Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine
- Physical state: Clear slightly yellowish liquid
- Analytical purity: 95.9 g/100 g
- Lot/batch No.: 000STD77L0
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

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: approx. 11 weeks
- Weight at study initiation: mean: females: 201 g, males: 318 g
- Housing: in groups of 5 animals/sex/cage
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance. No correction was made for the purity of the test substance.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1H-NMR spectroscopy
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 42-53 days 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).
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
daily
Details on study schedule:
The purpose of this study was to evaluate the potential toxic effects of the test substance when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of the dose range finding study (Project 499971; BASF Project 01R0402/04X033) where dose levels of 0, 150 and 400 mg/kg bw/day were assessed. Animals at 400 mg/kg bw/day had slightly lower body weight gains or weight loss, and slightly lower food consumption. Changes in haematology and clinical biochemistry parameters were noted, along with increased absolute and relative liver weights (both sexes) and higher relative kidney weights (males). Females at 150 mg/kg bw/day also had higher absolute and relative liver weights.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality, pain, distress or discomfort

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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 AND COMPOUND INTAKE: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight, epididymis weight
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, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals on day 29.
- Maternal animals: all surviving animals on lactation days 5 - 7

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology: Adrenal glands, Ovaries, (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, (Male and Female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach, Ileum, Testes, 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

