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Toxicological information

Toxicity to reproduction

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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
Study period:
2010-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline conform

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: not mentioned
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females
were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation : Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): pelleted diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 1 September 2010 (allocation) To: 18 October 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily, 30min prior dosing
- Storage temperature of food: ambient temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Amount of vehicle (if gavage): 5 ml
- Propylen glycol, specific gravity 1.036
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and
females, one female was cohabitated with one male of the same
treatment group, avoiding sibling mating. Detection of mating was
confirmed by evidence of sperm in the vaginal lavage or by the
appearance of an intravaginal copulatory plug. This day was
designated Day 0 post-coitum. Once mating occurred, the males
and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a
validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentrations) and accuracy
of preparation (all concentrations). Stability in the vehicle over 6 hours at room temperature was
also determined (highest and lowest concentration).
An extra stability analysis of the Group 4 formulation was conducted on Day 21 of the treatment
period. Measurements were conducted from the 50% position of the container and duplicate
samples were analyzed.
The accuracy of preparation was considered acceptable if the mean measured concentrations
were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of
variation was ≤ 10%. Formulations were considered stable if the relative difference before and
after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating,
during mating, and up to termination. Females were exposed for
42-46 days, i.e. during 2 weeks prior to mating, during mating,
during post-coitum, and during at least 4 days of lactation.
Female nos. 50 (Group 1), 77, 78 and 79 (Group 4) were not
dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each
day with a maximum of 6 hours difference between the earliest and
latest dose.
Details on study schedule:
Parturition
The females were allowed to litter normally. Day 1 of lactation was
defined as the day when a litter was found completed (i.e.
membranes and placentas cleaned up, nest build up and/or
feeding of pups started). Females that were littering were left
undisturbed.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 100 and 200 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
40
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on data obtained from the Material Safety Data Sheet, based on the
results of the 10-Day pilot study, and in consultation with the Sponsor, the dose levels for this reproduction/developmental toxicity
screening test were selected to be 50, 100 and 200 mg/kg.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of exposure, weekly thereafter, Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

-reproduction data:
mating index
fertility index
conception index
gestation index
Sperm parameters (parental animals):
Parameters examined in [all] male parental generations:
testis weight, epididymis weight, stage of spermatogenisis was examined by histopathology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
mortality, clinical signs, body weights, sex
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals , following completion of mating period (min day 28)
- Maternal animals: All surviving animals, either lactation day 5-7 or post-coitum day 26

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
- female: corpora lutea and implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively.
liver, kidney, spleen, testes, epidymides
Postmortem examinations (offspring):
SACRIFICE
killed by decaptation on days 5, 6 or 7

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach
was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
Dunnett-test, Steel-test, Fisher Exact-test,
Reproductive indices:
Mating index, Fertility index, Conception index, Gestation index, Duration of gestation,
Offspring viability indices:
Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Haematology
The following (statistically significant) changes in haematology parameters distinguished treated females from control animals:
-Lower relative monocyte counts (200 mg/kg)
-Lower red blood cell counts (50, 100 and 200 mg/kg)
-Higher reticulocyte counts (50, 100 and 200 mg/kg; not significant at 200 mg/kg)
-Higher red blood cell distribution width (RDW; 50, 100 and 200 mg/kg)
-Lower haemoglobin levels (200 mg/kg)
-Lower haematocrit levels (100 and 200 mg/kg).

Clinical Biochemistry
At 200 mg/kg the following changes in clinical biochemistry parameters distinguished treated females from control animals:
-Higher alanine aminotransferase (ALAT) levels
-Higher alkaline phosphatase (ALP) levels
-Higher total bilirubin levels
-Higher potassium levels
-Higher glucose levels (both sexes)

Macroscopic Examination
Accentuated lobular pattern of the liver was noted for two females at 100 mg/kg and for two females at 200 mg/kg. Microscopically, this correlated to slight vaculolation of the hepatocytes of the periportal area or hypertrophy of the hepatocytes of the centrilobular area at 100 mg/kg, and to congestion or minimal hypertrophy of the hepatocytes of the centrilobular area at 200 mg/kg, which constituted a toxicologically-relevant finding.

Organ weights
At 200 mg/kg absolute organ weights and organ to body weight ratios were significantly higher than controls for the liver (fresh) and kidneys (fresh and post-fixed) for females only. At 100 mg/kg, liver to body weight ratios (fresh) and kidney to body weight ratios (fresh and post-fixed) were significantly higher than controls for females. At 50 mg/kg, the female kidney to body weight ratios (fresh) were significantly higher than control values. Organ weights and organ weight ratios were unaffected for males at any dose level.

Microscopic examination
Treatment related microscopic findings were noted in the liver (both sexes) and kidneys (females). An increased incidence and severity of hypertrophy of the hepatocytes of the centrilobular area in the liver was recorded in 7/10 males and 6/10 females in Group 4 (200 mg/kg bw/day; minimal to slight). Vacuolation of the epithelium of the proximal tubule of the kidneys was recorded in 5/10 females of Group 4 (200 mg/kg bw/day; minimal to slight); males were not affected.

Hypertrophy of the hepatocytes of the centrilobular area was also seen in 1/10 males of Groups 1 (control), 2 (50 mg/kg bw/day) and 3 (100 mg/kg bw/day), and was not considered to be treatment related for these groups.

The severity (up to a moderate degree) of hematopoietic foci (primarily erythropoiesis) recorded in the spleen of females was above background but comparable for Groups 1 and 4. This was considered to be a physiological response to the pregnancy/lactation of these females.

There were no treatment related findings noted in any reproductive organs up to 200 mg/kg bw/day and the spermatogenic staging profiles were normal for all Group 1 and Group 4 males.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: changes in clinical biochemistry parameters, macroscopic findings, organ weights and microscopic findings in the liver (for both sexes) and kidneys at 200 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Gross pathological findings:
no effects observed

Details on results (F1)

Mortality
Two pups of the control group, five pups at 50 mg/kg, six pups at 100 mg/kg and two pups at
200 mg/kg were found dead or were missing during the first days of lactation. Pups missing
were most likely cannibalized. No toxicological relevance was attributed to these dead or
missing pups since the mortality incidence did not show a dose-related trend and remained
within the range considered normal for pups of this age.

Clinical signs
Incidental signs including a missing tail and small size were noted for individual pups at 50 and
200 mg/kg, respectively. The nature and incidence of these clinical signs remained within the
range considered normal for pups of this age, and were therefore not considered to be
toxicologically relevant.

Body weight
At 200 mg/kg, the body weights of both sexes were significantly reduced on both lactations
Days 1 and 4. Body weights of female pups at 50 and 100 mg/kg were both significantly lower
than controls on lactation Day 1.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: reduced body weight, considered secondary to maternal toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

Parental results:

Parental toxicity, primarily for females, was evident at 200 mg/kg. This was characterized by changes in clinical biochemistry parameters, macroscopic findings, organ weights and microscopic findings in the liver (for both sexes) and kidneys. For females at 200 mg/kg there were changes in clinical biochemistry parameters that are suggestive of liver cell membrane degradation. Additional findings were noted including higher absolute and relative liver weights, accentuated lobular pattern of the liver noted at macroscopic examination and the microscopic finding consisting of hypertrophy of the hepatocytes of the centrilobular area in the liver. These females also had higher absolute and relative kidney weights and vacuolation of the epithelium of the proximal tubule of the kidneys found at microscopic examination. Collectively, these findings are evidence of an effect of the test substance and were assessed as adverse.

At 100 mg/kg, accentuated lobular pattern of the liver was noted for a few females along with higher liver to body weight ratios. Taken together with the finding of hypertrophy of the hepatocytes of the centrilobular area in the liver at microscopic examination, these data collectively suggest the presence of a mild functional change in normal liver activity. Since there were no signs of any cellular damage and mild functional liver changes are reversible, there findings were considered to be adaptive and not adverse.

There were several statistically significant changes in haematology parameters that distinguished treated females from controls. At the high-dose level (200 mg/kg bw/day) decreased red blood cells (by 15%), haematocrit (by 12%) and, in particular, reduced haemoglobin (by 12%) indicate a regenerative anaemia which, because of the degree of the effects, is considered to constitute an adverse effect. However, the toxicological relevance of the haematological findings in the low- and mid-dose females (50 and 100 mg/kg bw/day, respectively) is questionable since the red blood cell and haematocrit changes remained below 10%, were within the range of available historical control data, and a significant effect on haemoglobin is missing.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption).

Reproductive results: No reproduction toxicity was observed at any dose level.

Developmental results: Significantly lower body weights (about 12-15%) were observed for both sexes on lactation Days 1 and 4 for pups at 200 mg/kg, indicating delayed offspring development. Significantly lower body weights were seen for female pups at 50 and 100 mg/kg on lactation Day 1 only; because the difference from controls was only slight, remained within the normal range of historical control data and normalized closer to control levels by Day 4, they were not considered toxicologically-relevant.

No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

In conclusion, treatment with Pergafast 201 by oral gavage in male and female Wistar Han rats at dose levels of 50, 100 and 200 mg/kg body weight/day revealed parental toxicity at 200 mg/kg body weight/day. No reproduction toxicity was observed at any dose level. However, a delay in offspring development was seen at the maternally toxic dose level of 200 mg/kg bw/day, characterized by lower pup body weights for both sexes on Days 1 and 4 of lactation. There were no effects on pup morphology or on pup survival indicative of frank developmental toxicity, and there were no adverse findings for offspring evident at doses not producing maternal toxicity.

Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:

Parental NOAEL: 100 mg/kg/day

Reproduction NOAEL: at least 200 mg/kg/day

Developmental NOAEL: 100 mg/kg/day