Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
2011-12-28 to 2012-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

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)
Version / remarks:
22 March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test as of July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
NOTOX B.V. Hambakenwetering 7 5231 DD's-Hertogenbosch The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 weeks
- Housing: in Macrolon plastic cages (MIV type, height 18 cm)
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 22.2°C
- Humidity (%): 25 - 74%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hours artificial light and 12 hours darkness per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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/density of the test substance. No correction was made for the purity of the test substance.

Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: 14 days
- 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.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase according to a validated method. 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. 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%.

The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation < 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

On 05-01-12, the Group 2, Group 3 and Group 4 formulations used for dosing on 04-01-12 were analysed. The concentrations analysed were below target (i.e. 78% - 89%).

On 26-01-12, the Group 2, Group 3 and Group 4 formulations used for dosing were analysed. The concentrations analysed in the formulation of Group 2 and Group 4 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 were below target (i.e. 58%).

On 06-02-12, the Group 2, Group 3 and Group 4 formulations used for dosing were analysed. The concentrations analysed in the formulation of Group 2 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 and Group 4 were below target (i.e. 68% and 82%).

On 22-02-12, the Group 2, Group 3 and Group 4 formulations were prepared for analysis only. The concentrations analysed in the formulation of Group 2 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 and Group 4 were below target (i.e. 75% and 88%).

All formulations were prepared correctly and no analytical or other practical reason could be given for the variability in the analyses or the low accuracies obtained. Concentrations over a greater range were determined to be accurate in the 14-Day pilot study NOTOX Project 498336; BASF Project 01R0658/01X035). As such, the results were ultimately accepted and the results were attributed to the nature of the test substance itself. Furthermore, the formulations were consistently accurate at the 5 mg/kg dose level, which was the determined NOAEL. There was no impact on the study’s integrity
Duration of treatment / exposure:
Males were exposed for 29-31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 38-43 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
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.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20, 60 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on results from a 14-day dose range finding study where 0, 10, 25, 60 and 120 mg/kg bw/day were administered. Several haematology and clinical biochemistry parameters were affected at 60 and/or 120 mg/kg bw/day, and higher liver and spleen weights (absolute and relative) were noted at these dose levels as well. Enlarged spleens were noted for all animals at 120 mg/kg bw/day, and foci on the stomach and thickened limiting ridge of the stomach were also commonly noted at this dose level. Collectively, the data at 120 mg/kg is suggestive of haemolytic anaemia. Based on these results, 5, 20 and 60 mg/kg bw/day were chosen for the main study.
Positive control:
not applicable

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were at least conducted between 2.5 and 4 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and 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.

FOOD CONSUMPTION
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. 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.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical observations, body weight, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
Pathology
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
- Females which delivered: Lactation Days 5-7
- Females which failed to deliver: Post-coitum Day 30
- Males: Following completion of the mating period (a minimum of 28 days of dose administration)
- Spontaneous deaths: As soon as possible after death and always within 24 hours
- Euthanized in extremis: When pain, distress or discomfort is considered not transient in nature or is 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. The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin: Ovaries, Adrenal glands, Adrenal glands, Peyer's patches, 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, Spinal cord -cervical, midthoracic, lumbar, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum,Testes 2, Jejunum, Thymus,Kidneys, Thyroid including parathyroid if detectable, Lacrimal gland, exorbital, Tongue, Larynx, Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus,Lymph nodes - mandibular, mesenteric, Vagina, All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Organ weights
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
- Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix),Kidneys , Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

Histopathology
All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Of all the males of the control and high dose group and all males suspected to be infertile or which died before mating, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
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 all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups)
- Stomach, spleen and sternum of all animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in these organs in Group 4
- Liver and kidneys of all male animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs of all animals of Groups 1 and 4 and from male no. 22 and female no. 62 (20 mg/kg bw/day) who did not have live offspring.




Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. 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:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test 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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test 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). 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:
- Male mating index
- Male fertility index
- Female mating index
- Female fertility index
- Gestation index
Offspring viability indices:
- Live birth index
- Postimplantation loss

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One female was killed in extremis and one female was found dead at 60 mg/kg bw/day. Both of these animals were noted with necrosis of the glandular stomach at the microscopic examination, which was possibly related to treatment.
No clinical signs of toxicity were noted during the observation period. Piloerection was noted for a single high dose female on one day during the study. At the incidence observed, it was not considered to be toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Males at 60 mg/kg bw/day had significantly lower red blood cells (RBC), haemoglobin, and mean corpuscular haemoglobin concentration (MCHC), along with increased reticulocytes and mean corpuscular volume (MCV). Females at this dose level had significantly increased neutrophils (also seen for females at 20 mg/kg bw/day, but was not statistically significant) and decreased lymphocytes (also seen for females at 20 mg/kg bw/day, but did not reach statistical significance). These data collectively suggest a mild regenerative macrocytic hypochromic anemia.

There were no other toxicologically relevant changes in haematology parameters.

The statistically significant increase in prothrombin time (PT) seen for females at 20 mg/kg bw/day was attributable to slightly low control values and did not reflect a treatment related effect.

CLINICAL CHEMISTRY
Males at 60 mg/kg bw/day had significantly lower alkaline phosphatase (ALP) and significantly higher inorganic phosphate levels than controls. Clinical biochemistry parameters were unaffected for females at this dose level. The changes for males were not considered to be biologically relevant.

There were no other treatment related effects on clinical biochemistry parameters.