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

Organ weights: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid
Postmortem examinations (offspring):
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.
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) 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) 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.
Reproductive indices:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), duration of gestation
Offspring viability indices:
Percentage live males at first litter check, Percentage live females at first litter check, Percentage of postnatal loss days 0 - 4 of lactation, viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day there were three males that died spontaneously (nos. 31, 32 and 38) on study days 21, 25, and 29 and one that died after blood collection immediately prior to the scheduled necropsy (no. 37), and there was one female (no. 72) that was euthanized in extremis on day 7 post coitum.
Clinical signs noted for the animals that died prematurely or were euthanized in extremis included lethargy, hunched posture, piloerection and quick breathing. These were noted on single or limited occasions on the day(s) immediately preceding death of these animals. Alopecia was noted for a single female at 300 and chromodacryorrhea (of the right periorbital region) was recorded for a single male at 100 mg/kg bw/day. These were the only other clinical signs observed and were incidental in nature.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 300 mg/kg bw/day there were three males that died spontaneously (nos. 31, 32 and 38) on study days 21, 25, and 29 and one that died after blood collection immediately prior to the scheduled necropsy (no. 37), and there was one female (no. 72) that was euthanized in extremis on day 7 post coitum.
Clinical signs noted for the animals that died prematurely or were euthanized in extremis included lethargy, hunched posture, piloerection and quick breathing. These were noted on single or limited occasions on the day(s) immediately preceding death of these animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gains were significantly lower than controls for males at 300 mg/kg bw/day during the mating period. Males slightly lost weight during the second week of the study and during mating period. Body weights and/or body weight gains were significantly lower during the post coitum and lactation periods for females at 300 mg/kg bw/day. Absolute body weights were lower during the post coitum and lactation periods for females at 100 mg/kg bw/day as well, though the differences were not always statistically significant. Body weight gain was significantly lower for females at 100 mg/kg bw/day on Day 8 of the pre mating period. In the absence of a dose response effect it was not considered toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute food consumption was lower during the mating period, and relative food consumption was lower during the last part of the premating period and throughout the mating period for males at 300 mg/kg bw/day. Females at this dose level had significantly lower absolute food consumption during days 0-7 and 14-20 of the post coitum period and over days 1-4 of the lactation period. There were no other treatment related effects on absolute or relative food consumption up to 100 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were seen in several organs including:
Lung:
- Hemorrhage was noted in 4/8 males and 2/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Mucous cell hyperplasia of the bronchi/bronchioles epithelium was noted in 6/8 males (up to moderate) and in 2/6 females (up to marked) treated at 300 mg/kg bw/day.
- Bronchiolization of the alveoli wall was noted in 6/8 males (up to moderate) and in 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Alveolar inflammation was noted at increased incidence and severity in 7/8 males (up to moderate) and 5/6 females (up to marked) treated at 300 mg/kg bw/day, compared to 2/5 males (minimal) in the 0 mg/kg bw/day, 2/5 males and 4/5 females (minimal) in the 30 mg/kg bw/day and 4/5 males and 2/5 females (minimal) in the 100 mg/kg bw/day treated rats.
- Interstitial inflammation was noted at increased incidence and severity in 8/8 males (up to moderate) and 4/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 1/5 males and 1/5 females (minimal) in the 30 mg/kg bw/day and 2/5 males (minimal) in the 100 mg/kg bw/day treated rats.
The mixed inflammatory infiltrate consisted of lymphocytes, granulocytes and macrophages.
- Fibrin deposition was noted in 3/8 males (up to moderate) treated at 300 mg/kg bw/day.
- Fibrosis was noted in 6/8 males (up to moderate) and in 3/6 females (up to slight) treated at 300 mg/kg bw/day.
Stomach:
- Ulceration of the forestomach was noted in 4/8 males (up to marked) and 2/6 females (up to moderate) in the 300 mg/kg bw/day treated rats.
- Hyperplasia of the forestomach was noted in 7/8 males and 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Lymphogranulocytic inflammation of the forestomach was noted in 5/8 males and 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Lymphogranulocytic inflammation of the limiting ridge was noted at increased incidence and/or severity in 6/8 males (up to slight) and 4/6 females (minimal) at 300 mg/kg bw/day, compared to minimal degree in 1/5 males treated at 0 mg/kg bw/day, 2/5 males treated at 30 mg/kg bw/day and in 1/5 females in the 100 mg/kg bw/day group.
- Vacuolation of the limiting ridge was noted at increased incidence and/or severity in 3/8 males (up to moderate) and 4/6 females (up to slight) treated at 300 mg/kg bw/day compared to 3/5 males at minimal degree in the 100 mg/kg bw/day.
- Lymphogranulocytic inflammation of the glandular stomach was noted at increased severity in 4/8 males and 2/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 2/5 males with a minimal degree in the 100 mg/kg bw/day treated males.
- Increased apoptosis in the mucosa of the glandular stomach was noted in 1/6 females (moderate) treated at 300 mg/kg bw/day.
- Hemorrhage of the glandular mucosa was noted at slight degree in 1/8 male treated at 300 mg/kg bw/day.
Thymus:
- Lymphoid atrophy was noted at increased severity (moderate) in 1/7 females treated at 300 mg/kg bw/day compared to minimal in 1/5 females treated at 100 mg/kg bw/day. This increased severity was considered to be secondary due to the poor condition of this unscheduled death.
Spleen:
- An increase in severity of hemopoietic foci (3 minimal, 3 slight, 2 moderate) was recorded in males treated at 300 mg/kg bw/day, compared to 5/5 minimal treated at 0 mg/kg bw/day, 5/5 minimal treated at 30 mg/kg bw/day and 2/5 minimal and 3/5 slight treated at 100 mg/kg bw/day.
Kidney:
- Corticomedullary tubular basophilia was noted at increased incidence and severity in 6/6 males (slight to marked) and 4/6 females (up to slight) treated at 300 mg/kg bw/day, compared to minimal degree in 1/5 males and 1/5 females treated at 0 mg/kg bw/day, 2/6 males and 1/5 females treated at 30 mg/kg bw/day and 2/7 males and 1/5 females treated at 100 mg/kg bw/day.
- Granular cast(s)/degeneration of tubular epithelium was noted in 5/6 males (up to slight) and 1/6 females (minimal, unscheduled death) treated at 300 mg/kg bw/day.
- Tubular dilatation was noted in 5/6 males (up to slight) and 5/6 females (minimal) treated at 300 mg/kg bw/day.
An increase in incidence of hyaline cast(s) was noted in 4/6 males (minimal degree) treated at 300 mg/kg bw/day, compared to 1/6 males treated at 30 mg/kg bw/day.
Adrenal gland:
- Lymphocytic inflammation of the distal part of the zona fasciculate and zona reticularis was noted in 3/5 females (all minimal) treated at 100 mg/kg bw/day and in 1/8 males (slight) and 5/6 females (slight to marked) treated at 300 mg/kg bw/day.
Eyes:
- Retinal dysplasia (rosettes) was recorded in 5/7 males (up to slight) and 5/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 1/5 females (minimal, only unilateral) treated at 100 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
There were significantly fewer corpora lutea and implantation sites for females at 300 mg/kg bw/day compared to controls, and the number of implantation sites was significantly lower for females at 100 mg/kg bw/day as well. A single female in the 100 mg/kg bw/day group had only 1 implantation site, and when recalculated discounting her data, the mean number of implantation sites was 10.9 for this group. The number of implantation sites was higher for controls (13.7) than the historical mean (11.7), though the mean discounting female no. 67 remained slightly below available historical control data (p5 = 11.3). However, as females at 100 mg/kg bw/day produced viable litters, the slight reduction in implantation site number at 100 mg/kg bw/day was not considered to be adverse. The number of corpora lutea were lower for females in the 30 and 100 mg/kg bw/day groups as well (not statistically significant). However, these were attributable to a relatively high mean noted for control females and was not considered treatment related for females in these groups.
The mating, fertility and conception indices and precoital time were unaffected by treatment.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): At 300 mg/kg bw/day there were three males that died spontaneously (nos. 31, 32 and 38) on study days 21, 25, and 29 and one that died after blood collection immediately prior to the scheduled necropsy (no. 37), and there was one female (no. 72) that was euthanized in extremis on day 7 post coitum.
Clinical signs noted for the animals that died prematurely or were euthanized in extremis included lethargy, hunched posture, piloerection and quick breathing. These were noted on single or limited occasions on the day(s) immediately preceding death of these animals. Alopecia was noted for a single female at 300 and chromodacryorrhea (of the right periorbital region) was recorded for a single male at 100 mg/kg bw/day. These were the only other clinical signs observed and were incidental in nature.

BODY WEIGHT (PARENTAL ANIMALS): Body weights and body weight gains were significantly lower than controls for males at 300 mg/kg bw/day during the mating period. Males slightly lost weight during the second week of the study and during mating period. Body weights and/or body weight gains were significantly lower during the post coitum and lactation periods for females at 300 mg/kg bw/day. Absolute body weights were lower during the post coitum and lactation periods for females at 100 mg/kg bw/day as well, though the differences were not always statistically significant. Body weight gain was significantly lower for females at 100 mg/kg bw/day on Day 8 of the pre mating period. In the absence of a dose response effect it was not considered toxicologically relevant.

FOOD CONSUMPTION AND COMPOUND INTAKE (PARENTAL ANIMALS): Absolute food consumption was lower during the mating period, and relative food consumption was lower during the last part of the premating period and throughout the mating period for males at 300 mg/kg bw/day. Females at this dose level had significantly lower absolute food consumption during days 0-7 and 14-20 of the post coitum period and over days 1-4 of the lactation period. There were no other treatment related effects on absolute or relative food consumption up to 100 mg/kg bw/day.

REPRODUCTIVE FUNCTION (PARENTAL ANIMALS): There were significantly fewer corpora lutea and implantation sites for females at 300 mg/kg bw/day compared to controls, and the number of implantation sites was significantly lower for females at 100 mg/kg bw/day as well. A single female in the 100 mg/kg bw/day group had only 1 implantation site, and when recalculated discounting her data, the mean number of implantation sites was 10.9 for this group. The number of implantation sites was higher for controls (13.7) than the historical mean (11.7), though the mean discounting female no. 67 remained slightly below available historical control data (p5 = 11.3). However, as females at 100 mg/kg bw/day produced viable litters, the slight reduction in implantation site number at 100 mg/kg bw/day was not considered to be adverse. The number of corpora lutea were lower for females in the 30 and 100 mg/kg bw/day groups as well (not statistically significant). However, these were attributable to a relatively high mean noted for control females and was not considered treatment related for females in these groups.
The mating, fertility and conception indices and precoital time were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS): Terminal body weights were significantly lower for animals of both sexes at 300 mg/kg bw/day (not statistically significant for females). Males at this dose level also had significantly higher absolute and relative spleen weights (relative spleen weights were also higher for males at 100 mg/kg bw/day), higher relative liver and kidney weights and significantly lower absolute testes and seminal vesicle weights. Females at this dose level had significantly higher thymus (absolute and relative) and significantly lower thyroid (absolute and relative) and adrenal (absolute) weights. The significantly increased brain to body weight ratio seen for males at 300 mg/kg bw/day was secondary to the lower terminal body weights and was not considered to be toxicologically relevant.
Similarly, the significantly higher relative thyroid weights that were noted for females at 30 mg/kg bw/day occurred in the absence of a dose response relationship and were not considered to be toxicologically relevant.
Other organ weights and organ to body weight ratios were similar between control and treated groups.

GROSS PATHOLOGY (PARENTAL ANIMALS): Macroscopic findings noted for the animals that died spontaneously or were killed in extremis included beginning or advanced autolysis, dark red, reddish, or grey-white discoloration of the lungs, many dark red foci on the lungs, dark red contents of the small intestines, irregular surface of, and a tan and/or reddish focus on the stomach or forestomach, reddish foci on the stomach glandular mucosa, stomach limiting ridge thickened, greenish foci on the caecum, reddish discoloration of the thymus, black and/or dark red discoloration of the mesenteric or mandibular lymph node, agenesis and/or reduced size of the seminal vesicles.
Relevant macroscopic findings noted for animals at 300 mg/kg bw/day that survived to the scheduled necropsy included many dark red foci or an isolated tan focus on the the lungs, reddish foci on the stomach glandular mucosa and irregular surface of the forestomach. Additionally, female no. 73 was noted with a dark red and hard nodule in the right uterine horn. Incidental findings noted for control and treated animals included reddish or dark red foci on the thymus, reddish discoloration of the thymus, yellowish focus on the right medial lobe of the liver, alopecia, yellowish or greenish soft nodules on the epididymides, pelvic dilation of the kidney(s), watery-clear cyst on the right kidney, tan discoloration of the right clitoral gland, and uterus contains fluid. These findings occurred without a treatment related distribution and remained within the background range of findings seen for rats of this age and strain, and were thus not considered to be treatment related or toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS): Treatment-related microscopic findings were seen in several organs including:
Lung:
- Hemorrhage was noted in 4/8 males and 2/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Mucous cell hyperplasia of the bronchi/bronchioles epithelium was noted in 6/8 males (up to moderate) and in 2/6 females (up to marked) treated at 300 mg/kg bw/day.
- Bronchiolization of the alveoli wall was noted in 6/8 males (up to moderate) and in 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Alveolar inflammation was noted at increased incidence and severity in 7/8 males (up to moderate) and 5/6 females (up to marked) treated at 300 mg/kg bw/day, compared to 2/5 males (minimal) in the 0 mg/kg bw/day, 2/5 males and 4/5 females (minimal) in the 30 mg/kg bw/day and 4/5 males and 2/5 females (minimal) in the 100 mg/kg bw/day treated rats.
- Interstitial inflammation was noted at increased incidence and severity in 8/8 males (up to moderate) and 4/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 1/5 males and 1/5 females (minimal) in the 30 mg/kg bw/day and 2/5 males (minimal) in the 100 mg/kg bw/day treated rats.
The mixed inflammatory infiltrate consisted of lymphocytes, granulocytes and macrophages.
- Fibrin deposition was noted in 3/8 males (up to moderate) treated at 300 mg/kg bw/day.
- Fibrosis was noted in 6/8 males (up to moderate) and in 3/6 females (up to slight) treated at 300 mg/kg bw/day.
Stomach:
- Ulceration of the forestomach was noted in 4/8 males (up to marked) and 2/6 females (up to moderate) in the 300 mg/kg bw/day treated rats.
- Hyperplasia of the forestomach was noted in 7/8 males and 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Lymphogranulocytic inflammation of the forestomach was noted in 5/8 males and 3/6 females (up to moderate) treated at 300 mg/kg bw/day.
- Lymphogranulocytic inflammation of the limiting ridge was noted at increased incidence and/or severity in 6/8 males (up to slight) and 4/6 females (minimal) at 300 mg/kg bw/day, compared to minimal degree in 1/5 males treated at 0 mg/kg bw/day, 2/5 males treated at 30 mg/kg bw/day and in 1/5 females in the 100 mg/kg bw/day group.
- Vacuolation of the limiting ridge was noted at increased incidence and/or severity in 3/8 males (up to moderate) and 4/6 females (up to slight) treated at 300 mg/kg bw/day compared to 3/5 males at minimal degree in the 100 mg/kg bw/day.
- Lymphogranulocytic inflammation of the glandular stomach was noted at increased severity in 4/8 males and 2/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 2/5 males with a minimal degree in the 100 mg/kg bw/day treated males.
- Increased apoptosis in the mucosa of the glandular stomach was noted in 1/6 females (moderate) treated at 300 mg/kg bw/day.
- Hemorrhage of the glandular mucosa was noted at slight degree in 1/8 male treated at 300 mg/kg bw/day.
Thymus:
- Lymphoid atrophy was noted at increased severity (moderate) in 1/7 females treated at 300 mg/kg bw/day compared to minimal in 1/5 females treated at 100 mg/kg bw/day. This increased severity was considered to be secondary due to the poor condition of this unscheduled death.
Spleen:
- An increase in severity of hemopoietic foci (3 minimal, 3 slight, 2 moderate) was recorded in males treated at 300 mg/kg bw/day, compared to 5/5 minimal treated at 0 mg/kg bw/day, 5/5 minimal treated at 30 mg/kg bw/day and 2/5 minimal and 3/5 slight treated at 100 mg/kg bw/day.
Kidney:
- Corticomedullary tubular basophilia was noted at increased incidence and severity in 6/6 males (slight to marked) and 4/6 females (up to slight) treated at 300 mg/kg bw/day, compared to minimal degree in 1/5 males and 1/5 females treated at 0 mg/kg bw/day, 2/6 males and 1/5 females treated at 30 mg/kg bw/day and 2/7 males and 1/5 females treated at 100 mg/kg bw/day.
- Granular cast(s)/degeneration of tubular epithelium was noted in 5/6 males (up to slight) and 1/6 females (minimal, unscheduled death) treated at 300 mg/kg bw/day.
- Tubular dilatation was noted in 5/6 males (up to slight) and 5/6 females (minimal) treated at 300 mg/kg bw/day.
An increase in incidence of hyaline cast(s) was noted in 4/6 males (minimal degree) treated at 300 mg/kg bw/day, compared to 1/6 males treated at 30 mg/kg bw/day.
Adrenal gland:
- Lymphocytic inflammation of the distal part of the zona fasciculate and zona reticularis was noted in 3/5 females (all minimal) treated at 100 mg/kg bw/day and in 1/8 males (slight) and 5/6 females (slight to marked) treated at 300 mg/kg bw/day.
Eyes:
- Retinal dysplasia (rosettes) was recorded in 5/7 males (up to slight) and 5/6 females (up to moderate) treated at 300 mg/kg bw/day, compared to 1/5 females (minimal, only unilateral) treated at 100 mg/kg bw/day.
The remainder of microscopic findings recorded were within the normal range of background pathology encountered in Wistar (Han) rats of this age.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs noted for any pup.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Pup body weights were higher for all treated groups compared to controls, though it was only statistically significant for pups at 30 mg/kg bw/day. The higher body weights for the smaller litters of females 52, 54, 56 and 58 contributed to this. The slightly smaller litters at 100 mg/kg bw/day also contributed to their higher (not statistically significant) means. The litters at 300 mg/kg bw/day all had fewer pups than controls. The five litters available had 3, 6, 4, 2, and 2 pups, and with the exception of the litter with 4 pups (female no. 75) the pups of the other litters did not have higher body weights that would be expected to coincide with litters of such small size. However, the treated pups gained a comparable or higher percentage of weight than controls from postnatal Days 1-4 (49, 63, 58 and 54% gain for pups from the control, 30, 100, and 300 mg/kg bw/day groups, respectively). As such, the differences in body weights were not considered to be toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No milk in the stomach was the only macroscopic finding seen and was incidental since it was noted for animals that survived until the scheduled necropsy, indicating they were sufficiently fed the days prior to the scheduled necropsy. This was noted for a single pup (no. 2) from litter 43 (control) and the entire litter from female no. 62 (100 mg/kg bw/day).

Details on results (F1)

VIABILITY (OFFSPRING): One pup of the control group and two pups at 100 mg/kg bw/day went missing during the first days of lactation. These pups were most likely cannibalized. No toxicological relevance was attributed to these 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. There were no dead or missing pups in the 30 and 300 mg/kg bw/day groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion