Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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:
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
Sex:
male/female
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
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
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.
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
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

Parental animals: Observations and 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); Week 4 – All males; 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: week 4 - males; on or day 4 of lactation - females
- Anaesthetic used for blood collection: No data
- Animals fasted: males were fasted
- How many animals: 5 animals per sex 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: week 4 - males; on or day 4 of lactation - females
- Animals fasted: males were fasted
- How many animals: 5 animals per sex 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 sex per group once (first 5 males and females per group); Week 4 – 5 selected males per group (prior to blood sampling); 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
Litter observations:
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.
Postmortem examinations (parental animals):
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, epididymis (2x), eyes, adrenal gland, clitoral gland, lacrimal glands, mammary gland, parathyroid glands, pituitary gland, prostate, preputial glands, salivary glands, seminal vesicle including coagulation gland, thyroid glands (2x), gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys (2x), 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 (2x), oviduct (2x), pancreas, pharynx, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes (2x), 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 male and female 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, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, 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, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicle including coagulation gland, thyroid gland, heart, kidney, liver, ovary, spleen, testis, thymus, uterus.
Postmortem examinations (offspring):
Where practicable, animals found dead or killed were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal pups were discarded.
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.
Reproductive 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
Offspring viability indices:
- 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: P0 (first parental generation)

Details on results (P0)

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) in both sexes at 750 mg/kg/day and males at 250 mg/kg/day. At both levels, these findings were noted immediately post dose and were no longer evident 1 hour after dosing; noted generally only one occasion at 250 mg/kg/day and 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 in both sexes throughout study.

FOOD CONSUMPTION
Exposure group mean food consumption was comparable to controls in both sexes prior to mating and in females during gestation and lactation. Statistically significant differences in male food consumption on Day 8 did not follow a dose related pattern and were considered too small too be attributed to treatment; therefore, these differences were considered to be incidental.

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

HAEMATOLOGY AND COAGULATION
- At 750 mg/kg/day, white blood cell, lymphocyte and basophil counts were approximately 40-60% higher in males at the end of the treatment period when compared to controls, and neutrohpil count was approximately 2.5-fold higher in males than controls.
- There were no other changes in any of the haematology and coagulation parameters in males or any changes in females which were considered to be related to treatment with the test substance.

CLINICAL CHEMISTRY
- In males, group mean glucose levels were higher at the end of the treatment period in all treated groups when compared to Controls (up to 30%) and in females at 250 and 750 mg/kg/day, alanine aminotransferase was up to 40% higher in females when compared to controls.
- Lower group mean lactate dehydrogenase and creatine phosphokinase in males at 750 mg/kg/day were not positively attributed to treatment as these parameters were variable between individual animals throughout the groups.
- There were no other changes in any of the clinical chemistry parameters in either sex 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 in both sexes, 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
- In treated males, higher kidney and liver weight were noted when compared to controls, with increases of up to 14% and 20% in absolute, relative and covariate kidney and liver weights respectively.
- There were no other treatment related organ weight changes in males, or any 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, in either sex. 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, in either sex. 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 (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion