Registration Dossier

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 December 2014 and 19 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No.421: “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
None
Specific details on test material used for the study:
None

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 292 to 362 g, the females weighed 170 to 204 g, and were approximately twelve weeks old (see deviations from Study Plan).

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Due to the aggressive behavior of Male 26 from the low dose group (Cage 7), this animal was housed separately from Day 3 of the treatment period. During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages; an attempt was made to re-introduce Male 26 to its original cage on Day 20 of treatment, however, the animal was moved back to individual housing immediately thereafter due to persisting aggressive behavior. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as solution in distilled water. The formulations were assessed to be homogeneously prepared. The stability of the test item formulations was determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services and these formulations were found to be stable for up to twenty-eight days when stored at +4 °C, in the dark. Formulations were therefore prepared approximately every three weeks and stored at approximately 4 °C in the dark before use.

Samples of the test item formulation were taken and analyzed for concentration of FAT 40868/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 98 to 105% of the nominal concentration.

Procedure
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of distilled water.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC / UV) using an external standard technique.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in water were prepared for external calibration. An aliquot, approximately 0.02 g of test item was accurately weighed into a 200 mL volumetric flask and brought to volume with water to yield a solution with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with water. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with water this was then shaken to dissolve. Where necessary, sample solutions were further diluted with water to achieve the working concentration.

Accuracy of samples
Samples of distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in water from a stock solution of 1.039 mg/mL by serial dilution covering the concentration range 0.0520 to 0.2078 mg/mL.

Instrumental setup
HPLC: Agilent technologies 1200, incorporating autosampler and workstation
Column: Gemini C18 (100 x 4.6 mm id)
Column temp.: 30 °C
Mobile phase:
Eluent A: 2 g TRAB in 100 mL acetonitrile + 900 mL water
Eluent B: 2 g TRAB in 100 mL acetonitrile
Time %B
0 0
5 90
10 90
Flow rate: 1 mL/min
UV detector wavelength: 590 nm
Injection volume: 10 µL
Retention time: ~ 6 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.998 and considered to be acceptable.

Accuracy
The fortified samples of distilled water were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The test item was found to be stable in the formulations when kept for 28 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and distilled water as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Duration of treatment / exposure:
Approximately 6 weeks
Frequency of treatment:
Daily
Duration of test:
Approximately 6 weeks
No. of animals per sex per dose:
Twelve male and twelve females
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offsprings until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all littering females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Examinations

Maternal examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing (see deviations from Study Plan). This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Pathology
Necropsy
All surviving adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:

Coagulating gland, Prostate, Epididymides ♦, Seminal vesicles, Ovaries, Testes ♦, Mammary gland, Uterus/Cervix, Pituitary, Vagina, Gross lesions*

* = As gross lesions were confined to skin and/or kidney discoloration in most animals of either sex from the high dose group and two females from the intermediate dose group, skin and kidneys from five control males and females were also preserved as above; these were not processed.

All tissues were dispatched to the histology processing Test Site (Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk, IP23 7PX) (Principal Investigator: D Roberts). The tissues from control (excluding skin and kidneys) and 1000 mg/kg bw/day dose group animals and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Macroscopic abnormalities from the high dose animals were also examined; as discolouration of skin and/or kidneys from the high dose animals was not associated with any microscopic changes, the remaining macroscopic abnormalities which were confined to two intermediate dose females showing dark kidneys were not processed or microscopically examined. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Microscopic examination was conducted by the Study Pathologist (W Henderson).
Ovaries and uterine content:
Not assessed
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offsprings born
ii. Number of offsprings alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offsprings from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offsprings were assessed for surface righting reflex on Day 1 post partum.
Statistics:
See below
Indices:
See below

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no treatment-related deaths during the study.

Male 9 from the control group was killed on Day 35 of dosing due to a physical injury. Prior to death, this animal was observed to have an open wound close to the left eye with yellow discharge from the wound whilst the eye appeared to be dark with the surrounding tissue swollen. At necropsy, there were no internal macroscopic findings for this animal and the injury was considered likely to have resulted from fighting amongst cage animals.

Clinical Observations
There were no clinical signs for any of the animals considered to be related to the toxicity of the test item.

At 1000 mg/kg bw/day, instances of blue fur staining by the test item were frequently observed in both males and females from Day 4 of dosing and generally persisted throughout the remainder of the treatment period. Individual animals of both sexes receiving 300 mg/kg bw/day and some males treated with 100 mg/kg bw/day also showed similar clinical observations on some days. Blue or dark colored faeces were also observed for animals of both sexes treated with 300 or 1000 mg/kg bw/day from the first week of dosing with the latter also showing blue staining of the cage bedding. Animals of either sex receiving 100 mg/kg bw/day also showed occasional blue colored faeces from Week 3 of dosing.

A small number of males treated with 100 or 300 mg/kg bw/day also showed clinical signs of physical injury (open wound and/or scab formation) on some days; these were considered likely to have resulted from fighting amongst cage animals.

Body Weight
Over the first two weeks of dosing period, group mean body weight gains in males given 1000 mg/kg bw/day were slightly lower than controls albeit without attaining statistical significance. Overall mean body weight gain for these animals was similar to controls and any initial effect of treatment on body weight development was considered not to be of an adverse nature. For males receiving the test item at 100 or 300 mg/kg bw/day, occasional body weight fluctuations were observed but their weekly body weight gains generally remained comparable with controls throughout the treatment period.

During the pre-pairing and initial gestation phases of the study, there was no indication of an effect of treatment with FAT 40868/A TE at any dose levels on body weight development in females. Over Days 14 to 20 of gestation, females treated with 300 or 1000 mg/kg bw/day showed slightly lower group mean body weight gains in relation to controls which resulted in slightly lower cumulative body weight gains over the entire gestation period for these females. These differences were, however, neither dose-related nor statistically significant and were considered to have resulted from the slightly lower litter size, i.e. less fetuses, for these females (see section 4.1.3.1) and as such were considered not to be treatment-related. During the lactation phase of the study, the mean body weight gains in females receiving 100 or 300 mg/kg bw/day were slightly lower than controls. There was no dose-dependence or statistical significance and since lactation body weight gains for the high dose females were similar to control, this finding was considered to be incidental.

Food Consumption
Throughout the treatment period, dietary intake across all test item-treated groups of males remained similar to controls. Over the first two weeks of dosing, food conversion efficiency in males from the 1000 mg/kg bw/day dose group was marginally lower than controls reflecting intergroup differences in body weight gains for these animals over the corresponding period. For the remaining treatment period, food conversion efficiency for these males was similar to controls.

During the pre-pairing phase of the study, there was no effect of treatment with the test item at any dose level on food consumption or food conversion efficiency in females. Throughout gestation, food consumption for test item-treated females also remained similar to controls. Over Days 1 to 4 of lactation, females given 100 or 300 mg/kg bw/day showed slightly lower food intake in relation to controls; however, these differences did not achieve statistical significance and the corresponding value for the high dose females was comparable with controls.

Water Consumption
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of both sexes given the test item in relation to controls.

Reproductive Performance
Mating
There was no effect of treatment on mating performance with all animals mating within four days after pairing.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with FAT 40868/A TE at any dose level.

Female 92 treated with 1000 mg/kg bw/day did not achieve pregnancy following evidence of successful mating. Histopathological examination of the reproductive tissues from this female showed it to be cycling normally and there was no obvious reason for the lack of pregnancy in this female nor in the corresponding male (Animal Number 80). Due to the isolated nature of this incident, it was considered unlikely to be related to treatment with the test item.

All the remaining females on the study were found to be pregnant.

Gestation Length
With the exception of Female 93 from the high dose group which showed a gestation length of 24 days, gestation length was between 22 and 23½ days for the remaining pregnant females and their distribution appeared to be similar to controls. A statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.

Pathology
Necropsy
Adults
At 1000 mg/kg bw/day, most animals of either sex showed blue discoloration of the skin and/or kidneys with the latter appearing to be dark in some animals. Dark kidneys were also observed in two females from the 300 mg/kg bw/day dose group. Following microscopic examination of these tissues from the high dose animals, these were considered not related to the toxicity of the test item.

No such effects were detected in 300 mg/kg bw/day males or animals of either sex treated with 100 mg/kg bw/day.

Organ Weights
When compared with controls, group mean absolute and body weight-related testes and epididymides weights in males given the test item at all dose levels were similar to controls.

Histopathology
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

There was no histopathological correlation with the macroscopic discoloration of the tissues noted at necropsy. Expected minor findings were apparent in the kidneys and/or skin examined but these were not considered to be attributed to treatment with the test item.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

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): no effects observed
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
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In total, twelve females each from the control and 300 mg/kg bw/day dose groups and eleven females each from the 100 and 1000 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. Female 42 from the low dose group showed a total litter loss on Day 2 post partum. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
At 300 or 1000 mg/kg bw/day, the mean number of corpora lutea was slightly lower than controls in a dose-related manner. Although individual values for 1/12 intermediate dose females and 3/11 high dose females were below the historical control data ranges, inter-individual variation was particularly high for the high dose females and statistical significance was not achieved for these differences. There was no detrimental effect of treatment on pre- or post-implantation losses at any dose level and in the absence of any histopathology correlate, this finding was considered unlikely to be treatment-related. It is worth noting that whilst 2/12 intermediate dose females showed higher pre-implantation losses than the background data ranges resulting in slightly higher mean pre-implantation loss for this dose group, the difference was not statistically significant. The corresponding value in the high dose females was statistically significantly lower than controls (p<0.01) and as such this finding was deemed to be incidental. At 100 mg/kg bw/day, the mean number of corpora lutea, implantation sites and pre- or post-implantation losses were similar to controls.

The mean number of implantation sites in females treated with 300 or 1000 mg/kg bw/day was slightly lower than controls due to the above-mentioned factors. Of the litters born to these females and surviving to Day 5 post partum, this resulted in slightly lower litter size at birth and subsequently on Day 1 and 4 post partum. As there were no dose-relationships and statistical analysis did not reveal any significant differences these differences were considered not to be treatment-related. At 100 mg/kg bw/day, litter sizes were comparable with controls. At all dose levels, there was no effect of treatment on survival indices.

There was no effect of treatment with the test item at any dose level on sex ratio on Days 1 or 4 post partum.

Offspring Growth and Development
There was no indication of a detrimental effect of treatment with the test item on offspring body weights and body weight development. Group mean litter weights for the intermediate and high dose groups were slightly lower than controls on Days 1 and 4, but without any dose-relationship or statistical significance and this was likely due to the slightly lower litter sizes for these dose groups. Due to the slightly lower litter sizes in these dose groups, group mean pup weights on Days 1 and 4 post partum were marginally higher than controls, but again without achieving statistical significance.

Surface righting reflex data did not reveal any intergroup differences when compared with controls.

Clinical signs detected in pups from all treated groups included small size, cold, weak, no milk in stomach, physical injury, found dead or missing. Female 42 from the low dose group showed a total litter loss on Day 2 post partum whereas Female 61 from the 300 mg/kg bw/day dose group showed 10 pups missing on the same day; there were no similar incidents amongst litters from the high dose group and these findings were considered unlikely to be treatment-related. Any other clinical observations were considered to be low incidence findings observed in offspring in studies of this type and as such unrelated to the toxicity of the test item.

Pathology
Necropsy
Offspring
Macroscopic necropsy findings for offsprings on the study did not indicate effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.

Effect levels (fetuses)

Key result
Dose descriptor:
other: No observed effect
Sex:
not specified
Basis for effect level:
other: No observed effect
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guideline 421.

….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (distilled water). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Surviving males were terminated on Day 43, followed by the termination of all littering females and their surviving offspring on Day 5 post partum. Female 92 which did not produce a pregnancy was terminated on Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

There were no treatment-related deaths on the study. One control male was killed in extremis on Day 35 of dosing due to a physical injury. Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item. There was no adverse effect of treatment with FAT 40868/A TE on male body weight development during the study. There was also no effect of treatment with the test item on body weight development in females during the pre-pairing, gestation or lactation phases of the study. Food intake across all of the test item-treated male groups remained similar to controls during the study. There was also no effect of treatment with FAT 40868/A TE on food consumption in females during the pre-pairing, gestation or lactation phases of the study. Visual inspection of water bottles did not reveal any treatment-related differences in water intake. There was no effect of treatment on mating performance. All animals mated within four days of pairing. There were no treatment-related effects in conception rates for test item-treated animals in relation to controls. There were no differences in gestation lengths in animals receiving the test item when compared with controls. There was no detrimental effect of treatment with FAT 40868/A TE on corpora lutea count, pre-implantation loss, number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day. There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs up to Day 5 of age at 100, 300 or 1000 mg/kg bw/day. Most animals of either sex treated with 1000 mg/kg bw/day showed blue discoloration of the skin and/or kidneys with the latter appearing to be dark in some animals. Dark kidneys were also observed in two females from the 300 mg/kg bw/day dose group. No such effects were evident in males treated with 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day. There was no effect of treatment with the test item on absolute or body weight-related organ weights when compared with controls. There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. There was no histopathological correlation with the macroscopic discoloration of the tissues from the high dose animals noted at necropsy. Expected minor findings were apparent in the kidneys and/or skin examined but these were not considered to be attributed to treatment with the test item.

In summary, the oral (gavage) administration of FAT 40868/A TE to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. Based on the results of the study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.