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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 260 - 390 g (males), 175 - 240 g (females)
- Fasting period before study:
- Housing: in groups of three to five in Makrolon type-4 cages with wire mesh tops during acclimatisation and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment
- Diet (ad libitum): pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: daily. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours). Towards the end of the study, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and stored at -20 ± 5 °C until analysis.
The samples were analyzed by derivatisation with MSTFA and analysis by GC-FID. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: Minimum 28 days
Females: Approximately 6 weeks
Frequency of treatment:
once daily (within four hours)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on single dose toxicity data of the test compound and repeated dose toxicity data of chemically similar compounds, the results of a 14-day range-finding study the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum).
- detailed clinical observations were performed outside the home cage in a standard arena and the animals were observed for the following changes in: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples will be obtained on the day before or on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group will be obtained on day 5 postpartum.
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes, for approximately 18 hours
- How many animals: 5 males randomly selected from each group and from 5 lactating females from each group
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular hemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count: platelet count, reticulocytes; prothrombin time (= thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes, for approximately 18 hours
- How many animals: see haematology
- Parameters checked: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bile acids, bilirubin (total), cholesterol (total), creatinine, gamma-glutamyl-transferase, glucose, triglycerides, and urea

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at one time during the study following the daily dose administration (males shortly before the scheduled sacrifice and females on day 3 or 4 postpartum)
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group
- Battery of functions tested:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The following organs were trimmed from any adherent tissue, as appropriate, and their wet weight was taken: adrenal glands (weighed as pairs), brain, epididymides, heart, kidneys (weighed as pairs), liver, ovaries (weighed as pairs), prostate, seminal vesicles (inclusive coagulating gland), spleen, testes, thymus, thyroid (after fixation), uterus (including cervix)
HISTOPATHOLOGY: Yes
The following tissues from all parental animals were preserved in neutral phosphate buffered 4% formaldehyde solution if not otherwise described: adrenals, brain, bone marrow (femur), epididymides (in Bouin’s fixative), gross lesions, heart, kidneys, liver, lymph nodes (axillary and mesenteric), ovaries (with oviduct), peripheral nerve (sciatic), prostate, seminal vesicles with coagulating gland, small and large intestines (incl. Peyer’spatches; duodenum, jejunum, ileum, colon, caecum, rectum), spinal chord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes (in Bouin’s fixative), thymus, thyroids, and parathyroids if possible, trachea and lungs (preserved by inflation with fixative and then immersion), uterus (with vagina), urinary bladder
The following tissues were only examined by histopathology in case of macroscopic findings indicative of potential toxicity: aorta, esophagus, eyes with optic nerve and harderian gland, femur with knee joint, lacrimal gland, larynx, mammary gland (male and female), nasal cavity, pancreas, pharynx, pituitary gland, salivary glands – mandibular, sublingual, skeletal muscle, sternum with bone marrow.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied if the variables could be dichotomized without loss of information
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed in males. Incidentally, one control female showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Furthermore, one female treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. These isolated findings were not related to treatment with the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 1000 mg/kg bw/day, one male died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded. The death was considered not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain in males was lower at 1000 mg/kg bw/day during the pre-pairing period. This was a result of a minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse. No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean food consumption was reduced during the first 11 days of treatment in males. Thereafter, the food consumption remained slightly lower than in controls. Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse. There were no effects on mean food consumption at any dose level and in any study phase in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 1000 mg/kg bw/day resulted in higher absolute and relative liver and kidney weights in animals of both genders. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse. Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
VIABILITY/MORTALITIY
At a dose level of 1000 mg/kg bw/day, one male (no. 171, third animal delivery) died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded until day 13 of the paring period. One day before he died he showed a body weight loss of 3%. During necropsy, a dark red discolored thymus was recorded.

DAILY AND DETAILED WEEKLY CLINICAL OBSERVATIONS
No clinical signs were noted in males during the course of this study. Incidentally, one control female (no. 45) showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Necropsy of this female revealed masses in the uterus and vagina. Furthermore, one female (no. 66) treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. During necropsy reddish discolored ovaries and enlarged axillary lymph nodes were noted. Both females were pregnant.

These isolated findings were not related to treatment with the test item.

