Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-propylheptyl) phthalate
EC Number:
258-469-4
EC Name:
Bis(2-propylheptyl) phthalate
Cas Number:
53306-54-0
Molecular formula:
C28H46O4
IUPAC Name:
bis(2-propylheptyl) phthalate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 5 wks
- Weight at study initiation: (P) Males: 120.6 - 161.0 g; Females: 110.9 - 145.7 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages; pregnant animals and their litters were housed together until PND 21 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment, wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): Drinking water; ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15x
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly during premating period for F0 and F1 males and females as well during gestation, lactation and post weaning period for F0 males; once for F0 and F1 females and females during mating period and for F0 and F1 females during gestation, lactation and post weaning period
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): pregnant animals and their litters were housed together until PND 21 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in the diet for a period of 37 days at room temperature were carried out before the study was initiated.
Homogeneity and concentration control analyses by GC analysis were carried out at the beginning and toward the end of the premating periods. Additionally, at least one analysis of the test substance preparations for female animals was carried out during the gestation and lactation periods. In addition to each sample another one was kept in reserve.
Duration of treatment / exposure:
F0: 126 days
F1: 131 days
Frequency of treatment:
continuously by diet
Details on study schedule:
- F1 parental animals not mated until at least 75 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: y request of the sponsor

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of the premating period and then once a week at the same time of the day (in the morning). The F0 and F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. Females were not weighed during pairing until there was positive evidence of sperm in vaginal smears. Females without litter were not weighed during lactation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: a few days before terminal sacrifice of the animals, i.e. after approx. 16 (males) or 18 (females) weeks of treatment; in the morning
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No
- How many animals: 10
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: a few days before terminal sacrifice of the animals, i.e. after approx. 16 (males) or 18 (females) weeks of treatment; in the morning
- Animals fasted: No
- Anaesthetic used for blood collection: Yes, isoflurane
- How many animals: 10/sex/group
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, cyanide-insensitive palmitoyl-CoA-oxidation, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 and F1 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in F0/F1 male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


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


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animal (after weaning of pups the parental animals (F0 and F1) were sacrificed)
- Maternal animals: All surviving animal (after weaning of pups the parental animals (F0 and F1) were sacrificed)

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


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in were prepared for microscopic examination and weighed, respectively:
- Organ weights: Anesthetized animals, Liver, Kidneys, Adrenal glands, Testes, Epididymides, Cauda epididymis, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Spleen, Brain, Pituitary gland,Thyroid glands (with parathyroid glands)
- Microscopic examination: Vagina, Cervix uteri, Uterus, Ovaries, Oviducts, Left testis, Left epididymis, Seminal vesicles, Coagulation glands, Prostate, Pituitary gland, Adrenal glands, Liver, Kidneys, Spleen, Brain, Thyroids (with parathyroids), All gross lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations macroscopic and/or microscopic examination) as follows: All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted



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


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in were prepared for microscopic examination and weighed, respectively:
- Organ weights: Liver, Kidneys, Adrenal glands, Testes, Epididymides, Cauda epididymis, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Spleen, Brain, Pituitary gland,Thyroid glands (with parathyroid glands)
- Microscopic examination: Vagina, Cervix uteri, Uterus, Ovaries, Oviducts, Left testis, Left epididymis, Seminal vesicles, Coagulation glands, Prostate, Pituitary gland, Adrenal glands, Liver, Kidneys, Spleen, Brain, Thyroids (with parathyroids), All gross lesions
Statistics:
- Dunnett-test (two sided): Food consumption, body weight and body weight change, estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, anogenital distance, anogenital index, duration of sexual maturation
- Fisher's Exact Test: Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data, males with a certain amount of abnormal sperm
- Wilcoxon-Test: Proportions of affected pups per litter with necropsy observations, nipple/ areola anlagen, total spermatids/g testis, total sperm/g cauda epididymides, Sperm motility (%)
- Kruskal-Wallis-Test: Pup organ weights (absolute and relative)
Reproductive indices:
- Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
- Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100
- Female mating index (%) = number of females mated / number of females placed with males x 100
- Female fertility index (%) = number of females pregnant / number of females mated x 100




