Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Charles River Laboratories, Preclinical Services, Tranent (PCS-EDI), Edinburgh, EH33 2NE UK
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Decycloxirane
- Batch (Lot) No.: 0011684820
- Physical description: colourless, clear liquid
- Purity: 97.55 - 97.82%
- Storage conditions: Ambient at laboratory temperature
- Expiration data: 3 August 2015

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: males 11-12 weeks, females 10-11 weeks
- Weight at study initiation: males 284-332 g, females 167-218 g
- Housing: 2 or 3 animals per cage by sex, in suspended polycarbonate/polypropylene cages with solid bottoms; bedding material was sterilised white wood shaving; A few days prior to mating males were transferred to individual cages, females were transferred to individual cages after mating
- Diet: SDS VRF-1 breeder diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 25, 62.5, 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed in week 1, 4, and 6 by GC using a validated analytical procedure. For concentration, the criteria for acceptability was mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: During the evening of Day 15 of the study, females were housed with their allocated co-group male partner, for a maximum of 4 nights.
- Proof of pregnancy: Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
- After successful mating mated females were transferred to individual solid bottomed cages. Females with litters were retained in this type of cage until termination
Duration of treatment / exposure:
- Males were treated for 2 weeks prior to mating until necropsy after 4 weeks of treatment.
- Females were treated for 2 weeks prior to mating, then throughout mating, gestation and until at least Day 4 of lactation.
Frequency of treatment:
once daily
Duration of test:
Until day 5-8 of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 250, 750 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a dose-range finding study, in which rats received the test item at the dose levels of 300 or 1000 mg/kg/day for a period of 14 days. On that study, 300 mg/kg/day was well tolerated and was not associated with any test item related findings. At 1000 mg/kg/day, reduced group mean weight gain and higher liver weights than controls were noted in both sexes. These findings were considered unsuitable for use in pregnant/lactating females. Therefore, 750 mg/kg/day was chosen as the high dose level on this study to explore toxicity.

Examinations

Maternal examinations:
MORTALITY
- Frequency: Twice daily, once early morning and as late as possible each day.
- Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS
- Frequency: Weekly, beginning Week -1.

PRE/POSTDOSE OBSERVATIONS
- Frequency: Prior to dosing and regularly throughout the day.
- Procedure: All the animals were examined for reaction to treatment. The onset, intensity and duration of these signs were recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT
- Frequency: Weekly during pretreatment, daily during dosing period during pre-mating period, for all animals. Weights for females were also measured on Days 0, 7, 14, 16 and 20 of gestation and Days 1 and 4 of lactation.

FOOD CONSUMPTION
- Frequency: Starting Week -1, weekly for both sexes until pairing for mating. Mated females: over the periods Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation – with the exception one animal in the control group where consumption was not measured over Days 0-4 of lactation in error.

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Pretreatment – All animals once (including spare animals); Prior to sacrifice – All females
- Procedure: The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The anterior, lenticular and fundic areas were examined.

HAEMATOLOGY
- Time schedule for collection of blood: day 4 of lactation
- Anaesthetic used for blood collection: No data
- How many animals: 5 animals per group
- Haematology parameters checked: red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, reticulocytes, reticulocyte count (absolute), red blood cell distribution width, platelets, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells
- Coagulation parameters checked: activated partial thromboplastin time, fibrinogen, prothrombin time

CLINICAL CHEMISTRY
- Time schedule for collection of blood: day 4 of lactation
- How many animals: 5 animals per group
- Parameters checked: urea, glucose, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase, sodium, potassium, chloride, total protein, albumin, globin, albumin/globulin ratio, cholesterol, creatinine, total bilirubin, calcium, inorganic phosphate, triglycerides, total bile acids, thyroid stimulating hormone, thyroid hormones (triiodothyronine (T3), thyroxine (T4))

NEUROBEHAVIOURAL EXAMINATION
- Time schedule: Pretreatment – 5 selected rats per group once; During Lactation – the first 5 females per group which rear their litter to at least Day 3 of lactation (prior to blood sampling)
- Battery of functions tested: sensory activity (reaction to sudden sound and to touch on the rump with a blunt probe) / grip strength / pain perception / Landing foot splay / motor activity / other abnormalities

GROSS PATHOLOGY
- Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- The reproductive tracts of all females were examined for signs of implantation and the number of any implantation sites were recorded. The total number of corpora lutea graviditas were recorded for each female.