No findings were noted at detailed weekly clinical observation during the whole course of the study.

FUNCTIONAL OBSERVATIONAL BATTERY
There were no treatment-related changes noted at functional observational battery.

One male at 100 mg/kg bw/day had increased faeces-balls and one female at 300 mg/kg bw/day showed decreased rearings. These isolated findings showing no dose relationship were deemed unrelated to treatment with the test item. A statistically significantly lower mean body temperature was noted in males of the first and second delivery at 1000 mg/kg bw/day (37.8 °C compared to 38.5 °C in the control group). No similar effect was noted for males treated at 100 and 300 mg/kg bw/day or in females. Since the value of 37.8°C was borderline to the range of the historical control data (38.0°C-38.6°C), and no change was noted in females, this finding was considered to be incidental.

LOCOMOTOR ACTIVITY
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.

Locomotor activity was not affected by the treatment with the test item in animals of both genders at any dose level.

FOOD CONSUMPTION
At 1000 mg/kg bw/day, mean food consumption was statistically significantly reduced in males during the first 11 days of treatment in males of the first and second delivery (-9.3% on day 11 of prepairing when compared with controls). Thereafter, the food consumption remained slightly lower than in controls, but without reaching statistical significance. Food consumption was also lower in males of the third delivery, but not to a statistically significant degree.

Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse.

There were no effects on mean food consumption of females at any dose level and in any study phase.

BODY WEIGHTS
Mean body weight gain of males was statistically significantly lower at 1000 mg/kg bw/day during the pre-pairing period, resulting in slightly reduced mean body weights (-3% at the end of this period compared to the controls). This was a result of the minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse.

No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level.

Towards the end of the gestation period, body weights and body weight gain were reduced in females at 1000 mg/kg bw/day (statistically significantly different body weight gains from day 17 post coitum onwards). These differences were considered to be a result of the increased post implantation loss and low number of pups, which were noted for dams at this dose level.

HEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.

CLINICAL BIOCHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.

Slightly higher urea levels were noted in males at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day. Additionally, lower alkaline phosphatase levels were noted in females at 300 mg/kg bw/day, higher creatine levels in males and lower glucose levels in females at 1000 mg/kg bw/day, respectively. However, in the absence of dose-dependency and/or since values were additionally within the historical control range, a test item-related effect can be excluded.

ORGAN WEIGHTS
Treatment with the test item at 1000 mg/kg bw/day resulted in statistically significantly higher absolute and relative liver and kidney weights in animals of both genders, which was confirmed in animals of the third delivery except for kidney weights in females. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse.

Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental.

There were no effects of treatment at 100 and 300 mg/kg bw/day.

MACROSCOPICAL FINDINGS
There were no treatment-related necropsy findings.

All findings were typical of this strain and age of rat and were considered to be incidental.

HISTOPATHOLOGICAL FINDINGS
There were no treatment-related findings. In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.

All findings were typical of this strain and age of rat and were considered to be incidental.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL is highest dose tested
Critical effects observed:
no

Please refer to the attached pdf document for detailed summary tables of relevant parameters.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 22 March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
63705-03-3
EC Number:
613-333-7
Cas Number:
63705-03-3
IUPAC Name:
63705-03-3

Test animals

Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 260 - 390 g (males), 175 - 240 g (females)
- Fasting period before study:
- Housing: in groups of three to five in Makrolon type-4 cages with wire mesh tops during acclimatisation and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment
- Diet (ad libitum): pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no, if mating was not recorded during the additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
- 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:
On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours). Towards the end of the study, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and stored at -20 ± 5 °C until analysis.
The samples were analyzed by derivatisation with MSTFA and analysis by GC-FID. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: Minimum 28 days
Females: Approximately 6 weeks
Frequency of treatment:
once daily (within four hours)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: - Dose selection rationale: Based on single dose toxicity data of the test compound and repeated dose toxicity data of chemically similar compounds, the results of a 14-day range-finding study the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum).
- detailed clinical observations were performed outside the home cage in a standard arena and the animals were observed for the following changes in: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation
No food consumption was recorded during the pairing period.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, daily sperm production, seminal vesicles, stages of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The following organs were trimmed from any adherent tissue, as appropriate, and their wet weight was taken: adrenal glands (weighed as pairs), brain, epididymides, heart, kidneys (weighed as pairs), liver, ovaries (weighed as pairs), prostate, seminal vesicles (inclusive coagulating gland), spleen, testes, thymus, thyroid (after fixation), uterus (including cervix)
HISTOPATHOLOGY: Yes
The following tissues from all parental animals were preserved in neutral phosphate buffered 4% formaldehyde solution if not otherwise described: adrenals, brain, bone marrow (femur), epididymides (in Bouin’s fixative), gross lesions, heart, kidneys, liver, lymph nodes (axillary and mesenteric), ovaries (with oviduct), peripheral nerve (sciatic), prostate, seminal vesicles with coagulating gland, small and large intestines (incl. Peyer’spatches; duodenum, jejunum, ileum, colon, caecum, rectum), spinal chord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes (in Bouin’s fixative), thymus, thyroids, and parathyroids if possible, trachea and lungs (preserved by inflation with fixative and then immersion), uterus (with vagina), urinary bladder
The following tissues were only examined by histopathology in case of macroscopic findings indicative of potential toxicity: aorta, esophagus, eyes with optic nerve and harderian gland, femur with knee joint, lacrimal gland, larynx, mammary gland (male and female), nasal cavity, pancreas, pharynx, pituitary gland, salivary glands – mandibular, sublingual, skeletal muscle, sternum with bone marrow.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied if the variables could be dichotomized without loss of information

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed in males. Incidentally, one control female showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Furthermore, one female treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. These isolated findings were not related to treatment with the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 1000 mg/kg bw/day, one male died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded. The death was considered not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain in males was lower at 1000 mg/kg bw/day during the pre-pairing period. This was a result of a minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse. No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean food consumption was reduced during the first 11 days of treatment in males. Thereafter, the food consumption remained slightly lower than in controls. Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse. There were no effects on mean food consumption at any dose level and in any study phase in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Poor mating performance has been observed. However, this poor mating performance of animals was considered to be due to the disturbance of the light / dark cycle during the conduct of this study and not a test item-related effect. Therefore, additional animals have been tested (third delivery of animals).

Details on results (P0)

VIABILITY/MORTALITIY
At a dose level of 1000 mg/kg bw/day, one male (no. 171, third animal delivery) died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded until day 13 of the paring period. One day before he died he showed a body weight loss of 3%. During necropsy, a dark red discolored thymus was recorded.

DAILY AND DETAILED WEEKLY CLINICAL OBSERVATIONS
No clinical signs were noted in males during the course of this study. Incidentally, one control female (no. 45) showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Necropsy of this female revealed masses in the uterus and vagina. Furthermore, one female (no. 66) treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. During necropsy reddish discolored ovaries and enlarged axillary lymph nodes were noted. Both females were pregnant.

These isolated findings were not related to treatment with the test item.

No findings were noted at detailed weekly clinical observation during the whole course of the study.

FUNCTIONAL OBSERVATIONAL BATTERY
There were no treatment-related changes noted at functional observational battery.

One male at 100 mg/kg bw/day had increased faeces-balls and one female at 300 mg/kg bw/day showed decreased rearings. These isolated findings showing no dose relationship were deemed unrelated to treatment with the test item. A statistically significantly lower mean body temperature was noted in males of the first and second delivery at 1000 mg/kg bw/day (37.8 °C compared to 38.5 °C in the control group). No similar effect was noted for males treated at 100 and 300 mg/kg bw/day or in females. Since the value of 37.8°C was borderline to the range of the historical control data (38.0°C-38.6°C), and no change was noted in females, this finding was considered to be incidental.

LOCOMOTOR ACTIVITY
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.

Locomotor activity was not affected by the treatment with the test item in animals of both genders at any dose level.

FOOD CONSUMPTION
At 1000 mg/kg bw/day, mean food consumption was statistically significantly reduced in males during the first 11 days of treatment in males of the first and second delivery (-9.3% on day 11 of prepairing when compared with controls). Thereafter, the food consumption remained slightly lower than in controls, but without reaching statistical significance. Food consumption was also lower in males of the third delivery, but not to a statistically significant degree.

Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse.

There were no effects on mean food consumption of females at any dose level and in any study phase.

BODY WEIGHTS
Mean body weight gain of males was statistically significantly lower at 1000 mg/kg bw/day during the pre-pairing period, resulting in slightly reduced mean body weights (-3% at the end of this period compared to the controls). This was a result of the minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse.

No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level.

Towards the end of the gestation period, body weights and body weight gain were reduced in females at 1000 mg/kg bw/day (statistically significantly different body weight gains from day 17 post coitum onwards). These differences were considered to be a result of the increased post implantation loss and low number of pups, which were noted for dams at this dose level.

HEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.

CLINICAL BIOCHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.

Slightly higher urea levels were noted in males at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day. Additionally, lower alkaline phosphatase levels were noted in females at 300 mg/kg bw/day, higher creatine levels in males and lower glucose levels in females at 1000 mg/kg bw/day, respectively. However, in the absence of dose-dependency and/or since values were additionally within the historical control range, a test item-related effect can be excluded.

ORGAN WEIGHTS
Treatment with the test item at 1000 mg/kg bw/day resulted in statistically significantly higher absolute and relative liver and kidney weights in animals of both genders, which was confirmed in animals of the third delivery except for kidney weights in females. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse.

Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental.

There were no effects of treatment at 100 and 300 mg/kg bw/day.

MACROSCOPICAL FINDINGS
There were no treatment-related necropsy findings.

All findings were typical of this strain and age of rat and were considered to be incidental.

HISTOPATHOLOGICAL FINDINGS
There were no treatment-related findings. In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.

All findings were typical of this strain and age of rat and were considered to be incidental.

MATING PERFORMANCE AND FERTILITY
For three females in the control group (nos. 45, 47 and 53), one female at 100 mg/kg bw/day (no. 58) and three females at 1000 mg/kg bw/day (nos. 79, 80 and 85), no mating was noted during the first pairing period and therefore these females were paired with a second male. A body weight gain indicating pregnancy was observed in female nos. 47 and 74 in the second and first pairing period, respectively, and therefore pairing of this female was stopped. Female no. 58 also did not mate during the second pairing period. All remaining females mated successfully during the second pairing period.

Percentage of mating was 100% at the dose levels 0, 300 and 1000 mg/kg bw/day and 93.3% at the dose level of 100 mg/kg bw/day. The lower value at 100 mg/kg bw/day was due to female no. 58 that did not mate during both pairing periods. Because it was an isolated case and occurred at the lowest dose level, this was considered not to be related to the treatment with the test item. The fertility index was 78.9%, 66.7%, 71.4% and 75.0% in order of ascending dose levels due to 4 not-pregnant females at 0, 100, 300 and 1000 mg/kg bw/day, respectively.

The gestation index was: 86.7%, 90.0%, 100.0% and 91.7% with increasing dose levels. The lower gestation index in the control group was due to two pregnant females (nos. 45 and 54), for which no birth was recorded until day 25 post coitum. The same applied to female 66 treated at 100 mg/kg bw/day.

The poor mating performance of animals was considered to be due to the disturbance of the light / dark cycle during the conduct of this study and not a test item-related effect. Therefore, additional animals have been tested (third delivery of animals).

MATING PERFORMANCE AND FERTILITY OF THIRD DELIVERY OF ANIMALS
For one female in the control group (no. 175) no pairing was noted during the first pairing period. A body weight gain of 11% indicated a pregnancy and therefore pairing of this female was stopped. However, towards the end of gestation it was observed that the female was not pregnant. As a consequence percentage of mating was 91.7% and 100% at the dose levels 0 and 1000 mg/kg bw/day.

The fertility index was 75.0% and 91.7%, respectively, due to 3 and 1 not-pregnant females at 0 and 1000 mg/kg bw/day, respectively.

The gestation index was 100.0% in both groups.

CORPORA LUTEA COUNT
The mean number of corpora lutea per dam was 17.3, 16.7, 14.6 and 15.0 in order of ascending dose levels.

Third Delivery of Animals
The mean number of corpora lutea per dam was 15.1 and 14.1 in the control group and at 1000 mg/kg bw/day, respectively.

