition The females will be allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that are littering will be left undisturbed.
Number of pups Approximately 480 pups (40 litters x 12 pups).
Identification of pups On PND 1, all pups will be randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
When general hair growth blurs the identification, the pups will be identified by tattoo on the feet.
Dose / conc.:
650 ppm
Remarks:
48 mg/kg bw/day in males and 88 mg/kg bw/day in females.
Dose / conc.:
1 000 ppm
Remarks:
151 mg/kg bw/day in males and 226 mg/kg bw/day in females.
Dose / conc.:
2 000 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
Parental animals: Observations and examinations:
Mortality / Viability At least twice daily. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death will be recorded as precisely as possible.
Clinical signs Once prior to first administration and at least once daily from start of treatment onwards, detailed clinical observations will be made for all animals.

Body weights Males and females will be weighed on the first day of exposure and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. In order to monitor the health status animals may be weighed more often. This will be documented in the study raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, except for males and females which are housed together for mating and for females without evidence of mating.
Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption Subjective appraisal will be maintained during the study period
and a quantitative assessment introduced in the event of a suspected treatment related effect.
Oestrous cyclicity (parental animals):
Estrous cycle Daily vaginal lavage will be performed to determine the stage determination of estrus beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of
copulation is observed. Vaginal lavage will continue for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage will be taken to determine the stage of estrus.
Sperm parameters (parental animals):
No
Litter observations:
Each litter will be examined to determine the following, if practically possible:
Mortality / Viability The numbers of live and dead pups will be determined on PND 1 and daily thereafter. If possible, defects or cause of death will be evaluated. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
Clinical signs At least once daily, detailed clinical observations will be made for all animals. Only days on which clinical signs are present between first and last litter check will be given in the respective report tables.
Body weights Live pups will be weighed on PND 1, 4, 7 and 13.
Sex Sex will be determined for all pups on PND 1 and 4.
Anogenital distance Anogenital distance (AGD) will be measured for all live pups on PND 1. The AGD will be normalized to the cube root of body weight.
Areola/nipple retention On PND 13, all males in each litter will be examined for the number of areola/nipples.
Postmortem examinations (parental animals):
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).
Postmortem examinations (offspring):
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.
Dermal irritation (if dermal study):
not examined
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, non-treatment-related
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:
effects observed, non-treatment-related
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, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
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.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Reproductive performance:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
Remarks on result:
other: based on an acyclic estrous cycle in 6/10 females at 6000 ppm
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of male and female pups at 6000 ppm were reduced. Compared to controls, mean body weights of 6000 ppm pups were about 10% lower at birth and about 30% lower on PND 13. The magnitude of this change was considered to represent an adverse effect on pup development, and occurred in the presence of clinical signs of toxicity and considerably reduced food consumption (associated with reduced body weights) of the dams. However, there was no apparent correlation between lower body weights and food intake of the dams and lower pup body weights. Also, the dams did not show lack of maternal care or obvious target organ toxicity.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
2 000 ppm
Treatment related:
yes
Conclusions:
In conclusion, based on the results of this combined 28-day toxicity study with the reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect levels (NOAELs) of FRESKOMENTHE were established:
Parental NOAEL:
650 ppm, based on renal toxicity in males starting at 2000 ppm (hyaline droplet accumulation, accompanied by indicators oftubular 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 andfood consumption at 60003ppm in both sexes.

Reproduction NOAEL:
2000 ppm, based on an acyclic estrous cycle in 6/10 females at 6000 ppm.

Developmental NOAEL:
2000 ppm, based on reduced pup body weights at 6000 ppm.
Executive summary:

Parental NOAEL: 650 ppm.

Reproduction NOAEL: 2000 ppm.

Developmental NOAEL: 2000 ppm.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
226 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good

Justification for classification or non-classification