Offspring viability indices:
- Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
- Postimplantation loss (%) = (number of implantations – number of pups delivered) / number of implantations x 100
- Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x 100
- Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4 after birth x 100







Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: statistically significantly decreased mean body weights of males during study weeks 7-16 (up to 12%); statistically significantly decreased mean body weight gain of males during study weeks 4-10 and 13-14 (up to 45%); decreased overall body weight of males (-16%; weeks 0 -16); statistically significant lower body weight of females at the end of gestation (GD 20, about 6%) and statistically significantly decreased body weight gain in every gestational week; average decrease of weight gain during gestation (GDs 0-20) in females was about 18%. The body weights of the high dose F0 females remained statistically significant lower (about 8%) than control during the entire lactation period, whereas the lactation body weight gain was comparable to the control group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher food consumption of males during study weeks 3-10 and 12-16; statistically significant lower food consumption of females on GDs 14 -20 (about 7%) during gestation, and statistically significant below control on PNDs 7-14 (about 10%) during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: decrease red blood cell counts, hemoglobin values as well as hematocrit values in the F0 rats of both sexes of the 600 mg/kg bw/d dose group (i.e. after approx. 16-18 weeks of treatment).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: increase of the alkaline phosphatase activity in females and males; marginal, but statistically significant increase of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; bilirubin levels increased in female; decreased globulin values in both sexes; decreased total protein levels in males and females; albumin values were increased in the females (not statistically significant because of a high standard deviation); urea levels were statistically significant increased in the females and there was the same trend in the urea values of the males in this dose group; the triglyceride as well as the cholesterol values in the rats of both sexes were decreased; the calcium levels were decreased in the males
- 200 mg/kg bw/d: increase of the alkaline phosphatase activity in males; marginal, but statistically significant increase of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity (for AST not statistically significant in the 200 mg/kg bw/d dose group because of a greater standard deviation); decreased globulin values in both sexes; decreased total protein levels in the males; albumin values were increased in the females; the triglyceride as well as the cholesterol values in the rats of both sexes were decreased; the calcium levels were decreased in the males
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: liver of all male and female animals of 600 mg/kg test group as well as of all male and 2 female animals of the 200 mg/kg test group revealed a minimal (grade 1) to slight (grade 2) diffuse, centrolobular pronounced hepatocytic hypertrophy comprising cytoplasmic eosinophilia and a fine granular structure, indicative for the proliferation within the microsomal compartment (most likely peroxisomes). The liver discoloration was found to be most likely linked to the hepatocellular peroxisome proliferation.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: There were no test substance-related mortalities in any of the male and female F0 and F1 parental animals in any of the groups.
- Clinical signs: There were no test substance related clinical findings in the F0 females during the gestation and lactation period for F1 litter. For all dose levels, no clinical signs or changes in general behavior, which may be attributed to the test substance, were detected in male or female F1 generation parental animals. No test substance-related clinical findings were noted in all treated F1 females during the gestation and lactation and period for F2 litter.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0:
- 600 mg/kg bw/d: statistically significantly decreased mean body weights of males during study weeks 7-16 (up to 12%); statistically significantly decreased mean body weight gain of males during study weeks 4-10 and 13-14 (up to 45%); decreased overall body weight of males (-16%; weeks 0 -16); statistically significant lower body weight of females at the end of gestation (GD 20, about 6%) and statistically significantly decreased body weight gain in every gestational week; average decrease of weight gain during gestation (GDs 0-20) in females was about 18%. The body weights of the high dose F0 females remained statistically significant lower (about 8%) than control during the entire lactation period, whereas the lactation body weight gain was comparable to the control group; statistically significant higher food consumption of males during study weeks 3-10 and 12-16; statistically significant lower food consumption of females on GDs 14 -20 (about 7%) during gestation, and statistically significant below control on PNDs 7-14 (about 10%) during lactation
- 200 and 40 mg/kg bw/d: Mean body weights and body weight gain of males were comparable to the control group throughout the entire study. Food consumption of male and female rats was generally comparable to the respective controls throughout the entire study.

F1:
- 600 mg/kg bw: statistically significantly decreased mean body weights of males during study weeks 5-16 (up to 13%); statistically significantly decreased mean body weight gain of males during study weeks 3-11 (up to 31%); reduced overall weight gain of males (-14%; weeks 0-16); sporadically higher (statistically significant during study weeks 4-5) food consumption of males during the study; higher food consumption of female (statistically significant during study weeks 4-5, 6-7, 8-10) for the most part of the study; statistically significant higher food consumption of females on GDs 0 – 7 (about 6%) during gestation
- 200 and 40 mg/kg bw/d: Mean body weights and body weight gain of males were comparable to the control group throughout the entire study. During premating, the mean body weights and body weight gain of all treated parental F1 females (40, 200 and 600 mg/kg bw/d) were comparable to the control group, despite of some sporadical increases/decreases of weight/weight gain in all groups. Also during the gestation and lactation periods body weights and body weight gain of female animals of all treated groups were comparable to the concurrent control group, taking normal biological variability into account. Food consumption of the F1 parental male and female rats was generally comparable to the respective controls throughout the entire study.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
For all test groups the intake of the test substance correlated well with the desired target doses. The actual test substance intake was calculated on the basis of interpolated mean body weights of each test group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0:
The mean estrous cycle duration in the different test groups was similar: 5.4 days in the mid-dose group, 5.6 days in the low-dose group, 6.0 in the control group and 6.1 in the high-dose group.

F1:
The mean estrous cycle duration in the different test groups was similar: 4.0 days in the low-dose group, 4.1 days in the control and 4.2 days in the mid and high-dose groups

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related effects were noted for the sperm parameters, examined at or after the sacrifice of the F0 and F1 parental males.
The number of animals with more than 6.5% abnormal sperm in the F0 high-dose group (600 mg/kg bw/d) was slightly but statistically significant higher. Since the average rate of abnormal sperms as well as the morphology of the testicular tubules were not affected and this parameter was not changed in the F1 generation, this apparent increase is regarded as an incidental finding and biologically not relevant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- the male mating index was 100% in all groups including the controls for F0 males and 96% in the low-dose group and 100% in the remaining test groups for F1 males.
- the male fertility index ranged between 96% and 100% for F0 males and between 92% and 96% for F1 males without showing any relation to dosing
- the female mating index calculated after the mating period for F1 litter was 100% in all test groups and 100% in test groups 10, 12 and 13 and 96% in test group 11 for the F2 litter
- the fertility index varied between 96% (600 mg/kg bw/d) and 100% for F0 females and 96% in the control, 92% in the mid and high-dose groups and 100% in the low-dose group for F1 females. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility and do not show any relation to dosing.
- the gestation index was 100% in all groups including controls (0, 40, 200 and 600 mg/kg bw/d), indicating that all pregnant F0 and F1 females had live offsprings.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0:
- 600 mg/kg bw/d: statistically significant decrease of absolute terminal body weight in males; statistically significant increase of absolute kidney and liver weights; statistically significant increase in absolute cauda epididymis weights, statistically significant decrease of absolute prostate weights statistically significant decrease of absolute seminal vesicle weights and statistically significant decrease of absolute ovary weights were considered as not treatment-related; statistically significant increase of relative kidney weights in males; statistically significant increase of relative liver weights
- 200 mg/kg bw/d: treatment-related effect in the males cannot be excluded; statistically significant increase of absolute kidney and liver weights; statistically significant increase of relative kidney weights in males; statistically significant increase of relative liver weights
- 40 mg/kg bw/d: statistically significant decrease of absolute seminal vesicle weights was considered as not treatment-related; the minimal statistically significant increase of relative liver weights in 40 mg/kg males is disregarded as a treatment-related effect

The statistically significant increase of relative brain weights in 40 mg/kg females and 600 mg/kg males and females, of relative cauda epididymis and epididymides weights in 200 mg/kg and 600 mg/kg males, of relative kidney weights in 40 mg/kg and 600 mg/kg females, of thyroid gland weights in 600 mg/kg females and of testes weights in 600 mg/kg males as well as the statistically significant decrease of seminal vesicle weights in 40 mg/kg males is considered incidental and not treatment-related due to missing histopathological correlates, decrease of absolute terminal body weights and/or missing dose-response relationship
All other mean absolute weight parameters did not show significant differences compared to the control group.

F1:
- 600 mg/kg bw/d: statistically significant decrease of absolute terminal body weight in males; statistically significant increase of absolute kidney weights in males; the statistically significant increase of absolute liver weights in males and females; statistically significant increase of absolute weight of thyroid glands in females; treatment-related effect on the weight development of thyroid glands of males; statistically significant decrease of absolute spleen weights in 600 mg/kg males was considered incidental and not treatment-related; statistically significant increase of relative kidney and liver weights in males and females; statistically significant increase of relative weight of thyroid glands in females; the statistically significant weight increase of brain, cauda epididymis, epididymides, pituitary gland, seminal vesicle, and testes in 600 mg/kg males was considered incidental and not treatment-related due to missing histopathological correlates and explainable by the decrease of terminal body weight.
- 200 mg/kg bw/d: statistically significant increase of absolute kidney weights in males; statistically significant increase of absolute liver weights in males and females; treatment-related effect on the weight development of thyroid glands of males; statistically significant increase of relative kidney weights in males; statistically significant increase of relative liver weights in males and females
- 40 mg/kg bw/d: no treatment-like effect on the weight development of thyroid glands of males


GROSS PATHOLOGY (PARENTAL ANIMALS)
F0:
- 600 mg/kg bw/d: treatment-related liver enlargement of 23 males and 20 females and brown to dark-brown liver discoloration of 15 males;
- 200 mg/kg bw: treatment-related liver enlargement of 2 males

F1:
- 600 mg/kg bw: liver enlargement of 23 males and 14 females and brown to dark-brown liver discoloration of 17 males and 1 female animal
- 200 mg/kg bw: treatment-related liver enlargement of 1 male

All other gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.


HISTOPATHOLOGY (PARENTAL ANIMALS)
F0:
- Liver: liver of all male and female animals of 600 mg/kg test group as well as of all male and 2 female animals of the 200 mg/kg test group revealed a minimal (grade 1) to slight (grade 2) diffuse, centrolobular pronounced hepatocytic hypertrophy comprising cytoplasmic eosinophilia and a fine granular structure, indicative for the proliferation within the microsomal compartment (most likely peroxisomes). The liver discoloration was found to be most likely linked to the hepatocellular peroxisome proliferation.

All other findings in the treatment groups were either single observations, or were similar to the control rats in distribution pattern and severity. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.