The significant increase in glucose seen for males at 20 mg/kg bw/day was not considered to be toxicologically relevant as it occurred in the absence of a dose related distribution

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Total movements were significantly lower for females at 60 mg/kg bw/day. Total activity was lower than controls during intervals 6-8, though the difference from controls was only slight, there were no significant effects on ambulatory activity, and in the absence of any relevant clinical observations like lethargy or reduced activity, the difference between controls was not considered to be toxicologically relevant.

ORGAN WEIGHTS
Absolute and relative liver and spleen weights were higher for animals for both sexes at 60 mg/kg bw/day (absolute liver weights were not significantly higher for females at this level). At 20 mg/kg bw/day, absolute liver weights were higher for animals of both sexes, though the difference was not significantly different from controls. Liver to body weight ratios were significantly higher for males at this dose level, however

GROSS PATHOLOGY
At 60 mg/kg bw/day one female was found dead and at the macroscopic examination was noted with the GI tract distended with gas, beginning autolysis, gelatinous contents of the GI tract, many greenish foci on the stomach, enlarged adrenal glands, two fetuses and two resorptions in the right uterine horn, and seven fetuses in the left uterine horn.
Enlarged lungs, pale discoloration of the lungs and watery clear fluid in the thoracic cavity were noted for one female that was euthanized in extremis. These are indicative of a gavage error. This female was also noted with four living fetuses in the left uterine horn and eight living fetuses in the right uterine horn.

Macroscopic findings noted for animals at 60 mg/kg bw/day that survived to the end of the scheduled treatment period included black discoloration of the spleen, noted for five males and two females. Additionally, dark red foci on the stomach glandular mucosa, enlarged spleen and/or liver were noted for a few animals at this dose level. Reduced size and black brown discoloration of the liver papillary process was seen for one male.
There were no treatment related effects on the macroscopic examination up to 20 mg/kg bw/day


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were present in:
- Stomach: hyperplasia of the limiting ridge was present at an increased incidence and severity in male and female rats treated at 20 (Group 3; 5/5 and 3/5 rats respectively) and 60 (Group 4; 8/8 and 5/6 rats respectively) mg/kg bw/day up to a moderate degree compared to minimal degrees in control (Group 1; 2/5 male rats) and 5 mg/kg bw/day (Group 2; 2/5 and 2/5 rats respectively) treated rats
- Stomach: hyperkeratosis of the limiting ridge was present in male and female rats treated at 20 (2/8 and 2/5 rats respectively) and 60 (2/5 and 5/6 rats respectively) mg/kg bw/day.
- Spleen: hemopoietic foci, primarily erythropoiesis were present at an increased severity in male and female rats treated at 60 mg/kg bw/day (7/7 and 8/8 respectively) up to a marked degree. The severity was higher compared to gradings noted up to a moderate degree in control (5/5 rats both sexes), in 5 (5/5 rats both sexes) and in 20 (5/5 rats both sexes) mg/kg bw/day treated rats. This was the microscopic correlate to the macroscopically enlarged spleens.
- Spleen: hemosiderin pigment was present at an increased incidence and severity in male rats treated at 60 mg/kg bw/day (7/7) up to a marked degree compared to a minimal degree noted for control (3/5), 5 mg/kg bw/day (4/5) and 20 mg/kg bw/day (4/5) treated rats. There was an increased mean severity seen for female rats treated at 60 mg/kg bw/day (8/8) of slight to moderate degree compared to a minimal to slight degree in control (5/5), in 5 mg/kg bw/day (5/5) and 20 mg/kg bw/day (5/5) treated rats. This was the microscopic correlate to the macroscopic black discolouration.
- Sternal bone marrow: erythroid hyperplasia was present in male and female rats treated at 60 mg/kg bw/day (5/5 and 5/6 respectively) up to a slight degree
- Liver: centrilobular hepatocellular hypertrophy, was present at a slight degree in the 60 mg/kg bw/day treated female that was found dead. In males, this was present at an increased incidence and severity in 20 (4/5) and 60 (5/5) mg/kg bw/day treated rats up to a slight degree, compared to minimal degrees in control (1/5) and 5 mg/kg bw/day (2/5) treated male rats.
- Kidneys: hyaline droplets were present at an increased incidence and severity in males treated at 60 mg/kg bw/day (5/5) up to a moderate degree, compared to minimal to slight degrees present in control (3/5), 5 (3/5) and 20 (5/5) mg/kg bw/day treated male rats.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: - changes in haematology parameters, macroscopic findings and organ weights, - adverse microscopic alterations in the stomach, spleen and liver - microscopic findings in the kidneys and bone marrow
Remarks on result:
other: Generation: P general systemic toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: Generation: P: reproductive performance and fertility (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The number of dead pups at first litter check represented 0 and 2.2% for controls and animals at 60 mg/kg bw/day, respectively while the postnatal loss represented 3.1 and 0% of living pups for controls and high dose animals, respectively.
Two pups of the control group went missing and one pup of the control group and two pups at 60 mg/kg bw/day were found dead during the first days of lactation. No toxicological relevance was attributed to these dead/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 (OFFSPRING)
Small size, an incidental finding, was the only clinical sign noted. This was not toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 60 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING)
The only incidental macroscopic findings for pups that were found dead was the absence of milk in the stomach. Wound on the left front leg and shoulder were the only findings noted at macroscopic examination for surviving pups. These were incidental in nature and no toxicological relevance was attributed to any of these findings

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion