Fyroflex SOL-DP

Developmental toxicity / teratogenicity

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 study, conducted according to standardized guidelines, with analytical verification of test compound concentrations

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

SOL-DP (Batch No. 869090084), a white powder. Stored at room temp, dry, closed container.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animals' age allocated for the study (Day 0 of gestation): 20 to 27 weeks for the preliminary phase and 24 to 29 weeks for the main phase.
Bodywights: 3.19 to 4.03 kg for the preliminary phase and 3.05 to 4.57 kg for the main phase.

Housing and Husbandary:
The animals were housed individually in type LCP cages.
Temp: 16-20 Deg C
RH: protocol 40-70%
14 hours continious light and 10 hours continious dark per 24 hours, with the lights on at 6:00 GMT.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose, in water for formulation

Details on exposure:

The test substance was prepared for administration as a series of graded concentrations in the vehicle. The required amounts of test material were wighed out. Starting with the lowest concentration, the test material was transferred to a mortar and ground to a fine powder. Small amounts of vehicle were added and mixed with the test material to form a smooth paste. The suspension was poured into a measuring cylinder. This was then made to the required volume to provide the correct concentration. The suspension was then mixed using a high shear homogeniser and
transferred to the final issue containers, via syringe, whilst magnetically stirring. The remaining concentrations were made in ascending concentration order. Dosing formulations were prepared weekly and stored at room temperature, protected from light, prior to administration.

The exposure of the animals to the test material was by gavage and was not mixed with the food.

VEHICLE: A similarly constituted Control group received the vehicle 1% MC (methylcellulose, in water for formulation), at the same volume-dose throughout the same period.

The animals were allowed access to a standard rabbit diet, StanRab diet. Diet availability was restricted to 150 g per animal per day during acclimatisation up to one week prior to the onset of mating, or 200 g/animal/day thereafter. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. In addition, once daily, each rabbit was given a handful of autoclaved hay to promote gastric motility. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration during the first and last weeks of treatment (i.e first week in the preliminary phase and last week in the main phase) were analysed for achieved concentration of the test substance. Four samples (nominally 1 mL accurately weighed) were taken from all formulations; two samples were assayed from each formulation. The remaining samples from each formulation were retained frozen (nominally -20°C) as contingency for analysis if required.
The samples were analysed in accordance with the validated Huntingdon Life Sciences Analytical Procedure FIA/M093/10. The analytical method involved dissolution in acetonitrile and further dilution using acetonitrile/water 50/50 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (261 nm). Sample concentrations were determined with reference to external standards prepared in the concentration range 2.0 - 10.0 μg/mL.
At Week 1 and the last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis.Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. The contingency samples for Week 1 Group 2 were analysed as the coefficient of variation (CV) was outside the applied limit of 5%.

Details on mating procedure:

Females were allowed a minimum of two weeks acclimatisation before being selected for natural mating with stock males.
The males were checked for general health before pairing and were used for pairing on up to four consecutive days. The colony of stock males used was maintained specifically for the purpose of pairing. These males were considered not to be part of the study, and were
returned to stock after pairing. On each day of pairing, sufficient numbers of healthy females, which showed evidence of vaginal oestrus, were paired on a 1:1 basis with identified stock New Zealand White bucks. Pairs were observed to naturally mate at least twice Individual mating records were kept, and any reason for premature separation was documented.
Mated females were removed after the mating process was complete and 25 i.u. of luteinising hormone (Chorulon) was administered intravenously to promote successful ovulation. The day of mating was designated Day 0 of gestation. Females were allocated to group and cage position in sequence of mating on Day 0 of gestation. The animals were given unique new identity numbers, using an ear tag. Females mating on any one day were evenly distributed amongst the groups.

Duration of treatment / exposure:

22 days - from implantation (Day 6 after mating) until Day 28 after mating (shortly before the end of pregnancy).

Frequency of treatment:

daily

Duration of test:

29 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Initially, a preliminary phase of three groups, each comprising six female rabbits, received SOL-DP by gavage at doses of 100, 300 or 1000 mg/kg/day from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle 1% MC (methylcellulose, in water for formulation), at the same volume-dose throughout the same period. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. This preliminary phase of the study was designed to initially investigate the effects of treatment in a small number of animals.

As no adverse effects of treatent were observed, an additional 16 animals were allocated to each group to form the main phase of the study in order to form the full complement of 22 females at each dose level. The main phase females followed the same treatment regime as the preliminary phase.
For each phase, clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 29 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

For reporting purposes, data for the two phases were combined.

Examinations

Maternal examinations:

Clinical observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health of the occupant.

Detailed observations were recorded daily throughout the treatment period, at the following times in relation to dose administration:
Immediately before dosing, between one and two hours after completion of dosing of all groups and as late as possible in the working day.
In addition, a more detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 24 and 29 after mating to monitor general health.

Bodyweight:
The weight of each rabbit was recorded weekly during acclimatisation, on the day of mating (Day 0), and on Days 3 and 6-29 after mating.

Food consumption:
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded on a daily basis from Day 1 after mating.

Time of necropsy:
Animals surviving until the end of the scheduled study period were killed on Day 29 after
mating. The Control female animal exhibiting pregnancy loss was killed on the same day
that abortion was detected.

Macroscopic pathology:
All animals were subjected to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral midline incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was
recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The following reproductive assessment was made:
The gravid uterus was weighed prior to removal of the concepti; this weight included the weight of the ovaries. For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn.
For animals exhibiting pregnancy loss, expelled uterine contents were identified and examined, as appropriate.
The retained tissues were checked before disposal of the carcass.

Fetal examinations:

All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were
recorded. The neck and thoracic and abdominal cavities of all fetuses from each litter were dissected, the contents examined for visceral abnormalities and sex recorded.
Following visceral examination, one third of the fetuses in each litter were decapitated and their heads initially stored in Bouin’s fluid. The eviscerated fetuses and torsos were fixed in industrial methylated spirits prior to processing and staining with Alizarin Red.

Pathology:
Free-hand serial sections were prepared from the Bouin’s fixed heads and were examined under the microscope for soft tissue abnormalities within the head. Fetuses and decapitated torsos stained with Alizarin Red were assessed for skeletal development and abnormalities.

Statistics:

Parametric analysis, Bartlett's test, Two-way analysis of variance (ANOVA), t-tests, Williams’ test, Dunnett's test, Kruskal-Wallis’ test, Wilcoxon rank sum tests, Shirley's test, Steel's test
For more detailes, see attached on Statistics

Historical control data:

n/a

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no effect

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

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)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effect

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that daily oral gavage administration of SOL-DP to female New Zealand White rabbits up to and including 1000 mg/kg/day did not cause adverse effects upon maternal parameters or embryo-fetal survival, growth and development. The highest dose of 1000 mg/kg/day is therefore the No Observed Adverse Effect Level (NOAEL) in this study.

Executive summary:

The influence of SOL-DP on embryo-fetal survival and development in New Zealand White rabbits was assessed following oral administration from implantation (Day 6 after mating) until Day 28 after mating (shortly before the end of pregnancy).

Initially a preliminary phase of three groups, each comprising six female rabbits, received SOL-DP by gavage at doses of 100, 300 or 1000 mg/kg/day from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle 1% MC (methylcellulose, in water for formulation), at the same volume-dose throughout the same period. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. This preliminary phase of the study was designed to initially investigate the effects of treatment in a small number of animals. As no adverse effects of treatment were observed, an additional 16 animals were allocated to each group to form the main phase of the study in order to form the full complement of 22 females at each dose level.

The main phase females followed the same treatment regime as the preliminary phase. For each phase, clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 29 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination. For reporting purposes, data for the two phases were combined.

Administration of SOL-DP by oral gavage at dose levels of 100, 300 or 1000 mg/kg/day was well tolerated by pregnant New Zealand White rabbits, with no treatment-related mortalities observed. One Control female was killed for reasons of animal welfare on Day 17 of gestation due to signs of abortion and subsequent respiratory distress but this was related to the low implantation count and not to treatment with SOL-DP. There were no dosing signs observed and no clinical signs or macroscopic necropsy findings that were related to treatment. Maternal bodyweight (including bodyweight adjusted for the contribution of the gravid uterus) and food consumption were unaffected by treatment. Embryo-fetal survival was unaffected by treatment with no effects upon litter data and placental, litter and fetal weight, and there were no fetal findings observed that were considered to represent any adverse effect upon fetal development.

In conclusion, based on the results of this study it is concluded that daily oral gavage administration of SOL-DP to female New Zealand White rabbits up to and including 1000 mg/kg/day did not cause adverse effects upon maternal parameters or embryo-fetal survival, growth and development. The highest dose of 1000 mg/kg/day is therefore the No Observed Adverse Effect Level (NOAEL) in this study.