Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2010 to 06 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): AMICURE PACM
- Molecular weight: 210.4
- Substance type: Clear colourless liquid (determined at NOTOX)
- Physical state: liquid
- Analytical purity: 100.0%
- Lot/batch No.: 725502
- Expiration date of the lot/batch: 11 June 2011
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark.
- Other:
Specific Gravity :0.96
pH : 12
Stability at higher temperatures: Not indicated
Cis-trans Isomer content:
4,4'-Methylenedicyclohexanamine >95%
4,4-PACM T,T: 21%
4,4-PACM C,T >40
4,4-PACM C,C <30
2,4'-Methylenedicyclohexanamine <5%


Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 21.5°C
- Humidity (%): 38 - 81%
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room.

Temporary deviations from the maximum and minimum level of relative humidity occured in the animal room. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 19 April 2010 To: 06 Augustus 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(Specific Gravity : 1.036)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Based on trial formulations performed at NOTOX. Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information provided by the sponsor.
- Concentration in vehicle: 3, 10 and 20 mg/mL

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Dose levels: 0, 15, 50 and 100 mg/kg. For Group 4 animals, the dose level was changed form 150 mg/kg to 100 mg/kg on 26 June 2010 (Day 12 of the treatment period).

Preventive measures: As the test substance has a pH of 12 and is a severe irritant, AMICURE PACM might cause local esophagal and gastrointestinal effects. Therefore, the following preventive measures were taken:
-Wiping of the flexible catheter prior to dosing of each animal.
-Additional flushing of the catheter with 0.5 mL water (Elix, Millipore S.A.S., Molsheim, France) after dosing each animal with the test formulations.


Inadvertantly, on Days 8 and 9 of the study, the dose volume administered to one control animal was 1.05 mL instead of 1.59 mL due to a weighing error. As this was a control animal, a slightly lower dose volume for 2 days has no adverse affect on the study integrity. The weight and dose volume were corrected for this animal so all subsequent weights and dosing were correct.
Details on mating procedure:
- M/F ratio per cage: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
- Length of cohabitation: Following a minimum of 14 days of exposure for the animals, one female was cohabitated with one male of the same treatment group, avoiding sibling mating.

- Proof of pregnancy: 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.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion the treatment phase, according to a validated method (NOTOX project 493845). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. 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%.

Results:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

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. Animals were dosed up to the day prior to scheduled necropsy.
For Group 4 animals, the dose level was changed form 150 mg/kg to 100 mg/kg on 26 June 2010 (Day 12 of the treatment period).

After dosing on Day 9, one Group 4 female expelled some test substance, after which the technician intubated the animal a second time to administer a second water flush. This was done to prevent local irritation by the test substance. Because the test substance is irritating and corrosive, the biotechnician was taking protective emasures to minimize the chance of local esophogeal irritation by flushing with more water after the animal had expelled some test substance. This does not adversely affect the study.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 14 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10 and 20 mg/mL/day (0, 15, 50 and 100 mg/kg/day)
Basis:

No. of animals per sex per dose:
10 sex/group + 398 pups
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were based on results of a 10-Day dose range finding study (NOTOX Project 493843).
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, at least 2 hours after dosing (+/- 30 min) detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. At weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

On 20 June 2010 (Day 6 of treatment) clinical signs were documented in the raw data for one Group 4 female. There was no degree noted for the observation 'pale.' Sufficient information is available for a thorough evaluation and eliminating the grade of paleness has no adverse effect on the study's integrity.
On eight days the time between dosing and observations was longer than 2.5 hours, by a range of 3-32 minutes over the alloted time. The slight deviation in time has no adverse impact on the integrity of the data.

BODY WEIGHT: Yes
- Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. To more closely monitor the health of the high dose animals after changing the dose from 150 to 100 mg/kg, all animals were weighed twice during Week 3 of the pre-mating period. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. On Day 21 (5 June 2010) of the treatment period, an additional body weight was collected for one Group 4 female due to bad health. This was collected on paper (not on-line) and was reported separately.

FOOD CONSUMPTION: Yes
Weekly, except for males and females which were housed together for mating. To more closely monitor the health of the high dose animals after changing the dose from 150 to 100 mg/kg, all animals had food consumption measured twice during Week 3 of the pre-mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
(Average food consumption (per animal per day)/average body weight per cage) X 1000

WATER CONSUMPTION : Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes,eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

Oestrous cyclicity (parental animals):
not determined.
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testes weight, epididymides weight.

Of the selected 5 males of the control and high dose group, and all males suspected of infertility, additional slides of the testes were prepared to examine staging of spermatogenesis.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined
Litter observations:
PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
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.
Postmortem examinations (parental animals):
All males and the selected 5 females/group were fasted overnight (with a maximum of 21.5 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to the scheduled necropsy and all moribund animals were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Nine animals were necropsied after approximately 21.5 hours of fasting which was longer than the maximum of 20 hours allowed. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7.
Females which failed to deliver: Post-coitum Day 25-27 (females with evidence of mating)
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Animals killed in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Selected 5 animals/sex/group (see Allocation) and the females which were killed in extremis:
Identification marks: not processed, Ovaries, Adrenal glands, Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides *, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland) *, (Skin), Spinal cord -cervical, midthoracic, lumbar, Female mammary gland area, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach, Ileum, Testes *, Jejunum, Thymus, Kidneys, Thyroid including parathyroid (if detectable)
(Larynx), (Tongue), (Lacrimal gland, exorbital), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

All remaining animals and females which failed to deliver :
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes 1, Epididymides 1, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions, Identification marks: not processed

* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights (and terminal body weight) were recorded from the surviving animals:

Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix) , Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.
* weighed when fixed for at least 24 hours.

From all remaining Main males: Epididymides, Testes.

Histotechnology: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males of the control and high dose group additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Histopathology: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which were killed in extremis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals that failed to mate, conceive, sire or deliver healthy pups:

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

One mandibular lymph node of one Group 3 male and one eye of one Group 4 female was not available for histopahtology because these tissues were not collected at necropsy or discernable at trimming. Sufficient data was available for evaluation.
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by decapitation on Day 5 or 6 of lactation.

GROSS NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGTHS
no.
Statistics:
The following statistical methods were used to analyze the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test; Ref. 2) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test; Ref. 3) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950; Ref. 4) was applied to frequency data.

An attempt was made to transform the number of corpora lutea and implantation sites by using 1/x, log x, x2 and √x. However, a normal distribution was not obtained. Therefore, the number of corpora lutea and implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 5) to determine inter-group differences, followed by the Wilcoxon test (Ref. 6) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group the following calculations were performed:

Percentage mating = Number of females mated/Number of females paired x 100

Fertility index = Number of pregnant females/Number of females paired x 100

Conception rate = Number of pregnant females/Number of females mated x 100

Gestation index = Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check = Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check = Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 lactation = Number of dead pups on Day 4 lactation/Number of live pups at First Litter Check x 100

Viability index = Number of live pups on Day 4 lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant clinical signs noted up to 50 mg/kg.
Prior to euthanasia, animal no. 73 had piloerection and rales over 2 and 3 days, respectively
and was also noted with flat posture, hunched posture and pale appearance on the day of
euthanasia. Animal no. 79 showed hunched posture, rales and yellow feces over several days
before necropsy and laboured respiration, lean appearance and piloerection over single days
prior to necropsy.
At 100 mg/kg, yellow feces were noted for nearly all animals beginning after approximately one
week of treatment which persisted through the rest of the treatment duration. Hunched posture
was also noted for most females and for several males throughout most of the treatment period.
Several animals at 100 mg/kg also displayed rales (rales were also transiently noted for a single
female at 50 mg/kg), laboured respiration, piloerection, lean appearance, ptosis, lethargy and
chromodacryorrhoea, but at a lower frequency and for a much shorter duration than noted for
the yellow feces and hunched posture.
Focal erythema of the right flank or left ear was noted for animals 42 and 45 (Group 1),
respectively. Because they were both control animals, these signs were not related to treatment
with AMICURE PACM.
Incidental findings that were noted for control and treated animals included alopecia, scales
and/or scabbing over various areas of the body. These findings occurred within the range of
background findings to be expected for rats of this age and strain which are housed and treated
under the conditions in this study. At the incidence observed, they were considered to be signs
of no toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female at 150 mg/kg (Group 4; no.73) received treatment up to Day 5 and was euthanized
in extremis on Day 6 of treatment, and one female at 100 mg/kg (Group 4; no. 79 was
euthanized in extremis after 21 days of treatment. No definitive cause of death could be
determined for both of these high dose animals, though both were related to treatment with the
test substance.
There were no other unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute body weights and body weight gains were significantly lower for animals at 100 mg/kg
beginning on Day 8 of the pre-mating period for females and males. These reduced weights
persisted throughout the rest of the treatment period and reduced weight gains persisted
through the rest of the treatment period for males and until Day 1 of the mating period for
females.
At 50 mg/kg, absolute body weights were reduced from Days 14-20 of the post coitum period.
No other body weights or body weight gains differed significantly from controls.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No reproduction toxicity was observed up to 15 mg/kg.
Reduced numbers of implantation sites were noted for females at 50 and 100 mg/kg.
Developmental results:
No developmental toxicity was observed up to 15 mg/kg.
At 50 mg/kg, the gestation index was reduced compared to controls due to two females with
implantation sites only, though there was no toxicologically relevant effect on the number of
living pups at first litter check.
A reduced number of viable fetuses were seen for females at 100 mg/kg, and females at 50
mg/kg had a higher incidence of postnatal loss with a corresponding reduction in the viability
index.
AMICURE PACM NOTOX Project 493842
- Page 32 -
In conclusion, treatment with AMICURE PACM by oral gavage in male and female Wistar Han
rats at dose levels of 15, 50 and 100 mg/kg body weight/day revealed parental toxicity at 50
mg/kg characterized by microscopic findings in various organs and at 100 mg/kg body
weight/day characterized by changes in body weights, food consumption, clinical biochemistry
parameters, organ to body weight ratio changes and histopathological findings in various
organs. Reproduction toxicity was observed at 50 and 100 mg/kg characterized by a reduction
in implantation site number. Developmental toxicity included a lower gestation and viability
indices and increased postnatal loss for females at 50 mg/kg, and a reduced number of living
pups at first litter check at 100 mg/kg.

Details on results (P0)

Mortality
One female at 150 mg/kg (Group 4) received treatment up to Day 5 and was euthanized in extremis on Day 6 of treatment, and one female at 100 mg/kg (Group 4) was euthanized in extremis after 21 days of treatment. No definitive cause of death could be determined for both of these high dose animals, though both were related to treatment with the test substance.

There were no other unscheduled deaths.

Clinical Signs
There were no toxicologically significant clinical signs noted up to 50 mg/kg.

Prior to euthanasia, one Group 4 female had piloerection and rales over 2 and 3 days, respectively and was also noted with flat posture, hunched posture and pale appearance on the day of euthanasia. One other Group 4 female, showed hunched posture, rales and yellow feces over several days before necropsy and laboured respiration, lean appearance and piloerection over single days prior to necropsy.

At 100 mg/kg, yellow feces were noted for nearly all animals beginning after approximately one week of treatment which persisted through the rest of the treatment duration. Hunched posture was also noted for most females and for several males throughout most of the treatment period. Several animals at 100 mg/kg also displayed rales (rales were also transiently noted for a single female at 50 mg/kg), laboured respiration, piloerection, lean appearance, ptosis, lethargy and chromodacryorrhoea, but at a lower frequency and for a much shorter duration than noted for the yellow feces and hunched posture.

Focal erythema of the right flank or left ear was noted for two Group 1 females. Because they were both control animals, these signs were not related to treatment with AMICURE PACM.

Incidental findings that were noted for control and treated animals included alopecia, scales and/or scabbing over various areas of the body. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, they were considered to be signs of no toxicological relevance.

Functional observations
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

The variation in motor activity did not indicate a relation with treatment.
The high sensor count for one group 1 male was high enough at one 5 minute time point to be biologically implausible, and as such it was excluded from calculations. Similarly, differences seen between treated animals and control females regarding counts of the high sensor are attributable to lower values for control females than normal, and do not reflect treatment related toxicity.
The extremely high counts for the high sensor seen for Group 4 males was attributable to a very high value obtained for one animal. If the mean is re-calculated without that data, a mean of 46 is obtained, which is in line with control values. Similarly, the higher counts obtained for the low sensor for females at 50 mg/kg were attributable to high values obtained for one female. If the mean is re-calculated without her data, a mean of 293 is obtained, which is in line with control values. In the absence of corroborative clinical signs, like hyper activity, noted for either animal during normal clinical or arena observations, in neither case do these high values reflect treatment related toxicity.

Body Weights
Absolute body weights and body weight gains were significantly lower for animals at 100 mg/kg beginning on Day 8 of the pre-mating period for females and males. These reduced weights persisted throughout the rest of the treatment period and reduced weight gains persisted through the rest of the treatment period for males and until Day 1 of the mating period for females.

At 50 mg/kg, absolute body weights were reduced from Days 14-20 of the post coitum period.

No other body weights or body weight gains differed significantly from controls.

Food Consumption
At 100 mg/kg, absolute and relative food consumption was reduced over Days 1-15 of the pre-mating period (not statistically significant). Absolute food consumption was also slightly reduced for females from Days 4-11 of the post coitum period and from Days 1-4 of the lactation period.

For males at 100 mg/kg, relative food consumption was reduced over Days 1-18 of the pre-mating period, though this is most likely attributable to relatively high food consumption for controls over the same period.

At 50 mg/kg, absolute and relative food consumption was reduced for females from Days 1-4 of the lactation period.

Food consumption was similar between control and treated animals at all other time points.

Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.

The significant increases in mean corpuscular haemoglobin (MCHC) and platelets noted for males and females at 100 mg/kg, respectively, and the significant reduction in haematocrit values noted for males at 50 mg/kg were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.


Clinical Biochemistry
At 100 mg/kg, aspartate aminotransferase (ASAT) was significantly increased and creatinine was significantly reduced for both sexes compared to controls. ASAT was also increased for males at 50 mg/kg compared to controls, though it was not statistically significant.

The statistically significant increase in alanine aminostransferase (ALAT) and the significant reductions seen in total protein and cholesterol seen for males at 100 mg/kg were not considered to be toxicologically relevant as the difference compared to controls was slight and they remained within the range considered normal for rats of this age and strain.

Macroscopic Examination
There were no toxicologically-significant macroscopic findings noted for animals up to 100 mg/kg that survived the duration of the treatment period.

The macroscopic findings noted for one Group 4 animal at 150 mg/kg that was euthanized in extremis included GI tract distended with gas, yellowish contents of the small intestine, reduced size of the left clitoral gland or of the thymus, and enlarged adrenal glands. Findings for the second Group 4 female at 100 mg/kg, who was also euthanized in extremis, included emaciated, caecum distended with gas, thymus reduced in size and alopecia on the right shoulder.

Red-brown, yellowish, greenish and/or several dark red foci on the clitoral glands were noted for two females each at 15, 50 and 100 mg/kg. At the incidence observed, it was not considered to be toxicologically relevant.

Incidental findings among control and treated animals included alopecia, scabbing or a sore found on various regions of the body, reduced size of the seminal vesicles and red-brown discoloration of the thymus. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

Organ Weights
At 100 mg/kg, significantly higher kidney to body weight ratios (males) and liver to body weight ratios (both sexes) were seen compared to control animals. Animals of both sexes had lower terminal body weights compared to controls.

The lower absolute brain weight seen for females at 100 mg/kg was attributable to higher than normal absolute brains weights seen for controls and was not a sign of treatment related toxicity. The lower thyroid weights for females were also not considered to be toxicologically relevant because it occurred in the absence of any correlating macroscopic or histopathological changes and the relative weight was normal. Similarly, the higher testes to body weight and epididymides to body weight ratios noted for males were not considered to be toxiclogically significant because there were no correlating changes in microscopic findings, nor were there any problems for these males in impregnating females or producing litters.

At 50 mg/kg, lower absolute seminal vesicle weights were noted for males without a corresponding reduction in body weight at necropsy. One group 3 male had a very low seminal vesicle weight, which could explain the low seminal vesicle weights, however, even discounting his data, the mean seminal vesicle weights at 50 mg/kg remained lower than controls. In the absence of corroborative microscopic findings, this was not considered to be toxicologically relevant.

The increased brain to body weight ratios noted for males at 100 mg/kg were attributable to their lower body weight at necropsy compared to controls, and was not a sign of toxicity.


Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Microscopic Examination
Treatment-related microscopic findings were present in:

100 mg/kg:
-Stomach: Vacuolation of the musculature seen for 5/5 males and 6/7 females.
-Liver: Centrilobular vacuolation noted for 5/5 males and in 6/7 females.
-Brain: Vacuolation of the choroid plexus was seen for 5/5 males and 7/7 females.
-Skeletal muscle: Vacuolar myofiber degeneration and myofiber degeneration was seen in 5/5 males and 7/7 females.
-Eyes: Vacuolation of the cuboidal epithelium of the iris was seen for 4/5 males and 5/7 females.

50 mg/kg:
-Stomach: Vacuolation of the musculature seen for 2/5 males and 5/5 females.
-Liver: Centrilobular vacuolation was recorded in 3/5 males and 1/5 females.
-Skeletal muscle: Vacuolar myofiber degeneration was recorded in 2/5 males and in 1/5 females, myofiber degeneration was seen in 5/5 males and 5/5 females.
-Eyes: Vacuolation of the cuboidal epithelium of the iris was recorded in 2/5 males and in 2/5 females.

Skeletal myofiber degeneration was also recorded in 3/5 males of Group 1 and in 1/5 males of Group 2 (15 mg/kg), though the incidence and severity of this finding was higher for both sexes at 50 and 100 mg/kg.

All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

No abnormalities were seen in the reproductive organs of non-fertile animals which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 15 - < 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproduction toxicity was observed at 50 and 100 mg/kg characterized by a reduction in implantation site number.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Four pups of the control group, two pups at 15 mg/kg, seven pups at 50 mg/kg and four pups at
100 mg/kg were found dead or missing during the first days of lactation. Pups missing were
most likely cannibalized. The number of dead pups at 50 mg/kg is relatively high, though no
toxicological relevance was attributed to these dead/missing pups since the mortality incidence
did not show a dose-related trend and they remained within the range considered normal for
pups of this age.
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Reproduction data
Reproductive endpoints were unaffected up to 15 mg/kg.

The number of implantation sites were 14.1, 13.0, 10.7 and 11.5 for Groups 1, 2, 3 and 4, respectively. Implantation site number was significantly lower than controls for females at both 50 and 100 mg/kg.

The mating, fertility and conception indices, precoital time, and the number of corpora lutea were unaffected by treatment.

Developmental data
No toxicologically relevant effects on the gestation duration, parturition, and maternal care were seen up to 100 mg/kg. The clinical signs, body weight and macroscopic endpoints of the early postnatal pup development assessment were also unaffected by treatment.

Gestation
The gestation index and duration of gestation was similar between controls and treated animals at 15 and 100 mg/kg. At 50 mg/kg, the gestation index was 77.8%; a reduction compared to 100% for controls. The gestation index was also 100% for animals at 15 and 100 mg/kg as well. There were 9, 10, 9 and 8 pregnant females in the control, 15, 50 and 100 mg/kg groups, respectively. At 50 mg/kg, two females were pregnant but had implantation sites only, thus only 7 litters with viable pups were produced at this dose level.

Parturition/Maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

Early postnatal pup development
There were 13.4, 11.7, 11.9 and 8.9 living pups at first litter check in the control, 15, 50 and 100 mg/kg groups, respectively, with a statistically significant reduction in the number of living pups at first litter check seen at both 50 and 100 mg/kg. The significant reduction at 50 mg/kg does not reflect treatment-related toxicity as the mean number of live pups is nearly identical to those for animals at 15 mg/kg, and both remain similar with the 11.7 living pups per litter seen in the available historical control data. The statistical difference at this dose level (50 mg/kg) may be attributable to the relatively high number of pups per litter in controls compared to historical controls. However, a treatment-related effect on the reduction in living pups seen at 100 mg/kg cannot be excluded.

There was an increased postnatal loss noted at 50 mg/kg of 7.2% of living pups and a corresponding reduction in the viability index to 92.8%. Females from the control group had a postnatal loss incidence of 1.7% of living pups and a viability index of 98.3%. While the difference between control values was not statistically significant, and no dose-response relationship was seen, a treatment related effect of the test substance could not be excluded as the number of living pups at first litter check at 100 mg/kg was decreased. The postnatal loss seen for females at 15 and 100 mg/kg was 1.7 and 1.4% of living pups, respectively and the viability index was 98.3 and 98.6% for the same respective groups.

The statistically significant difference in the sex ratio seen for animals at 50 mg/kg (57:43) was attributable to the low male:female ratio (38:62) seen for control animals and was not related to treatment with AMICURE PACM.

The number of dead pups at first litter check, and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Mortality
Four pups of the control group, two pups at 15 mg/kg, seven pups at 50 mg/kg and four pups at 100 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalized. The number of dead pups at 50 mg/kg is relatively high, though no toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and they remained within the range considered normal for pups of this age.


Clinical signs
No milk in the stomach, a red spot on the nose, small or very small size, and pale appearance were incidental clinical symptoms of pups that were noted. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Body weights
Body weights of pups were considered to have been unaffected by treatment.

Macroscopy
Incidental macroscopic findings of pups that were found dead included scabbing of the cheek or nose and no milk in the stomach. Incidental macroscopic findings among surviving pups included small or very small size, a red spot on the nose, and an indentation of the skull
The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 15 - < 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on lower gestation and viability indices and increased postnatal loss at 50 mg/kg reduced numbers of viable pups at first litter check at 100 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Reproduction toxicity in females was observed at 50 and 100 mg/kg characterized by a reduction in corpora lutea and implantation site number. Two outliers in the 50 mg/kg bw/d group throw off the group means for these two endpoints.At 50 mg/kg bw/d, two demales were pregnant but had implantation sites only, causing the next parameter, gestation index, to be low (77/8%). All the other groups including the 100 mg/kg bw/d group had gestation indices of 100%. This lack of a dose-response calls into question the relevance of these indices in evaluation this study. Developmental toxicity in the F1 generation was based on lower gestation and viability indices and increased postnatal loss at 50 mg/kg reduced numbers of viable pups at first litter check at 100 mg/kg.

Applicant's summary and conclusion

Conclusions:
AMICURE PACM was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 15, 50 and 100 mg/kg/day. Males were exposed for 3 weeks prior to mating, during mating, and up to termination (for 36 days). The females were exposed for 3 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 48-52 days). Animals of the high dose group were exposed for the first 11 days to 150 mg/kg, whereby the high dose was lowered to 100 mg/kg due to treatment-related toxicity.

Formulation analysis showed that the formulations were prepared accurately, were homogenous, and were stable for at least 6 hours at room temperature.

Parental results:
There were treatment-related and toxicologically relevant effects at 50 and 100 mg/kg.

100 mg/kg
At the high dose, two females were euthanized in extremis. One female was euthanized after 5 days of treatment at 150 mg/kg, and the second female was euthanized after 21 days of treatment at 100 mg/kg.
In general, toxicologically-relevant clinical signs including hunched posture, yellow feces, piloerection, lean appearance, ptosis, lethargy, chromodacryorrhoea, and rales were noted. Lower body weights and body weight gains were observed throughout the treatment period (both sexes) along with reduced food consumption over much of the treatment period (both sexes). Clinical biochemistry parameters were affected including increased aspartate aminotransferase (ASAT) and reduced creatinine levels (both sexes) which are related to histopathological changes seen in skeletal muscle and liver (see below), along with higher kidney to body weight (males) and liver to body weight ratios (both sexes). Treatment related microscopic findings of various organs at 100 mg/kg were noted, including: the stomach (vaculolation of the stomach musculature; both sexes), liver (centrilobular vacuolation; both sexes), brain (vacuolation of the choroid plexus; both sexes), skeletal muscle (vacuolar myofiber degeneration and myofiber degeneration; both sexes), and the eyes (vacuolation of the cuboidal epithelium of the iris; both sexes).

50 mg/kg
Treatment related and toxicologically relevant microscopic findings were noted for various organs, including the stomach (vaculolation of the stomach musculature; both sexes), liver (centrilobular vacuolation; both sexes), skeletal muscle (vacuolar myofiber degeneration and myofiber degeneration; both sexes), and the eyes (vacuolation of the cuboidal epithelium of the iris; both sexes).
Slight reductions in body weights and food consumption were noted on a few occasions, but at the degree observed were not considered to be biologically significant.

No toxicologically relevant changes were seen at 15 mg/kg.

Reproductive results
No reproduction toxicity was observed up to 15 mg/kg.

Reduced numbers of implantation sites were noted for females at 50 and 100 mg/kg.

Developmental results:
No developmental toxicity was observed up to 15 mg/kg.

At 50 mg/kg, the gestation index was reduced compared to controls due to two females with implantation sites only, though there was no toxicologically relevant effect on the number of living pups at first litter check.
A reduced number of viable fetuses were seen for females at 100 mg/kg, and females at 50 mg/kg had a higher incidence of postnatal loss with a corresponding reduction in the viability index.


In conclusion, treatment with AMICURE PACM by oral gavage in male and female Wistar Han rats at dose levels of 15, 50 and 100 mg/kg body weight/day revealed parental toxicity at 50 mg/kg characterized by microscopic findings in various organs and at 100 mg/kg body weight/day characterized by changes in body weights, food consumption, clinical biochemistry parameters, organ to body weight ratio changes and histopathological findings in various organs. Reproduction toxicity was observed at 50 and 100 mg/kg characterized by a reduction in implantation site number. Developmental toxicity included a lower gestation and viability indices and increased postnatal loss for females at 50 mg/kg, and a reduced number of living pups at first litter check at 100 mg/kg.

Based on these results, the parental, reproduction and developmental No Observed Adverse Effect Levels (NOAELs) were considered to be 15 mg/kg/day.