F1:
- Liver: The liver of all male and 23 female animals of 600 mg/kg test group as well as of 22 male animals of the 200 mg/kg test group revealed a minimal (grade 1) to slight (grade 2) diffuse, centrolobular pronounced hepatocytic hypertrophy comprising cytoplasmic eosinophilia and a fine granular structure, indicative for the proliferation within the microsomal compartment (most likely peroxisomes).
Several special stains were carried out to elucidate the origin of the macroscopically noted brown discoloration of the liver, which were concluded to be most likely linked to the hepatocellular peroxisome proliferation.
- Kidney: kidneys of 24 male and 6 female animals of 600 mg/kg test group as well as 11 males of 200 mg/kg test group revealed a minimal (grade 1) eosinophilia of proximal tubular epithelial cells. One male animal showed a massive (grade 5) diffuse degeneration of the tubular epithelium in the testis together with a total aspermia and a moderate (grade 3) diffuse atrophy in both epididymides, which corresponded to the gross findings (organ size reduction) and were responsible for the infertility. The female mating partner did not have any histopathological findings.
Another male animal showed, corresponding to the gross lesions, a slight (grade 2) oligospermia in the left epididymis, but the left testis revealed no histopathological findings. The female mating partner did not show any lesions affecting the fertility.
- Thoroid gland: a follicular hypertrophy/hyperplasia was seen in the thyroid glands of 16 males and 18 females of the 600 mg/kg dose group as well as in13 male and 6 female animals of 200 mg/kg dose group. The follicular hypertrophy/hyperplasia in 2 male and 2 female control animals and in 3 male and 3 female animals of the 40 mg/kg dose group are considered incidental and not treatment-related. Altered colloid was seen 9 male and 4 female control animals, 11 males and 5 females of 40 mg/kg group, 21 males and 9 females of 200 mg/kg group and 25 males and 17 females of 600 mg/kg group.
- Pituitary gland: pituitary glands of 7 males of 600 mg/kg dose group revealed a minimal (grade 1) multifocal increase of basophilic cells.

All other findings in the treatment groups were either single observations, or were similar to the control rats in distribution pattern and severity. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.


HEMATOLOGY (PARENTAL ANIMALS)
- 600 mg/kg bw/d: decrease red blood cell counts, hemoglobin values as well as hematocrit values in the F0 and F1 rats of both sexes of the 600 mg/kg bw/d dose group (i.e. after approx. 16-18 weeks of treatment); statistically significant prolonged prothrombin time in the F1 males
-200 mg/kg bw/d: decent but statistically significant decrease of the red blood cell counts in F0 females, but regarded as non-adverse effect; decreased red blood cell counts in the F1 males accompanied by an increase of the MCV and MCH values, which were regarded as incidental.

CLINICAL CHEMISTRY
F0:
- 600 mg/kg bw/d: increase of the alkaline phosphatase activity in females and males; marginal, but statistically significant increase of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; bilirubin levels increased in female; decreased globulin values in both sexes; decreased total protein levels in males and females; albumin values were increased in the females (not statistically significant because of a high standard deviation); urea levels were statistically significant increased in the females and there was the same trend in the urea values of the males in this dose group; the triglyceride as well as the cholesterol values in the rats of both sexes were decreased; the calcium levels were decreased in the males
- 200 mg/kg bw/d: increase of the alkaline phosphatase activity in males; marginal, but statistically significant increase of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity (for AST not statistically significant in the 200 mg/kg bw/d dose group because of a greater standard deviation); decreased globulin values in both sexes; decreased total protein levels in the males; albumin values were increased in the females; the triglyceride as well as the cholesterol values in the rats of both sexes were decreased; the calcium levels were decreased in the males

F1:
- 600 mg/kg bw/d: increased alkaline phosphatase activities in males and females; increased bilirubin values in females; decreased globulin values in males and females; increased albumin values in males and females; decreased total protein levels in males and females; increased urea values and decreased glucose levels in males; decreased cholesterol and the triglyceride levels in both sexes; decreased calcium levels in males and females; increased inorganic phosphate in males
- 200 mg/kg bw/d: increased alkaline phosphatase activities in males; increased bilirubin values in females; decreased globulin values in males and females; increased albumin values in males and females; decreased total protein levels in males; decreased cholesterol and the triglyceride levels in both sexes; decreased calcium levels in males
- 40 mg/kg bw/d: decreased globulin values in males; the significant decrease of the triglyceride levels in males and low globulin values in the males were seen as non-adverse due to lack of histopathological findings.

Other found changes were marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings were considered to be of no toxicological significance.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
bone
kidney
liver
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw: statistically significantly decreased mean body weights of males during study weeks 5-16 (up to 13%); statistically significantly decreased mean body weight gain of males during study weeks 3-11 (up to 31%); reduced overall weight gain of males (-14%; weeks 0-16).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw: sporadically higher (statistically significant during study weeks 4-5) food consumption of males during the study; higher food consumption of female (statistically significant during study weeks 4-5, 6-7, 8-10) for the most part of the study; statistically significant higher food consumption of females on GDs 0 – 7 (about 6%) during gestation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: decrease red blood cell counts, hemoglobin values as well as hematocrit values in the F1 rats of both sexes of the 600 mg/kg bw/d dose group (i.e. after approx. 16-18 weeks of treatment); statistically significant prolonged prothrombin time in the F1 males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: increased alkaline phosphatase activities in males and females; increased bilirubin values in females; decreased globulin values in males and females; increased albumin values in males and females; decreased total protein levels in males and females; increased urea values and decreased glucose levels in males; decreased cholesterol and the triglyceride levels in both sexes; decreased calcium levels in males and females; increased inorganic phosphate in males
- 200 mg/kg bw/d: increased alkaline phosphatase activities in males; increased bilirubin values in females; decreased globulin values in males and females; increased albumin values in males and females; decreased total protein levels in males; decreased cholesterol and the triglyceride levels in both sexes; decreased calcium levels in males
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw/d: statistically significant decrease of absolute terminal body weight in males; statistically significant increase of absolute kidney weights in males; the statistically significant increase of absolute liver weights in males and females; statistically significant increase of absolute weight of thyroid glands in females; treatment-related effect on the weight development of thyroid glands of males; statistically significant decrease of absolute spleen weights in 600 mg/kg males was considered incidental and not treatment-related; statistically significant increase of relative kidney and liver weights in males and females; statistically significant increase of relative weight of thyroid glands in females; the statistically significant weight increase of brain, cauda epididymis, epididymides, pituitary gland, seminal vesicle, and testes in 600 mg/kg males was considered incidental and not treatment-related due to missing histopathological correlates and explainable by the decrease of terminal body weight.
- 200 mg/kg bw/d: statistically significant increase of absolute kidney weights in males; statistically significant increase of absolute liver weights in males and females; treatment-related effect on the weight development of thyroid glands of males; statistically significant increase of relative kidney weights in males; statistically significant increase of relative liver weights in males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 600 mg/kg bw: liver enlargement of 23 males and 14 females and brown to dark-brown liver discoloration of 17 males and 1 female animal.
- 200 mg/kg bw: treatment-related liver enlargement of 1 male.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: The liver of all male and 23 female animals of 600 mg/kg test group as well as of 22 male animals of the 200 mg/kg test group revealed a minimal (grade 1) to slight (grade 2) diffuse, centrolobular pronounced hepatocytic hypertrophy comprising cytoplasmic eosinophilia and a fine granular structure, indicative for the proliferation within the microsomal compartment (most likely peroxisomes).
Several special stains were carried out to elucidate the origin of the macroscopically noted brown discoloration of the liver, which were concluded to be most likely linked to the hepatocellular peroxisome proliferation.
- Kidney: kidneys of 24 male and 6 female animals of 600 mg/kg test group as well as 11 males of 200 mg/kg test group revealed a minimal (grade 1) eosinophilia of proximal tubular epithelial cells. One male animal showed a massive (grade 5) diffuse degeneration of the tubular epithelium in the testis together with a total aspermia and a moderate (grade 3) diffuse atrophy in both epididymides, which corresponded to the gross findings (organ size reduction) and were responsible for the infertility. The female mating partner did not have any histopathological findings.
Another male animal showed, corresponding to the gross lesions, a slight (grade 2) oligospermia in the left epididymis, but the left testis revealed no histopathological findings. The female mating partner did not show any lesions affecting the fertility.
- Thoroid gland: a follicular hypertrophy/hyperplasia was seen in the thyroid glands of 16 males and 18 females of the 600 mg/kg dose group as well as in13 male and 6 female animals of 200 mg/kg dose group. The follicular hypertrophy/hyperplasia in 2 male and 2 female control animals and in 3 male and 3 female animals of the 40 mg/kg dose group are considered incidental and not treatment-related. Altered colloid was seen 9 male and 4 female control animals, 11 males and 5 females of 40 mg/kg group, 21 males and 9 females of 200 mg/kg group and 25 males and 17 females of 600 mg/kg group.
- Pituitary gland: pituitary glands of 7 males of 600 mg/kg dose group revealed a minimal (grade 1) multifocal increase of basophilic cells.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P1)

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

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
bone
kidney
liver
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during early lactation (PND 0-4) was 99% in the control and low dose groups for F1 pups. A statistically significant lower viability index in mid dose group (94%) and high dose group (95%) went along with statistically significantly increased numbers of died and cannibalized pups in the mid and high dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F1 male and female pups were statistically significant below control from PND 14 onwards (about 11% on PND 21). Body weight gain was statistically significantly decreased in these pups from PND 4 onwards, the average weight gain during PND 4-21 was about 13% below control.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
- The mean number of delivered F1 and F2 pups per dam and the rates of liveborn and stillborn F1 and F2 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- The viability index indicating pup mortality during early lactation (PND 0-4) was 99% in the control and low dose groups for F1 pups. A statistically significant lower viability index in mid dose group (94%) and high dose group (95%) went along with statistically significantly increased numbers of died and cannibalized pups in the mid and high dose groups.
For F2 pups, the viability index was between PND 0-4 was 98% in test group 13, 99% in control group 10 and 100% in low- and mid dose groups.
- The lactation index for F1 pups indicating pup mortality on PND 4-21 was 100% in control group and low-dose group and 99% in test groups with 200 and 600 mg/kg bw/d.
For F 2 pups, the lactation index was between PND 4-21 varied between 99% (40 mg/kg bw/d) and 100% for the other dose groups.

Sex ratio
The sex ratio was unaltered by test substance administration. The percentage of males was 43.6%, 49%, 50.8%, 45% in the control, low, mid, and high dose group.

CLINICAL SIGNS (OFFSPRING)
The F1 generation pups did not display any clinical signs until weaning.
Only one pup (of the high-dose group showed a kinked tail. All other F2 generation pups did not show any clinical signs up to weaning

BODY WEIGHT (OFFSPRING)
Mean body weights of the high-dose F1 male and female pups were statistically significant below control from PND 14 onwards (about 11% on PND 21). Body weight gain was statistically significantly decreased in these pups from PND 4 onwards, the average weight gain during PND 4-21 was about 13% below control.
No test compound-related influence on F1 pup body weights was noted in the low- and mid-dose groups.
Mean body weights of the high-dose F2 male and female pups were statistically significant below control from PND 14 onwards (about 8% on PND 21). Body weight gain was statistically significantly decreased in these pups from PND 7 onwards, the average weight gain during PND 4-21 was about 9% below control.
No test compound-related influence on F2 pup body weights was noted in the low- and mid-dose groups.


SEXUAL MATURATION (OFFSPRING)
- Females: The mean number of days to reach the criterion in the control and 40, 200 and 600 mg/kg bw/d test groups amounted to 30.7, 30.3, 30.2, and 30.9 days, indicating that female sexual maturation was not influenced by the test substance.
- Males: The mean number of days to reach the criterion in the control and 40, 200 and 600 mg/kg bw/d test groups was 41.8, 42.4, 42.2, and 42.2 days, indicating that male sexual maturation was not influenced by the test substance.
Neither anogenital distance (ADG)/anogenital index (AGI) nor the number and percentage of F1 and F2 male pups having areolae was influenced by the test substance in all treated groups

ORGAN WEIGHTS (OFFSPRING)
Mean absolute and relative pup organ weights of the F1 pups did not show statistically significant differences to the control group. However, the decreased absolute thymus (-15.8%) and spleen weights (-15.9%) as well as the increased relative brain weights (+10.8%) of the high-dose F1 pups were assessed as secondary to the lower pup body weights in this group. The findings are neither adverse nor toxicologically relevant.
Mean absolute and relative pup organ weights of the F2 pups did not show statistically significant differences to the control group.
The decreased absolute thymus weights (-12.3%) as well as the increased relative brain weights (+9.4%) of the high-dose F2 pups were assessed as secondary to the lower pup body weights in this group. The findings are neither adverse nor toxicologically relevant.


GROSS PATHOLOGY (OFFSPRING)
The number and percentage of male pups having areaolae was not influenced by the test substance when examined on PNDs 12-15.
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (40, 200 and 600 mg/kg bw/d).

HISTOPATHOLOGY (OFFSPRING)
At gross necropsy, a number of common findings were seen in F1 and F2 pups, such as post mortem autolysis, incisors sloped, hemorrhagic thymus, diaphragmatic hernia, dilated renal pelvis, hemorrhagic testis, small testis and hemorrhagic epididymis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or higher incidences. All these findings were not considered to be associated to the test substance.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F1
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Only one pup (of the high-dose group showed a kinked tail. All other F2 generation pups did not show any clinical signs up to weaning
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
For F2 pups, the viability index was between PND 0-4 was 98% in test group 13, 99% in control group 10 and 100% in low- and mid dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F2 male and female pups were statistically significant below control from PND 14 onwards (about 8% on PND 21). Body weight gain was statistically significantly decreased in these pups from PND 7 onwards, the average weight gain during PND 4-21 was about 9% below control.
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
no effects observed

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Generation:
F2
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Absolute organ weights:

Males Females
40 mg/kg bw day 200 mg/kg bw day 600 mg/kg bw day 40 mg/kg bw day 200 mg/kg bw day 600 mg/kg bw day
F0 Terminal body weight 99% 96%** 87%**
Cauda epididymis 102% 106% 110%**
Kidneys 100% 110%** 114%**
Liver 101% 121%** 155%** 96% 111%** 147%**
Ovaries 98% 97% 90%**
Prostate 103% 100% 91%**
Seminal vesicle 92%* 99% 92%*
F1 Terminal body weight 98% 97% 86%**
Kidneys 101% 109%* 107%*
Liver 100% 120%** 146%** 105% 112%** 146%**
Spleen 101% 98% 84%**
Thyroid glands 115%** 119%** 107%* 103% 106% 117%**

*: p <= 0.05

**: p <= 0.01

Relative organ weights:

Males Females
40 mg/kg bw day 200 mg/kg bw day 600 mg/kg bw day 40 mg/kg bw day 200 mg/kg bw day 600 mg/kg bw day
F0 Brain 99% 103% 113%** 104%* 102% 104%**
Cauda epididymis 104% 111%** 128%**
Epididymides 103% 105%* 118%**
Kidneys 101% 115%** 132%** 104%* 103% 107%**
Liver 103%* 126%** 178%** 98% 113%** 151%**
Pituitary gland 100% 100% 100%**
Seminal vesicle 93%* 103% 106%
Testes 102% 105% 118%**
Thyroid glands 111% 100% 111%**
F1 Brain 103% 103% 115%**
Cauda epididymis 101% 106% 126%**
Epididymides 101% 103% 120%**
Kidneys 103% 112%** 125%** 101% 100% 107%**
Liver 102% 124%** 171%** 103% 111%** 146%**
Pituitary gland 100% 150% 150%**
Seminal vesicle 100% 105% 113%**
Testes 101% 103% 119%**
Thyroid glands 133%** 133%** 133%** 100% 100% 111%**

*: p <= 0.05

**: p <= 0.01

Applicant's summary and conclusion

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