HISTOPATHOLOGY
- The following tissues were collected from all animals: aorta, bone marrow smear, bone marrow femur, bone marrow sternum, femur bone, sternum bone, brain, cervix, eyes, adrenal gland, clitoral gland, lacrimal glands, mammary gland, parathyroid glands, pituitary gland, preputial glands, salivary glands, thyroid glands, gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys, caecum, colon, rectum, larynx, liver, lung, axillary lymph node (2x), mesenteric lymph node, skeletal muscle, nasal cavity, optic nerve (2x), sciatic nerve (2x), oesophagus, ovaries, oviduct (2x), pancreas, pharynx, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, thymus, tongue, trachea, ureter (2x), urinary bladder, uterus, vagina.
- The tissues collected (except the bone marrow smears) were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. Bone marrow smears were stained with May-Grunwald-Giemsa
- Full tissues of 5 animals used for haemotology and clinical chemistry were examined, as well as an additional 4 females from the control group and 4 females from the 250 mg/kg bw group.
- The reproductive organs (cervix, clitoral gland, coagulation gland, ovaries, preputial gland, uterus, and vagina) of all Group 1 and Group 4 animals.
- Gross lesions were checked in all animals

ORGAN WEIGHTS
Organ weights of the following organs were weighed at necropsy: brain, adrenal gland, pituitary gland, thyroid gland, heart, kidney, liver, ovary, spleen, thymus, uterus.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition were recorded. The day of birth of the litter (day on which first pups are born) was designated Day 0 of lactation. The duration of gestation in days was calculated.
The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined daily for the presence of milk in the stomach and for any externally visible abnormalities. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved; externally normal pups were discarded.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Pairwise comparisons were only performed against the control group. Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation and clinical chemistry were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test). In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results are reported. Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis. In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (i.e. less than 3 animals in any group), the untransformed parametric ANCOVA results are reported. Detailed functional observation incidence data was analysed by Fisher’s Exact Test.
Indices:
- Fertility Index (male) = number siring a litter / number paired
- Fertility Index (female) = number pregnant / number paired
- Gestation Index = number bearing live pups / number pregnant
- Birth Index = total number of pups born (live and dead) / number of implantations scars
- Live Birth Index = number of pups live on Day 0 of lactation / total number of born (live and dead)
- Viability Index = number of pups alive on Day 4 of lactation / number of pups alive on Day 0

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
- There were no premature deaths during the course of this study.
- Dosing of the test substance was associated with transient excess salivation and ploughing behaviour (animal burrowing through bedding with its head) at 750 mg/kg/day. These findings were noted immediately post dose and were no longer evident 1 hour after dosing; noted only sporadically throughout the dosing period at 750 mg/kg/day. One female at 750 mg/kg/day showed observations of hunched posture, pale eyes, reduced activity and rolling gait on Day 1 of lactation, however, it was considered that these signs were more likely to be related to parturition than treatment and were, therefore, incidental.

BODY WEIGHT
Exposure group mean body weights were similar to controls throughout study.

FOOD CONSUMPTION
Exposure group mean food consumption was comparable to controls prior to mating and during gestation and lactation.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopy findings which were considered to be related to treatment
with the test substance.

HAEMATOLOGY AND COAGULATION
There were no changes in any of the haematology and coagulation parameters which were considered to be related to treatment with the test substance.

CLINICAL CHEMISTRY
- In the 250 and 750 mg/kg/day dose groups, alanine aminotransferase was up to 40% when compared to controls.
- There were no other changes in any of the clinical chemistry parameters which were considered to be related to treatment with the test substance.

NEUROBEHAVIOUR
- Throughout the study, the type and distribution of neurotoxicity observations did not indicate an association with treatment with the test substance.
- Detailed functional observations were generally similar between controls and treated groups. Intergroup difference in grip strength, tail flick and foot splay did not follow dose related patterns and were not present, therefore, it was considered that these were incidental and unrelated to treatment with the test substance.
- There were no clear differences in motor activity between treated animals and controls which were considered to be related to treatment with test substance. Lower overall values at 250 and 750 mg/kg/day, when compared to controls at the end of the dosing period, were considered to be incidental as pretrial values for these groups were generally also lower.

REPRODUCTIVE PERFORMANCE
Mating performance, fertility, duration of gestation and litter size were similar in controls and treated groups.

ORGAN WEIGHTS
There were no changes in female organ weights at any dose level. Isolated organ weight values which differed from controls did not follow any patterns, trends or have any correlating data to indicate that they were of any toxicological significance; therefore, these changes were considered to be incidental or due to differences in sexual maturity and unrelated to treatment with test substance.

GROSS PATHOLOGY
There were no treatment related gross necropsy findings at any dose level. All findings were considered to be incidental or of the nature commonly observed in this species, and/or were of similar incidence and severity in controls and treated groups, therefore, they were considered not to be associated with treatment with the test substance.

HISTOPATHOLOGY
There were no treatment related microscopic findings in at any dose level. All findings were considered to be incidental or of the nature commonly observed in this species, and/or were of similar incidence and severity in controls and treated groups, therefore, they were considered not to be associated with treatment with the test substance.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
LITTER SURVIVAL, LITTER AND PUP WEIGHTS
Survival indices and litter and pup weights were generally similar between controls and treated groups. Slight intergroup differences did not follow a dose related pattern and were too small to be attributed to treatment with the test substance.

OBSERVATIONS AMONGST DAMS AND PUPS
The type and distribution of observations noted in dams and pups during lactation did not indicate any association with treatment with the test substance.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion