Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation Date: 02 October 2017 - Experimental Completion Date: 19 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
yes
Remarks:
But none of the deviations affected the integrity or interpretation of the results of the study
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test Article: The test article, a clear, very pale yellow liquid, was identified as Cyrene (alternative names Cyrene™/Dihydrolevoglucosenone).
Storage: 15 to 25°C, in a sealed container protected from the light.
Expiration Date: 07 July 2019
Purity: 99.8%
CAS Number: 53716-82-8
EC Number: 807-130-4
Molecular Formula: C6H8O3
Molecular Weight: 128.13 g/mol
Chemical class: Cyclic ketone

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
Details on species / strain selection
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. Crl:WI(Han) was selected as the Lab has experience with this type of stain and has been using in such studies.
Sex: male/female

Details on test animals and environmental conditions
Animal Specifications and Acclimation:
Forty-three male and 46 female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for study selection. Upon arrival, males were approximately 9 to 10 weeks of age, while females were approximately 7 to 9 weeks of age.
Males weighed between 290.0 and 436.8 g and females weighed between 170.5 and 217.1 g at the start of dosing. Animals were 10 to 12 weeks of age and considered sexually mature. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).

Housing:
Animals were housed in cages. During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom).
Water: Water from the main tap supply was provided ad libitum via water bottles.
Diet: Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). A 50 g sample of each batch of diet used on study was collected and stored at ambient temperature, and discarded after finalization of the study report.

Environment:
Animals were housed in a single, exclusive room. The room was air conditioned to provide a minimum of 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 22°C +/- 3°C and 30 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated.

Environmental Enrichment:
Animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation, nesting materials were provided as forms of environmental enrichment.
Animal Identification and Assignment to Study: Upon arrival, animals were assigned to dose groups using a total randomization procedure. Animals
were individually identified by electronic implant. Cages were placed in dose group order across the batteries.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Rats were administered Cyrene™ once daily by oral gavage. The control (vehicle) article was corn oil, and formulations were administered at a dose volume of 4 mL/Kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and Homogeneity:
Formulations of 4 and 250 mg/mL concentration were found to be homogenous and stable for 4 days at room temperature (15 to 25°C) in validation study 8367720 (non Covance).
Formulations of 7.5 and 250 mg/mL were prepared after the initial sampling for homogeneity; formulations were split into two aliquots. The second aliquot was stored refrigerated (2 to 8°C) until report finalization.
Samples for stability were collected from each formulation as soon as possible after preparation and after 4 and 10 days.
Homogeneity assessments were performed on samples used on Day 1 of dosing; samples were taken in triplicate from the top, middle, and bottom.
Achieved Concentration:
Samples (2 x 5 mL [random] aliquots from all formulations) prepared for use during Weeks 1 and 6 of dosing were taken for achieved concentration analysis.
Samples were dispatched at room temperature (15 to 25°C) for analysis.
Details on mating procedure:
During the pairing phase, one male was housed for up to 15 days with one female of the same dose group. Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal lavage was discontinued, and the male was removed from the cage. The day on which mating was confirmed was designated GD 0.
Duration of treatment / exposure:
Male rats were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43. Female rats were dosed for up to 55 days (2 weeks prior to pairing, during pairing, throughout gestation, and upto Lactation Day [LD] 13) and were sent to necropsy on LD 14.
Frequency of treatment:
Once daily
Duration of test:
Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43. Females were dosed for up to 55 days (2 weeks prior to pairing, during pairing, throughout gestation, and upto Lactation Day [LD] 13) and were sent to necropsy on LD 14.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control (corn oil) Group
Dose / conc.:
30 mg/kg bw/day
Remarks:
Low Dose Group
Dose / conc.:
300 mg/kg bw/day
Remarks:
Intermediate Dose Group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High Dose Group
No. of animals per sex per dose:
Four groups of 10 male and 10 female rats were administered 0, 30, 300, or 1000 mg/kg bw/day Cyrene™ once daily by oral gavage.
Control animals:
yes, concurrent vehicle
Details on study design:
A dose range-finding study was previously performed with once daily oral gavage administration of 0, 250, 500, or 1000 mg/kg bw/day Cyrene™ for 14 consecutive days. The test article was well tolerated at the highest dose level. The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.
Before study initiation, estrous cycle data were reviewed to ensure all females allocated to the study showed regular estrous cycles. Any female that did not show a regular estrous cycle was replaced by a spare female showing a regular estrous cycle.
The high-dose level of 1000 mg/kg bw/day was selected as the limit dose as this dose level was well tolerated in the previous dose range-finding study and was expected to be well tolerated over the longer dosing duration in this study.
The intermediate-dose level of 300 mg/kg bw/day allowed a 3-fold increase to the high dose. The lowdose level of 30 mg/kg bw/day was anticipated to be the no observed adverse effect level (NOAEL).
The prior to dose initiation body weights were calculated and inspected to ensure no unacceptable differences occurred between groups.
Cages were appropriately identified with study information, including study number and animal number(s).

Examinations

Maternal examinations:
Clinical Observations: Animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.
Clinical Examinations: Each animal was given a detailed physical examination once during acclimation then once daily from the start of dosing, including the day of necropsy.
Postdose Observations: Animals were observed daily for the first 3 days of dosing; upon return to the home cage; and approximately 0.5, 1, 2, and 4 hours postdose. In the absence of any postdose observations recorded during the first 3 days of dosing, no further postdose observations were scheduled.
Body Weights: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing, at weekly intervals prior to pairing, and until confirmation of mating; on GD 0, 7, 14, and 20; and on LD 0 (where applicable), 1, 4, 7, 13, and 14 (prior to necropsy).
Food Consumption: The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumptions were recorded for females from GD 0 to 20 and from LD 1 to 13. Consumption was calculated as g/animal/day.
Functional Observational Battery (FOB): FOB assessments were performed to allow blind testing (in a manner so the observer did not know the dose group of animals during testing). Observations were performed at the same time on each occasion (approximately 2 hours postdose), where possible.
Detailed Clinical Observational Measurements: All males were assessed for detailed clinical observational measurements once prior to dose initiation and once weekly thereafter. All females were assessed once prior to dose initiation; once weekly during the pre-pairing and pairing phases; on GD
0, 7, 14, and 20; and on LD 1, 7, and 13. Locomotor Activity: Locomotor activity was assessed in an automated photocell activity recorder for
30 minutes and was undertaken for five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post Pairing Day 16) for males and on LD 7 for females. Activity counts were recorded at 5-minute intervals. The following parameters were determined:
- Total activity counts
- Total rears
- Total mobile counts
Quantitative Assessments: Animals were observed in the hand and in an arena. Assessments were performed during Week 6 of dosing (Post Pairing Day 20) for males and on LD 7 for females.
Quantitative assessment parameters were as follows:
- Hind limb foot splay
- Fore and hind limb grip strength
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 early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
Litter Size and Sex Determination:
On PND 1, 4, 7, and 13, litter size and pup sex were recorded. Pups were uniquely identified from PND 1.
Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition underwent a macroscopic necropsy.
On PND 4, litters were culled to 10 pups/litter, with five pups/sex, where possible. Pups were selected randomly with no bias to size or clinical status. The sex was determined for surplus PND 4 pups, which were discarded with no macroscopic examination. Culling created a uniformly sized litter, wh
ich reduced differences in pup body weights due to litter size.
Clinical Observations: Each pup underwent a detailed clinical examination daily from PND 1.
Body Weights: Pup body weights were recorded on PND 1, 4, 7, and 13.
Ano-genital Distance: The ano-genital distance of all pups was recorded on PND 4.
Nipple/Areolae Count: The number of nipples/areolae for male pups was counted on PND 13.

Postmortem examinations (parental animals)
Males were sacrificed on Study Day 43 (Post-Pairing Day 22). Females were sacrificed on LD 14 (those that achieved pregnancy) or Day 26 post coitum (those that did not litter). Animals were fasted overnight prior to sacrifice. Animals were sacrificed in a controlled randomization sequence, where possible, by isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. Blood sampling information is detailed in Section 3.6. After sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

Postmortem examinations (offspring)
Surplus pups culled on PND 4 and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anaesthesia was established, PND 4 pups were decapitated, and PND 13 pups were exsanguinated by severing a major blood vessel.
Surplus pups culled on PND 4 were discarded following blood sample collection and sex deter mination. Full macroscopic examinations were conducted for all decedents, and external examinations for gross abnormalities, with particular attention to the external reproductive genitals, were
conducted for all pups sent to necropsy on PND 13.
Statistics:
Data Evaluation and Statistical Analysis
Where tables/appendices are computer-generated, rounding of individual values may have occurred during the calculation of derived values. Therefore, recalculation of derived values from the individual data, as presented in this report, may have, in some instances, yielded minor variations.
Data from test article-treated animals were compared with control data. Statistical analyses were per formed, where appropriate.
Data for each sex were analyzed separately, unless stated otherwise.
The following data were analyzed using Tox Reporting:
• Continuous behavioural (FOB) data - latency to first step, number of rears, hindlimb footsplay, forelimb grip strength, hindlimb grip strength Procedure I (ANOVA)
• Clinical pathology and thyroid hormones (adult male and female) Procedure I (ANOVA)
• Locomotor activity - Procedure I (ANOVA)

The following data were analyzed using Pristima:
• Body weights (adult) - Procedure I (ANOVA)
• Body weight gains (adult) - Procedure I (ANOVA)
• Food consumption (gestation and lactation) - Procedure I (ANOVA)
• Absolute organ weights and organ to terminal body weight ratios - Procedure I (ANOVA)
• The mean number of estrous cycles and mean cycle length - Procedure III
• Male and female mating, fecundity, and fertility indices - Procedure IV (one sided lower tail)

The following data were analyzed using SAS:
• The duration of gestation; number of implantation sites; number of pups born; number of pups alive on PND 1 and 4 (before culling) and % male pups on PND 1; percent post implantation loss; and livebirth and survival indices, where appropriate - Procedure III
• Pup weights (male, female, and combined) - Procedure II (litter size as the covariate)
• Ano-genital distance (males only) - Procedure II (cube root of mean pup weight as the covariate)
• Thyroid hormone (male, female and combined pups), where appropriate - Procedure I (ANOVA) -Tox Reporting
Indices:
Fertility and reproductive performance observed.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A transient instance of hunched posture, increased activity, or raised hair was recorded between GD 8 and 9 for three dams administered 1000 mg/kg bw/day, with regression observed thereafter. One male administered 300 mg/kg bw/day also showed hunched posture for 5 days during the post-pairing phase, with regression to normal observed thereafter. These findings were isolated to a few animals and occurred without a dose-response; as such, they were considered incidental and of no toxicological importance.
Thin fur, skin/fur staining, and vocalization were noted throughout the dose groups, including controls, and were of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower body weight gain was noted on Post-Pairing Days 8 through 15 for males administered 300 or 1000 mg/kg bw/day, compared with controls. Furthermore, statistically significant changes in body weight were noted during the lactation phase for females administered 300 mg/kg bw/day. In the absence of a convincing dose-related response, these changes were considered to have arisen incidentally.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was noted.
Food consumption for dams administered 1000 mg/kg bw/day was statistically significantly lower than controls between GD 4 and 5; however, due to the transient nature of this observation it was considered not toxicologically significant.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food consumption for dams administered 1000 mg/kg bw/day was statistically significantly lower than controls between GD 4 and 5; however, due to the transient nature of this observation it was considered not toxicologically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males administered 1000 mg/kg bw/day showed a statistically significant reduction in percentage reticulocytes and red cell distribution width, compared with controls (P < 0.01). Platelets, platelet crit, and fibrinogen were also elevated for males administered 1000 mg/kg bw/day, compared with controls (P < 0.001), with the effect also evident for males administered 300 mg/kg bw/day (P < 0.01 to P < 0.001). A decrease in activated partial thromboplastin time was also observed for
males administered 1000 mg/kg bw/day, compared with controls (P < 0.05).
No test article-related effects were noted for females from any dose group. Remaining statistically significant changes observed (increased haematocrit distribution width for males administered 300 mg/kg bw/day; increased white blood cell counts, specifically in the lymphocyte fraction, for males administered 30 mg/kg bw/day; increased mean cell volume for females administered 30 mg/kg bw/day; and reduced neutrophil counts for females administered 1000 mg/kg bw/day), did
not show a convincing dose response or any supporting correlations with the parameters investigated and, as such, were considered to have arisen incidentally, with no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cholesterol was increased for males and females administered 1000 mg/kg bw/day, compared with controls (P < 0.001 and P < 0.01, respectively). Males administered 300 mg/kg bw/day were similarly affected (P < 0.001).
Other changes in the clinical chemistry parameters were evident for males administered 1000 mg/kg bw/day, compared with controls. These included increased alanine aminotransferase (P < 0.05) and increased alkaline phosphatase activity (P < 0.01). Total protein, globulin, and calcium levels were also increased (P < 0.001), with a corresponding reduction in albumin:globulin ratio (P < 0.05). Furthermore, chloride levels were significantly reduced (P < 0.001), whereas urea and bile acids were elevated for males administered 1000 mg/kg bw/day, compared with controls, although statistical significance was only achieved for the elevation in urea (P < 0.01). For males administered 300 mg/kg bw/day, total protein (P < 0.05) and globulin (P < 0.001) were increased, compared with controls, with a corresponding reduction in albumin:globulin ratio (P < 0.01).
No test article related change in clinical chemistry was evident for females administered 300 mg/kg bw/day or both sexes administered 30 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Hormone Assessments: No effect in thyroid hormones was noted for either sex administered Cyrene™, compared with controls. Furthermore, no effect on estradiol or testosterone was noted for males following DOBCO administration, compared with controls.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant differences were evident in mean number of implantation sites or pups born. Post-implantation survival index for dams administered 1000 mg/kg bw/day was 7% lower than controls; however, statistical significance was not achieved. This decrease in the post-implantation survival index was attribtable to the total in utero litter loss; this impacted the mean percentile post-implantation. Other live birth or survival indices were not affected. As such, the marginally lower implantataion survival index was considered to have arisen incidentally.
Sex ratio (% males) was slightly higher for litters of dams administered 1000 mg/kg bw/day, compared with controls. No statistical significance was achieved, as such, this slight increase in male sex ratio is not considered to be related to the test article.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No toxicologically significant differences were evident in mean number of implantation sites or pups born.
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): No intergroup differences in gestation length were noted.
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects in maternal animals

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other: No effects in maternal animals

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Lower body weights were evident for pups from litters of dams administered 1000 mg/kg bw/day, compared with controls. No adverse effect on litter weight was noted for litters of dams administered 30 or 300 mg/kg bw/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No changes in survival indices.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No overt differences in T4 or TSH levels were noted for pups exposed to Cyrene™, compared with controls.
No nipples/areolae were present for male offspring from all litters, including controls.
Ano-genital distance was not affected following dam exposure to Cyrene™.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for developmental toxicity was established at the dose level of 300 mg/kg bw/day due to the reduced body weights in pups at the dose level of 1000 mg/kg bw/day.
Executive summary:

In the key (OECD 422) study, Cyrene™ was administered oral by gavage daily at 0, 30, 300, or 1000 mg/kg bw/day to male rats for 42 consecutive days and to female rats for up to 55 days (pre pairing, throughout gestation, and during the first 2 weeks of lactation), no toxicologically significant differences were evident in mean number of implantation sites or pups born.

Post-implantation survival index for dams administered 1000 mg/kg bw/day was 7% lower than controls; however, statistical significance was not achieved. This decrease in the post-implantation survival index was attribtable to the total in utero litter loss; this impacted the mean percentile post-implantation. Other live birth or survival indices were not affected. As such, the marginally lower implantation survival index was considered to have arisen incidentally.

Sex ratio (% males) was slightly higher for litters of dams administered 1000 mg/kg bw/day, compared with controls. No statistical significance was achieved, as such, this slight increase in male sex ratio is not considered to be related to the test article.

Lower body weights were evident for pups from litters of dams administered 1000 mg/kg bw/day, compared with controls. No adverse effect on litter weight was noted for litters of dams administered 30 or 300 mg/kg bw/day. Therefore, the NOAEL for developmental toxicity was established at the dose level of 300 mg/kg bw/day due to the reduced body weights in pups at the dose level of 1000 mg/kg bw/day.