DURATION OF GESTATION
The mean duration of gestation was 21.3, 21.6, 21.2 and 22.1 days, in order of ascending dose level. The duration of gestation of females treated at 1000 mg/kg bw/day was statistically significantly increased when compared with controls. This was mainly due to female nos. 78 and 87, which had gestation of 23 days. Furthermore those two females gave birth to very small litters, with only two surviving pups per litter on day 4 post partum, respectively. However, with one exception having 21 days of gestation (no. 81), the remainder of females treated at 1000 mg/kg bw/day (9 animals) showed a gestation duration of 22 days. This effect noted at 1000 mg/kg was considered to be related to treatment with the test item, although no doserelationship is visible at 100 and 300 mg/kg bw/day. The range of historical control data covers day 21.2 until day 21.7.

Third Delivery of Animals
The mean duration of gestation was 21.3 and 21.7 days in the control group and at 1000 mg/kg bw/day. The duration of gestation of females treated at 1000 mg/kg bw/day was not statistically significantly different to the control group (female no. 186 at 1000 mg/kg bw/day had a gestation duration of 23 days and gave birth to a small litter of only 2 pups).

IMPLEMENTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam was 14.1, 14.0, 12.7 and 11.8 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 6.6%, 10.3%, 6.3% and 16.2%, in order of ascending dose level. On a litter basis, mean incidence of post-implantation loss was 6.5%, 10.2%, 5.7% and 16.4%, respectively. The animal of the high dose group with only dead fetuses at first litter check was excluded from the calculation.

The total number of implantation loss was statistically significantly increased in females at 1000 mg/kg bw/day when compared with controls (21 losses when compared to 12 in controls,both distributed over eight litters). At this dose levels, female no. 142 was excluded from thisanalysis, which had 17 implantation sites but only 10 dead pups at first litter check (no living pups at first litter check).

This increase in post-implantation loss at 1000 mg/kg bw/day was outside of the range of the historical control data (3 - 16 losses and 3.3% - 14.4% as a percentage of total implantations) and was therefore considered to be related to treatment with the test item.

Additionally, for control female no. 45 one fetal resorption was noted in the vagina and one placenta in the uterus. Female no. 54 in the controls had only embryonic resorptions and at 100 mg/kg bw/day, female no. 66 had only fetal resorptions. All these findings were noted at necropsy on day 25 post coitum. All these females were pregnant but did not litter.

Third Delivery of Animals
The mean number of implantations per dam was 13.7 and 12.2 in the control group and at 1000 mg/kg bw/day, respectively. The mean incidence of post-implantation loss as a percentage of total implantations was 3.3% and 14.2% in the control group and at 1000 mg/kg bw/day. On a litter basis, mean incidence of post-implantation loss was 3.3% and 16.0%, respectively.

The total number of implantation loss was statistically significantly increased in females at 1000 mg/kg bw/day when compared with controls (19 losses in 8 litters when compared to 4 losses in 3 litters in controls).

LITTER SIZE AT FIRST LITTER CHECK
Mean litter size at first litter check was 13.2, 12.6, 11.9 and 9.9 pups in order of ascending dose levels (statistically significant at the high dose level). There were 3, 1 and 0 dead pups at 0, 100 and 300 mg/kg bw/day. In controls, there was maximally 1 dead pup per litter. At 1000 mg/kg bw/day, there were 6 dead pups from two litters, at which in litter 78 five dead pups were noted (litter 78 that had also a gestation of 23 days). The reduced litter size was considered to be a result of the increased post-implantation loss.

Due to these findings, a statistically significantly reduced birth index was noted at 1000 mg/kg bw/day (83.8% compared to 93.4% in controls).

Litter no. 142, which had no live pups but all 10 pups were found dead at first litter check, was excluded from the analysis. For this female a body weight loss of 31g was recoded from day 20 post coitum until day 22 post coitum. Most probably this loss is due to start giving birth on day 20 post coitum but with cannibalization of the dead pups immediately (17 implantation sites were noted for this female).

Litter size was not affected by the treatment up to the dose level of 300 mg/kg bw/day.

Third Delivery of Animals
Mean litter size at first litter check was 13.2 and 10.5 pups in the control group and at 1000 mg/kg bw/day, respectively (statistically significant at 1000 mg/kg bw/day). There were no dead pups in both groups. Due to these findings, a statistically significantly reduced birth index was noted at 1000 mg/kg
bw/day (85.8% compared to 96.7 in controls).

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
There was no effect on postnatal loss at any dose level.

The overall mean number of postnatal loss per dam was 0.8, 0.1, 0.0 and 0.5 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

In the control group, two litters lost 4 pups and two litters lost 1 pup, respectively. At 1000 mg/kg bw/day, female no. 87 lost 3 pups (with only 2 surviving pups on day 4 post partum) and two other litters 1 pup, respectively.

Third Delivery of Animals
The overall mean number of postnatal loss per dam was 0.0 and 0.3 at the dose level of 0 and 1000 mg/kg bw/day, respectively.

At 1000 mg/kg bw/day, female no. 186 lost its only 2 pups (normal pup weight) on day 2 and 3 post partum.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic / reproduction
Effect level:
1 000 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: NOAEL is highest dose tested
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

Three control pups from the litter no. 129 showed no milk in the stomach on the first day post partum. Those three pups were then missing at litter check on day 2 post partum, along with a further female pup. One pup of this litter had already been found dead at first litter check. The remaining 10 pups from this litter survived until their necropsy on day 4 post partum.

At 1000 mg/kg bw/day, one pup showed a black discolored exophthalmos on the first day post partum. This animal was free from clinical signs from day 2 post partum onwards and survived until necropsy on day 4. A further pup showed a wound on the head on days 3 and 4 post partum.

SEX RATIONS
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.

The proportion of males at first litter check was 56, 53, 50 and 51%, in order of ascending dose level.

BODY WEIGHTS TO DAY 4 POST PARTUM
Mean pup weights on day 1 and day 4 post partum were unaffected by treatment with the test item.

On day 1 post partum mean pup weights were 5.7, 6.1, 6.0 and 5.9 g in order of ascending dose level. Also mean body weight gain was similar in all groups on day 4 post partum and no effects on mean body weights were recorded.

MACROSCOPICAL FINDINGS
No test item-related findings were noted at macroscopic examination of F1 pups.

One male pup treated at 100 mg/kg bw/day had a missing right testicle at planned necropsy and two pups treated at 1000 mg/kg bw/day showed sores on the skin. These isolated findings were incidental and unrelated to treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Please refer to the attached pdf document for additional detailed summary tables of relevant parameters for F1 pups.

Table 1: P Animals Breeding for F1 Litters - First and Second Delivery of Animals

Group

1

2

3

4

(mg/kg/day)

(0)

(100)

(300)

(1000)

Femalenumbers                           45 -55

56 - 66

67 - 77

78 - 88

 

124 - 131

132 - 135

136 - 138

139 - 143

Number of females paired

19

15

14

16

Number of females mated (A)

19

14

14

16

Number of females not pregnant (B)

4

4

4

4

Number of pregnant females which did not give birth to living pups (C)

 

2

 

1

 

0

 

1

Number of females with living pups at first litter check

 

13

 

9

 

10

 

11

Number of females which reared their pups until day 4post partum

 

13

 

9

 

10

 

11

(A) Female no. 58 did not mate during either of the two paring periods

(B) Female nos. 48, 50, 51, 53, 57, 63, 65, 135, 70, 71, 76, 77, 79, 83, 86 and 88 were not pregnant

(C) Female nos. 45, 54, 66 and no. 142 (only dead pups at first litter check and therefore excluded from analysis).

Table 2: P Animals Breeding for F1 Litters - Third Delivery of Animals

Group

1

4

(mg/kg/day)

(0)

(1000)

Female numbers

174 - 185

186 - 197

Number of females paired

12

12

Number of females mated (A)

11

12

Number of females not pregnant (B)

2

1

Number of females which lost its whole litter (C)

 

0

 

1

Number of females with living pups at first litter check

 

9

 

11

Number of females which reared their pups until day 4post partum

 

9

 

10

(A) For female no. 175 no mating was observed during the first pairing period but due to her body weight gain of 11% mating was assumed (=mating not detected). However, the female was not pregnant.

(B) Female nos. 175, 176, 182 and 197 were not pregnant

(C) Female no. 186 lost its litter

Applicant's summary and conclusion