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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no one- or two-generation study available for 2 -propylheptan-1 -ol, but a read-across can be made from Di-(2-propylheptyl) phthalate.

Di-(2-propylheptyl) phthalate neither influenced fertility nor reproductive performance in the parental animals. Viability, sex ratio and sexual development/maturation of offspring up to the top dose of 600 mg/kg bw/d were unaltered. Neither anogenital distance/anogenital index nor the number and percentage of F1 and F2 male pups having areaolae were influenced in all treated groups. There were no indications of estrogenic or anti-androgenic activity (BASF, 2019).

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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
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







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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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
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
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
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
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.
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
Critical effects observed:
no
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
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
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
Critical effects observed:
no
Reproductive effects observed:
not specified

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

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification in chapter 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
40 mg/kg bw/day
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
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
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
Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F1
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
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
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A two-generation study with the source substance Di-(2-propylheptyl) phthalate according to OECD/EU guideline and in compliance with GLP regulations is available. This study is considered reliable without restrictions.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no data concerning potential effects of 2-Propylheptan-1-ol on fertility available. Therefore, a read-across from di-(2-propylheptyl) phthalate to 2-Propylheptan-1-ol was conducted.

Toxicity to reproduction of di-(2-propylheptyl) phthalate was evaluated in a Two-Generation Reproduction Toxicity Study performed under GLP according guidelines OECD 416, EU Method B.35 and EPA OPPTS 870.3800 (BASF, 2009). The test substance was administered to groups of 25 male and 25 female young Wistar rats via the diet over two parental (F0 and F1) generations with dietary target dosages of 0, 40, 200 and 600 mg/kg bw/day. The test substance concentrations in the diet were adjusted regularly throughout the study to maintain the intended dose levels. It was found, that di-(2-propylheptyl) phthalate neither influenced fertility or reproductive performance in the parental animals, nor viability, sex ratio and sexual development/maturation of offspring, up to the top dose of 600 mg/kg bw/d. Also, neither anogenital distance/anogenital index nor the number and percentage of F1 and F2 male pups having areaolae were influenced in all treated groups. However, the mid and high dose parental animals showed clinical and/or pathological signs of systemic toxicity, while in the high dose F0 animals and F1 males, intermittent reductions of food consumption and body weights/body weight gain were noted, either during premating, mating, gestation and lactation phases of this study. Furthermore, the rats of both sexes in the F0 as well as in the F1 generation of the high dose group showed a mild anemia. Clinical pathological changes indicated a test substance related effect of the liver cell and bone metabolism beginning in the 200 mg/kg bw/d dose groups in both sexes in the F0 and F1 generation. This was further substantiated by a hepatocellular hypertrophy, which was centrolobular pronounced along with a cytoplasmic eosinophilia, indicative of peroxisome proliferation. Organ weight changes of liver and kidneys as well as corresponding morphological changes in the kidneys, thyroid and pituitary glands are regarded as secondary to the peroxisome proliferation. Xenobiotic metabolism induced in parallel results in an increased elimination of T3/T4 from rat serum by increased glucuronidation. This secondary mode of action is not relevant to humans as rodents are more sensitive to thyroid effects.Bhat et al. (2014), Regulatory Toxicology and Pharmacology 70, 65-74, published the comprehensive oral risk assessment undertaken by the US EPA chaired Health Advisory board of NSF International. Bhat reduced the interspecies extrapolation factor for derivation of the oral reference dose (RfD) to 1 applying the US EPA guidance. The lack of human relevance of these thyroid effects is also reflected in the opinion of theGerman MAK Commission (2015). The F1 and F2 offspring in the high dose group (600 mg/kg bw/d) had significantly reduced body weights and gained also less body weight than the control offspring from day 14 of lactation onwards. Secondary to the reduced body weight gain, lower weights of spleen and thymus were noted in the high dose offspring. Thus, the NOAEL for general, systemic toxicity was determined to be 40 mg/kg bw/d for the F0 and F1 parental rats, based on effects secondary to peroxisome proliferation.The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity in the F1 and F2 progeny is 200 mg/kg bw/day, based on slightly decreased pup body weights/pup weight gain in the second third of lactation. Importantly, the developmental effects do not occur in the absence of parental toxicity.

In summary, the available relevant testing data on the source substance, di-(2-propylheptyl) phthalate, do not give any indication for adverse effects on fertility. Therefore, no potential for toxicity to reproduction and is assumed for the target substance as well.

Effects on developmental toxicity

Description of key information

- rat, OECD 414: LOAEL(fetal toxicity) = 600 mg/kg bw; NOAEL(fetal toxicity) = 200 mg/kg bw; NOAEL(maternal toxicity) = 50 mg/kg bw (BASF, 2004; reliable)

- rat, OECD 414, Screening: LOAEL(fetal toxicity) = 790 mg/kg bw; NOAEL(fetal toxicity) = 158 mg/kg bw; NOAEL(maternal toxicity) = 158 mg/kg bw (BASF, 1995; reliable)

 

There is no developmental toxicity / teratogenicity study in a second species available for 2 -propylheptan-1 -ol, but a read-across can be made from Di-(2-propylheptyl) phthalate (CAS 53306-54-0):

- rabbit, OECD 414, Screening: NOAEL(fetal toxicity) ≥ 127 mg/kg bw; NOAEL(maternal toxicity) = 66 mg/kg bw (BASF, 2018; reliable)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Jan. 22, 2001)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Propylheptanol
- Physical state: liquid/colorless;
- Analytical purity: 99.8%
- Lot/batch No.: Tk 2012
- Test substance No.: 02/0356-1
- Stability: The stability under storage conditions was confirmed by reanalysis. The stability of Propylheptanol in doubly distilled water + Cremophor EL for a period of 7 days at room temperature was demonstrated.
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature, under N2
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 70 - 84 days at delivery
- Weight at study initiation: 145.5 - 187.9 g
- Housing: single (from day 0 - 20 p.c.)
- Diet: ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland; ad libitum;
- Water: drinking water of tap water quality from water bottles; ad libitum;
- Acclimation period: between the supply on day 0 and the first administration on gestational day 6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
other: doubly distilled water with Cremophor EL
Details on exposure:
- Concentration of the emulsion (mg/100 ml): 500, 2000, and 6000
- Dose volume: 10 ml/kg bw

PREPARATION OF DOSING SOLUTIONS:
The test substance emulsions in doubly distilled water were prepared at the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the emulsions, an appropriate amount of the test substance was weighed depending on the dose group in calibrated beakers, topped up with doubly distilled water and some drops Cremophor EL and subsequently thoroughly mixed using a high-speed homogenizer. A magnetic stirrer was used to keep the emulsions homogeneous during treatments.
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 doubly distilled water + Cremophor EL for a period of at least 7 days at room temperature were carried out before the study was initiated.
Samples of the test substance emulsions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analyses at the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 600 mg/kg body weight/day). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
The test substance emulsions were analyzed by GC. The analytical values of the samples corresponded to the expected values within the limits of the
analytical method, i.e. were always above 90% of the nominal concentration.
Details on mating procedure:
The animals were mated by the breeder (time-mated) and supplied on day 0 post coitum (= detection of vaginal plug / sperm).
Duration of treatment / exposure:
day 6 through day 19 p.c.
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
50 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, concurrent vehicle
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check for mortality was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results.
Corrected body weight gain (net maternal body weight change):
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (With the exception of day 0, the consumption of food was determined on the same days as was body weight.)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: With the exception of day 0, the consumption of drinking water was determined on the same days as was body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Weight of liver, kidneys and spleen, Weight of the unopened uterus
One test group 1 animal and two test group 3 animals that died or had to be sacrificed during the study period were examined according to the same procedures as the females killed on schedule (exception: no weight determinations of uterus, liver, kidneys and spleen).

Examinations of the dams at termination:
- Clinical Pathology:
Blood was taken from the retroorbital venous plexus in the morning from non-fasted animals without anesthesia. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence.
The following examinations were carried out in all animals per test group at the end of the administration period.
Hematology: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count;
Furthermore, differential blood smears were prepared and stained according to Wright without being evaluated.
Clotting analyses: prothrombin time (Hepato Quick's test)
Clinical chemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
- Food consumption, water consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Organ weights (liver, kidneys, spleen): Non-parametric one-way analysis using KRUSKAL-WALLIS-test (two-sided). If the p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON-test (two-sided) for the equal medians.
- Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
- Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Clinical pathology parameters, except differential blood count: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided) .If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Indices:
In the present study the glossary of WISE et al. (Wise et al., 1997) was used as much as, possible to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (Phahoud et al., 1999; Solecki et al., 2001):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that occurs also in fetuses of control animals and is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that has otherwise followed a normal pattern of development.
Moreover, the terms "unclassified observation" or "unclassified cartilage observation" were used for those fetal findings, which could not be classified as malformations or variations (e.g. focal liver necrosis in fetuses, isolated cartilage findings without any impact on the respective bony structure).
Historical control data:
The results were compared with historical control data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- clinically observed transient salivation after treatment in all animals and occasionally urine smeared fur in 6/25 animals

Test group 2 (200 mg/kg body weight/day):
- clinically observed transient salivation after treatment in 8/25 animals

The observed temporary salivation of the animals is considered to be substance-induced. It is very likely, that this finding was induced by bad taste of the test substance or local irritation of the upper digestive tract . This type of salivation is not assessed as an adverse systemically toxic effect .
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A number of low (1 animal) and high dose females (2 animals) either died or had to be sacrificed after gavage error. No relationship of these deaths to the test compound is assumed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- significantly reduced final body weight (7% below control )
- significantly reduced absolute (23% below control) and net (52% below control) body weight gain

Test group 2 (200 mg/kg body weight/day):
- significantly reduced final body weight (6% below control)
- significantly reduced absolute (15% below control) and net (19% below control) body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- significantly reduced food consumption (11 % below control)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- significantly increased water consumption (34% above control)
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- decreased platelets
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- decreased sodium, chloride, total protein, globulins and cholesterol
- increased inorganic phosphate and urea
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):
Test group 3 (600 mg/kg body weight/day):
- increased absolute (12% above control) and relative (20% above control) liver weight (due to peroxisomal proliferation)

Test group 2 (200 mg/kg body weight/day):
- increased relative (5% above control) liver weight (due to peroxisomal proliferation)
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: effects due to peroxisomal proliferation (no human hazard): increased relative liver weight
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- statistically significantly decreased fetal weights (11 % below control)

considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see above
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (600 mg/kg body weight/day):
- increased incidence of skeletal variations, in particular incompletely or non-ossified morphologically unchanged skeletal structures or unspecific variants of ossification (supernumerary thoracic vertebrae, supernumerary and/or wavy ribs)

considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity
Visceral malformations:
no effects observed
Other effects:
no effects observed
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
- statistically significantly decreased fetal weights (11 % below control)
- increased incidence of skeletal variations, in particular incompletely or non-ossified morphologically unchanged skeletal structures or unspecific variants of ossification (supernumerary thoracic vertebrae, supernumerary and/or wavy ribs)

These effects were regarded as secondary effects due to clear maternal toxicity.
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Dose (mg/kg bw) 0 50 200 600
Mated females 25 25 25 25
Pregnant females 22 22 24 22
Mortality of dams 0 1 0 1
Pregnant on C section 22 22 24 22
Clinical symptoms --- --- + +
Food consumption
day 6-19 p.c. (g)
20.6 19.9 19.7 18.4
Body weight day 0 (g) 174.4 171 170.9 175.5
Body weight day 6 (g) 204.3 268.8 259.6* 261.2*
Body weight gain (days 6-19), g 71.8 69.2 61.2** 55.5**
Uterus weight (g) 52.4 51.2 45.8 48.3
Liver weight rel. (%) 4.15 4.13 4.33* 4.97**
Kidney weight rel. (%) 0.15 0.51 0.52 0.53
Kidney weight rel. (%) 0.17 0.18 0.19* 0.16*
Platelets (giga/l) 788 797 761 682**
Na (mmol/l) 141 140.6 140.4 139.4*
Cl (mmol/l) 100.1 100.1 99.8 98.1**
Inorganic phosphate (mmol/l) 1.8 1.84 1.91 2.13**
Urea (mmol/l) 7 7.36 7.54 8.01**
Total protein (g/l) 66.1 65.1 65.3 60.7**
Globulin (g/l) 30.5 29.8 29.6 25.8**
Cholesterol (mmol/l) 2.29 2.15 5.15 1.84**
+ = salivation, urine smeared fur (high dose only)
* = p</=0.05; ** = p</= 0.01, Dunnett test or Kruskal-Wallis and Wilcoxon 
Caesarian section / fetal examination:
Dose (mg/kg bw) 0 50 200 600
Mated females 25 25 25 25
Pregnant on C section 22 22 24 22
Corpora lutea (mean) 10.8 10.8 10.8 11
Implantation sites (mean) 9.8 9.8 8.8 10
Post-implantation loss (mean %) 3.4 4.5 8.2 5.1
Dead fetuses/litter (mean %) 0 0 0 0
Resorption sites/litter (mean) 0.3 0.5 0.4 0.5
Mean No. of live fetuses/litter 9.5 9.3 8.8 9.5
Mean fetal weights, males (g) 3.6 3.7 3.6 3.2**
Mean fetal weights, females (g) 3.5 3.5 3.4 3.1**
% of fetuses with external malformations 0 0.5 0 0
% of fetuses with visceral malformations 0 0 1.1 0
% of fetuses with skeletal malformations 0 2.8 1.9 3.6
% of fetuses with external variations 0 0 0 0
% of fetuses with visceral variations 9.3 10 9.5 6.1
% of fetuses with skeletal variations 96 95 99 100
Executive summary:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Propylheptanol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (days 6 - 19 p.c.) elicited clear signs of maternal toxicity at dose levels of 200 mg/kg body weight/day and above. The test compound caused a number of clinical observations, reduced bodyweight gain, and caused a mild thrombocytopenia, slight changes in serum electrolytes, a mild impairment of kidney function, changes in protein metabolism and a slight induction of peroxisomal enzymes during pregnancy in the dams.

There were no substance-induced, dose-related influences on the gestational parameters and no test substance-induced indications of teratogenicity as well as prenatal developmental toxicity, up to and including the highest dose level (600 mg/kg body weight/day) . A slight retardation of embryo-/fetal development (delay of ossification and increased incidence of supernumerary thoracic vertebrae and supernumerary/wavy ribs) along with a slight decrease of the mean weight of the fetuses which exclusively was observed at 600 mg/kg body weight/day was considered

to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg body weight/day. The developmental NOAEL is 200 mg/kg body weight/day. No indications for a teratogenic potential of the test compound were noted.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(8 - 10 dams instead of 25)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Propylheptanol
- Physical state: liquid/colorless
- Analytical purity: 99.5%
- Lot/batch No.: CIW/E - Reg. No. 20 595
- Test substance No.: 94/279
- Stability: The stability of the test substance over the study period has been proven by reanalysis. The stability of the test substance emulsions over a period of up to 3 hours at room temperature was confirmed.
- Homogeneity: The homogeneity of the test substance was proven by visual inspection. Concerning the homogeneous distribution of the test substance in the vehicle analytical values corresponded to about 87.3% to 100.5%.
- Storage condition of test material: Room temperature; in tightly sealed container stored in darkness
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 70 days (day 0, detection of sperm)
- Weight at study initiation: mean: approximately 233 g
- Housing: singly
- Diet: ground Kliba 343 feed rat/mouse/ hamster supplied by KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water of tap water quality from water bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
other: doubly distilled water with approx. 0.005% Cremophor EL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Each day the test substance emulsions were freshly prepared shortly before the test substance was administered. For the preparation of the emulsions, an appropriate amount of the test substance was weighed and subsequently emulsified in doubly distilled water with about 0.005% Cremophor EL using a high speed sonicator. A magnetic stirrer was used to keep the emulsions homogeneous during treatment of the animals.

A standard dose volume of 5 ml/kg body weight was used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of active ingredient determined before the beginning of the treatment period was 99.5%. The results of the analyses of the emulsions of test substance confirmed the correctness of the prepared concentrations in general (the maximum deviation was about 13% from the target value).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 4 untreated females housed overnight with one adult male
- Length of cohabitation: about 15.5 h
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 through day 15 post coitum (pc)
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
158 mg/kg bw/day
Remarks:
= 1 mM/kg bw)
Dose / conc.:
790 mg/kg bw/day
Remarks:
= 5 mM/kg bw
Dose / conc.:
1 580 mg/kg bw/day
Remarks:
=10 mM/kg bw
No. of animals per sex per dose:
10
Control animals:
other: yes, group #1: bi-distilled water, group #2: emulsifier (bi-distilled water with approx. 0.005% Cremophor EL)
Details on study design:
- Dose selection rationale: The selection of doses for the present examination was based on the results of several preceding screening studies with different alcohols, which had a similar study design.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 pc). A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 pc).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 pc. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 pc minus weight of the uterus before it was opened minus body weight on day 6 pc).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (With the exception of day 0, the consumption of food was determined on the same days as was body weight.)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: liver, kidneys and the unopened uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Furthermore, calculations of conception rate and pre-and postimplantation losses were carried out.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
Examinations of dams and fetuses:

The DUNNETT-Test was used for a simultaneous comparison of several dose groups with both control groups. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters:
Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), liver and kidney weights (absolute and relative), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight.
FISHER's Exact Test was used for a pairwise comparison of each dose group with the controls for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON-Test was used for a comparison of each dose group with the controls for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations and/or unclassified observations in each litter.
Indices:
Classification of these findings was based in the present revision of the study report on the terms and definitions proposed by CHAHOUD et al. (Chahoud et al., 1999; Solecki et al., 2001):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that occurs also in fetuses of control animals and is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that has otherwise followed a normal pattern of development.
Moreover, the term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations (e.g. blood coagulum around placenta; focal liver necrosis in fetuses).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- numerous adverse clinical findings like apathy, unsteady gait, chromodacryorrhea, poor generaI/nutritionaI state, salivation and urine smeared fur before and/or after the daily intubation

Test group 3 (790 mg/kg body weight/day):
- salivation in all animals after the daily treatment (days 8 - 15 pc)
Mortality:
mortality observed, treatment-related
Description (incidence):
Test group 4 (1580 mg/kg body weight/day):
- intercurrent death of all except one rat between days 8 and 11 pc
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- statistically significantly lower body weights than the controls from day 8 to day 20 pc (again the high mortality during the treatment period has to be taken into consideration when the respective results are examined)
- considerable body weight loss during the treatment period; reduced corrected body weight gain in the only survivor

Test group 3 (790 mg/kg body weight/day):
- statistically significantly lower body weight gain if calculated for the total treatment period (about 21%)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- statistically significantly reduced food consumption during the treatment period and the first days post treatment (- 55%); due to the intercurrent death of all except one female of this dose group, however, the calculation of the food consumption from day 10 pc onwards was only based on one dam

Test group 3 (790 mg/kg body weight/day):
- slightly, but statistically significantly reduced food intake on days 6 - 8 pc and 13 - 15 pc

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
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):
Test group 4 (1580 mg/kg body weight/day):
- drastically lower mean gravid uterus weight in the only rat which survived until terminal sacrifice due to the resorption of all implants
- statistically significantly increased relative liver (61%) and kidney weights (45%) in the dam which survived until terminal sacrifice

Test group 3 (790 mg/kg body weight/day):
- statistically significantly reduced mean gravid uterus weight
- statistically significantly increased relative liver weight (5%) due to peroxisomal proliferation
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- stomach ulcerations or stomach erosions in 4 of the animals which died intercurrently; papillomatous hyperplasia in the forestomach of the dam which survived until terminal sacrifice
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- due to the resorption of all implants in the only surviving dam postimplantation loss of 100%
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- resorption of all implants in the only surviving dam
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Test group 4 (1580 mg/kg body weight/day):
- resorption of all implants in the only surviving dam (in contrast to the usual appearance of the uterine mucosa in case of late resorptions, in this case only very small amounts of fetal tissue in addition to placental tissue were still visible)
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The conception rate reached 100% in all test groups except the 790 mg/kg group were it reached 80%.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
organ weights and organ / body weight ratios
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- the mean fetal body weights in test group 3 (790 mg/kg body weight/day) were statistically significantly (about 10%) lower than in the two concurrent control groups
- since no fetuses were available in test group 4 (1,580 mg/kg body weight/day), no fetal weights could be evaluated in this group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see above
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Since no fetuses were available in test group 4 (1,580 mg/kg body weight/day), no sex distribution of the fetuses in this group could be calculated.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
- due to the premature death of all implants, no fetuses could be examined for extemal findings in test group 4 (1,580 mg/kg body weight/day)
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- due to the premature death of all implants, no fetuses could be examined for skeletal findings in test group 4 (1,580 mg/kg body weight/day)
- various malformations of the vertebral column (fused and/or misshapen vertebrae; absent cervical/lumbar vertebra; fused cervical arch; absent lumbar arch), the clavicle (deformed), the sternum (cleft sternum; malpositioned and bipartite sternebra) and/or the ribs (branched) were seen in test groups 0 - 3 (control #1, control #2, 158 and 790 mg/kg body weight/day); without any statistically significant differences berween the groups and without any relation to dosing
- observed skeletal variations were related to the skull (no or incomplete ossification of hyoid or all skull bones), the vertebral column (no ossification or incomplete, bipartite or dumbbell ossification of cervical arch, thoracic vertebra or centrum, lumbar arch or centrum and/or sacral arch or vertebra; supemumerary thoracic vertebra), the ribs (short or absent 13th, supemumerary 14th, cervical or wavy ribs), the sternum (misshapen sternebra; unilateral, bipartite, incomplete or missing ossification of sternebra), the fore and hindlimbs (incomplete ossification of metacarpal or metatarsal) and the pelvic girdle (incomplete ossification of pubis)
- the rate of skeletal variations was distinctly and statistically significantly increased at 790 mg/kg body weight/day. The main reason for this substance-induced effect is the increased occurrence of delays in the ossification process, which are actually a consequence of the reduced fetal body weights in this group
Visceral malformations:
no effects observed
Description (incidence and severity):
- due to the premature death of all implants, no fetuses could be examined for soft tissue findings in test group 4 (1,580 mg/kg body weight/day)
- one soft tissue malformation (dextrocardia) in one fetus of test group 2 (158 mg/kg body weight/day) and in one fetus of test group 3 (790 mg/kg body weight/day); This malformation of the heart occurs also sporadically in control fetuses. Therefore the isolated and disparate nature of this finding without a clear relation to dosing is not regarded to be treatment-related, but assessed to be spontaneous in nature.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The oral administration of Propylheptanol up to a dose of 790 mg/kg body weight to pregnant Wistar rats caused marginal effects on fetal morphology in the presence of matemal toxicity, but no indications for teratogenicity. The observable, statistically significantly increased skeletal variations of the mid dose fetuses mirror common findings on fetal morphology most probably due to fetal growth retardations and/or due to
matemal stress. They are, however, not indicative for selective effects on the fetal organism. No substance-induced effects on fetal morphology occurred at the low dose level (158 mg/kg body weight/day).
Dose descriptor:
LOAEL
Effect level:
790 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: secondary effects due to clear maternal toxicity
Dose descriptor:
NOAEL
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: secondary due to clear maternal toxicity
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
- reduced fetal body weights
- statistically significantly increased rate of skeletall variations (delayed ossification process)

These effects were regarded as secondary due to maternal toxicity.
Developmental effects observed:
yes
Lowest effective dose / conc.:
790 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Dose (mg/kg bw) 0# 0## 158 790 1580
Mated females 10 10 10 10 10
Pregnant females 10 10 10 8 10
Mortality of dams 0 0 0 0 9
Pregnant on C section 10 10 10 8 1
Clinical symptoms --- --- --- + ++
Body weight day 0 (g) 235.1 234.6 233.9 229.6 231.4
Body weight day 6 (g) 263.4 260.7 263.3 256.1 256.4
Body weight day 15 (g) 317.6 314.5 314.9 299 222**
Body weight day 20 (g) 391.7 391.4 392.4 366.1 265.1**
Body weight gain (days 6-15), g 54.2 53.8 51.6 42.9** **
Uterus weight (g) 82.9 82.4 83.9 66.9* 0.6**
* p< 0.05; ** p< 0.01 
Caesarian section / fetal examination:
Dose (mg/kg bw) 0# 0## 158 790 1580
Mated females 10 10 10 10 10
Pregnant on C section 10 10 10 8 1
Corpora lutea per dam 16.2 15.9 17.4 15.8 23**
Implantation sites per dam 18.8 15.1 16 14.4 19
Dams with viable fetuses 10 10 10 8 0
Live fetuses per dam 14.7 14.3 14.7 12.1 ---
Post-implantation loss (mean) 7.1 6 8.2 14.6 100**
Resorptions total (mean) 1.1 0.7 1.3 2.3 19**
Mean fetal weights, males (g) 3.9 4 3.9 3.5** ---
Mean fetal weights, females (g) 3.7 3.8 3.7 3.4** ---
No. of fetuses evaluated, live 147 143 147 97 ---
No. (%) fetus with malformations 3(2) 2(1.4) 3(2) 2(2.1) ---
No. (%) fetus with variations 68(46) 66(46) 77(52) 59(61) ---
0# control with bi-distilled water
0# control with bi-distilled water and 0.005% Cremophor EL
Executive summary:

Propylheptanol caused severest signs of maternal toxicity at 1580 mg/kg bw and was still clearly toxic to the dams at 790 mg/kg bw. No signs of maternal toxicity were noted at 158 mg/kg bw. Distinct signs of developmental toxicity were recorded at 790 and 1580 mg/kg bw, but no indications for teratogenicity occurred. The observed fetal variations were commonly noted in fetal morphology and were assessed as indications for fetal growth retardations and/or maternal stress, but not as selective effects on the fetal organism. No substance-induced prenatal developmental toxicity occurred at 158 mg/kg bw. The no observed effect level (NOAEL) on the maternal and fetal organism was 158 mg/kg bw. Since this screening study was performed in 1994 the original nomenclature and classification of fetal findings differed distinctly from the current procedures. To allow a comparison with a recent study or studies, the nomenclature and classification were revised according to the most recent recommendations of internationally accepted experts (Wise et al., 1997; Chahoud et al., 1999; Solecki et al., 2001 and in press (status: July 2003)).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Pre-natal Developmental Toxicity Study; Official Journal of the European Union, No. L 142
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
The analyses of the test item (= test substance) were carried out at Competence Center Analytics, BASF SE, 67056 Ludwigshafen, Germany.

Name of test substance: Palatinol 10-P
Test substance No.: 02/0183-6
Batch No.: F151_2017
CAS No.: 53306-54-0
Purity: 98.3 area-% (GC, DB-1)
98.8 area-% (GC, DB-5 HT)
Identity: confirmed
Homogeneity: given
Storage stability: Expiry date: 07 Dec 2018

The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.

ADDITIONAL TEST SUBSTANCE INFORMATION

Chemical name: 1,2-Benzenedicarboxylic acid, bis(2-propylheptyl) ester
Physical state / appearance:liquid / colorless, clear
Storage conditions: room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Species and strain:
Sexually mature, virgin New Zealand White rabbits (Crl:KBL(NZW)) were supplied at 16-18 weeks of age by Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France). Only animals free from clinical signs of disease were used for the investigations.

Animal identification:
The breeder produced unique identification of the rabbits by ear tattoo.

Reason for species selection:
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Housing and diet:
During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm).
For enrichment, wooden gnawing blocks were added (Typ KNH E-041, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
The animals were accommodated in fully air-conditioned rooms in which central air condi-tioning maintained a range of temperature of 19±2°C and a range of relative humidity of 45-65%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.
The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).
Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.
The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).

In-life dates:
17.01.18-08.03.2018
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test substance was administered daily as a homogeneous addition to the food with daily concentration adjustment on the basis of body weight and food consumption measurements (GD 6-29).

The test substance preparations were prepared once before the start of the study and were stored at room temperature.
The concentration of Palatinol 10-P in the diet was calculated with the following formula:

Food consumption: 70 g, 110 g or 160 g per day*
Mean body weight: 3900 g*

* = based on historical data

BW x D/FC = ppm

BW = mean body weight [g]
D = Dose [mg/kg body weight]
FC = daily food consumption [g]
ppm = dietary concentration of DPHP


For each concentration, the calculated amounts of the test substance were weighed out once before the start of the study and put in appropriate boxes at the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The boxes with the test substance were sent to Provimi Kliba SA (new name: Granovit AG), Kaiseraugst, Swizerland. Provimi Kliba SA mixed the respective amounts of test substance thoroughly with appropriate amounts of food in a “Diosna-Mischer” for 2 x 15 minutes. The mixtures of test substance and food were pelleted in a pelleting machine (size: 4.5 mm, round) using distilled water, thereafter put in a pellet dryer to reach ≤11% analytical moisture. The pellets were then stored at 9 - 16°C until shipment to BASF SE.
The pellets were shipped to BASF SE by truck.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study at the external test facility EAG Laboratories GmbH, Eiselauer Weg 4, D-89081 Ulm, Germany, under the responsibility of the Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice.

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the delivery of food and at the end of the administration period) for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the stability and the homogeneity of the samples of all concentrations. From each formulation, three samples were taken from the preparation vessel (one from the top, middle and bottom).

Results:
Stability analysis
The concentrations measured at the delivery of food and the end of administration period corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations. Therefore, the stability of the test substance preparations over the study period was demonstrated.

Homogeneity analyses of the test substance preparations
The homogeneous distribution of the test substance in the vehicle (pelleted Kliba maintenance diet rabbit and guinea pig “GLP”) was demonstrated.

Concentration control analyses of the test substance preparations
The results of the analyses of the test substance preparations in pelleted Kliba maintenance diet rabbit and guinea pig “GLP” confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
Details on mating procedure:
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

During the acclimatization period the animals were assigned to the different test groups ac-cording to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

Based on the pregnant animals the body weight on GD 0 varied between 3404 – 4261 g.
Duration of treatment / exposure:
The test substance was administered to the animals as a homogeneous addition to the food from GD 6-29. The animals of the control group were kept on basal diet.
Frequency of treatment:
ad libitum in the diet
Dose / conc.:
33.4 mg/kg bw/day (actual dose received)
Remarks:
The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was deter-mined during the administration period (GD 6-29). Nominal value: 40 mg/kg bw/day
Dose / conc.:
65.7 mg/kg bw/day (actual dose received)
Remarks:
The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was deter-mined during the administration period (GD 6-29). Nominal value: 80 mg/kg bw/day
Dose / conc.:
126.8 mg/kg bw/day (actual dose received)
Remarks:
The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was deter-mined during the administration period (GD 6-29). Nominal value 160 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
Based on results from preliminiary range-finding studies, the following target doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits:
40 mg/kg body weight/day: as low-dose level
80 mg/kg body weight/day: as mid-dose level
160 mg/kg body weight/day: as high-dose level

In order to achieve the desired dose levels in this feeding study as close as possible the following procedure was applied: three different mixtures of ground food and test substance were prepared for each dose and subsequently pelleted before the beginning of the admin-istration period. The test substance concentrations in these mixtures were based on the assumption of a mean body weight of 3.9 kg and an average food consumption of 160, 110 or 70 grams, respectively. Depending on the individual body weight and daily food consumption of each rabbit, the diet with the most appropriate concentration was applied each day.

The prepared concentrations were as follows:
•40 mg/kg body weight/day: 975 ppm (assumed daily food intake of 160 grams); 1418 ppm (assumed daily food intake of 110 grams); 2229 ppm (assumed daily food intake of 70 grams)
•80 mg/kg body weight/day: 1950/ 2836/ 4457 ppm (assumed daily food intake as specified before)
•160 mg/kg body weight/day: 3900/ 5673/ 8914 ppm (assumed daily food intake as specified before)

Applying this procedure the following effective doses were achieved:
33 mg/kg body weight/day: as low-dose level
66 mg/kg body weight/day: as mid-dose level
127 mg/kg body weight/day: as high-dose level.

These doses constitute about 80% of the target dose levels.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).

DETAILED CLINICAL OBSERVATIONS: Yes
- see cage-side observations

BODY WEIGHT: Yes
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The consumption of food was recorded daily during GD 0-29.
The intake of test substance was calculated from the amount of food consumed and ex-pressed in milligram per kilogram body weight per day.
The calculation of the group values/day was carried out according to the following formula:

ITx=FCx x C/BWy

ITx = intake of test substance on day x in mg/kg body weight/day
FCx = daily food consumption on day x in grams
C = concentration in ppm
BWy = body weight on day y in grams (last weighing before day x)


POST-MORTEM EXAMINATIONS: Yes
On GD 29, the surviving does were sacrificed in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal) and later, fetuses were removed from the uterus.

All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were deter-mined.

After the does had been sacrificed, they were necropsied and were assessed by gross pathology in randomized order to minimize bias. The uteri and the ovaries were removed.


OTHER:
Ovaries and uterine content:
The uteri and the ovaries were removed and the following data were recorded:

-Weight of the unopened uterus
-Number of corpora lutea
-Number and distribution of implantation sites classified as:
•Live fetuses
•Dead implantations:

a)Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b)Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c)Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology and organ weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
These data were used to calculate conception rate and pre- and postimplantation losses.

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations)/number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:

((number of implantations – number of live fetuses)/number of implantations) x 100
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).

Soft tissue examination of the fetuses:
After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.
The sex of the fetuses was determined by examination of the gonads in situ.
After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded.
All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination of the fetuses:
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.

Evaluation criteria for assessing the fetuses:
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.
In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:


Malformation: A permanent structural change that is likely to adversely affect survival or health.

Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
Statistics:
Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means (DUNNETT C W (1955): A multiple comparison proce¬dure for comparing several treatments with a control. JASA, Vol. 50, 1096-1121; DUNNETT C W (1964): New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482-491)

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions (SIEGEL S (1956): Non-parametric statistics for behavioral sciences. McGraw-Hill New York)

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians (NIJENHUIS A, and WILF H S (1978): Combinatorial Algorithms. Academic Press New York, 32-33; HETTMANSPERGER T P (1984): Statistical Inference based on Ranks. John Wiley & Sons New York, 132-142)
Indices:
see ovaries and uterine content
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female of test group 2 (No. 54 - 80 mg/kg bw/d) had blood in bedding on GD 12-13 and GD 16-19 (this animal had a 100% postimplantation loss, i.e. was pregnant by stain).

In total, reduced defecation was observed in ten control, sixteen low-dose, eighteen mid-dose and twenty high-dose females (0, 40, 80 and 160 mg/kg bw/d). No defecation was ob-served in one female, each, of the control, the low- and mid-dose group and in two high-dose females.

There were no further clinical findings in the other does in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 40, 80 or 160 mg/kg bw/d).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights (BW) and the average body weight change (BWC) of the high-dose rabbits (160 mg/kg bw/d) were statistically significantly reduced on GD 9-29 (BW) and on GD 6-9 (BWC). Furthermore, in test group 2 (80 mg/kg bw/d) the mean BW and average BWC were statistically significantly reduced on GD 14-21, GD 25 and GD 29 (BW) and on GD 6-9 (BWC). This is considered to be treatment-related. During the treatment period (GD 6-29) the high-dose does gained 67% less, the mid-dose does 54% less weight in comparison to the concurrent control rabbits (attaining statistical significance, respectively).
The mean BW and the average BWC of the low-dose group (40 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Corrected (net) body weight gain:
Mean carcass weight of the does of test group 3 (160 mg/kg bw/d) was statistically signifi-cantly reduced (-4%) in comparison to the control group.
Furthermore, the corrected body weight change (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was impaired in the high-dose group, attaining statistical significance (-359.9 g) in comparison to the concurrent control group ( 202.8 g).
Mean carcass weights and corrected body weight gain were not significantly different be-tween test groups 0-2 (0, 40 or 80 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in the sub-stance-treated groups was reduced during major parts of the treatment period, attaining sta-tistical significance on GD 6-19 (test group 3 - 160 mg/kg bw/d), on GD 6-11 and GD 12-14 (test group 2 - 80 mg/kg bw/d) and on GD 26-29 (test group 1 - 40 mg/kg bw/d). During the treatment period (GD 6-29), the high dose rabbits consumed 24% less, the mid-dose rabbits 15% less and the low-dose rabbits 10% less food.
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
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean gravid uterus weights of the rabbits of test groups 1-3 (40, 80 or 160 mg/kg bw/d) were not significantly different from controls. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some spontaneous findings were noted in individual females of test groups 1 and 3 (40 and 160 mg/kg bw/d). These gross findings were:
•watery feces, no feces in rectum in low-dose does Nos. 35 and 47 and in high-dose doe No. 83,
•a malpositioned kidney (right) and a short ureter (right) in high-dose doe No. 98.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):
•female No. 2 - not pregnant

Test group 1 (40 mg/kg bw/d):
•female No. 26 - not pregnant

Test group 2 (80 mg/kg bw/d):
•females Nos. 59, 64, 67 - not pregnant

Test group 3 (160 mg/kg bw/d):
•females Nos. 89, 90, 93 - not pregnant

Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 88% in the mid- and high-dose groups (80 and 160 mg/kg bw/d) and 96% in the control and low-dose groups (0 and 40 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study (according to the test guidelines).

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age (historical control data).

However, the number of late resorptions in the high-dose group (160 mg/kg bw/d) was statistically significantly higher than in the concurrent control group, caused by single does (Nos. 78 and 100) with more than one late resorption in their litters. As the overall resorption rate did not significantly differ from the concurrent control and was well within the historical control range, these findings were considered to be spontaneous in nature.

One female, each, of the mid- and high-dose groups (Nos. 54 and 96) had no viable fetuses, just early resorptions in their uteri (post-implantation loss = 100%). Furthermore, one dead fetus each was found in the litters of low-dose doe No. 47 and mid-dose doe No. 66. None of these findings is considered to be associated with the treatment.
Dose descriptor:
NOAEL
Effect level:
66 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (40, 80 and 160 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal external malformations

External malformations occurred in all test groups including the control (0, 40, 80 and 160 mg/kg bw/d), as listed in the following table. Many affected fetuses had associated soft tissue and/or skeletal malformations. Male fetus No. 60-08 had multiple external malformations, such as meningoencephalocele, misshapen upper jaw, absent nostrils, skin tag and anophthalmia, combined with soft tissue and skeletal malformations.

The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted.

Table: Individual fetal external malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 8-06 M b) short tail
8-07 M umbilical hernia
1 (40 mg/kg bw/d) 34-06 F b) spina bifida
49-01 M umbilical hernia
2 (80 mg/kg bw/d) 58-04 M a)b) short snout
60-08 M a)b) multiple external malformations
3 (160 mg/kg bw/d) 82-02 F a)b) microglossia, cleft palate
82-09 F a)b) cleft palate
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a) fetus with additional soft tissue malformation
b) fetus with additional skeletal malformation

Table: Total external malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 2 (0.9) 2 (1.0) 2 (1.2) 2 (1.1)
Litter incidence N (%) 1 (4.2) 2 (8.3) 2 (9.5) 1 (4.8)
Affected fetuses/litter Mean% 0.6 1.3 1.1 1.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent



Fetal external variations

One external variation, i.e. paw hyperflexion, was recorded in four fetuses of two high-dose litters (160 mg/kg bw/d). The incidence of this finding does not differ statistically from concurrent control, the finding itself is reversible postnatally and not considered biologically relevant. It can be found in the historical control data.

Table: Total external variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 0.0 0.0 0.0 4 (2.2)
Litter incidence N (%) 0.0 0.0 0.0 2 (9.5)
Affected fetuses/litter Mean% 0.0 0.0 0.0 1.9
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent


Fetal external unclassified observations

No external unclassified observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal skeletal malformations

Skeletal malformations were detected in single fetuses of all test groups including the control (0, 40, 80 and 160 mg/kg bw/d), as shown in the table below. The majority of affected fetuses had associated external and/or soft tissue malformations. Male mid-dose fetus No. 58-04 had multiple skeletal malformations affecting the whole fetal body (dwarfism), associated with external and visceral malformations. Male high-dose fetus No. 87-03 had multiple skeletal malformations in the area of sternum, cervical and thoracic vertebrae, ribs and scapula. All these findings were considered to be spontaneous in origin and not treatment-related.

No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.

Table: Individual fetal skeletal malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 8-06 M a) absent caudal vertebra
1 (40 mg/kg bw/d) 34-06 F a) splayed lumbar arch
46-04 M fused rib
2 (80 mg/kg bw/d) 58-04 M a)b) multiple skeletal malformations
60-08 M a)b) severely malformed skull bones
3 (160 mg/kg bw/d) 82-02 F a)b), 82-09 F a)b) severely malformed skull bones
86-03 M misshapen lumbar vertebra
87-03 M multiple skeletal malformations
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a) fetus with additional external malformation
b) fetus with additional soft tissue malformation


Table: Total skeletal malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 1 (0.5) 2 (1.0) 2 (1.2) 4 (2.2)
Litter incidence N (%) 1 (4.2) 2 (8.3) 2 (9.5) 3 (14)
Affected fetuses/litter Mean% 0.3 0.9 1.1 2.2
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent


Fetal skeletal variations

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Table: Total fetal skeletal variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 194 (91) 195 (93) 150 (87) 170 (92)
Litter incidence N (%) 24 (100) 24 (100) 21 (100) 21 (100)
Affected fetuses/litter Mean% 91.5 93.2 87.2 92.5
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside historical control ranges, were marked with X.

Table: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)
Test group 0 Test group 1 Test group 2 Test group 3 HCD
Finding 0 mg/kg 40 mg/kg 80 mg/kg 160 mg/kg Mean %
bw/d bw/d bw/d bw/d (range)
Incomplete ossification of hyoid; cartilage present 15.5 12.8 14.9 25.7* 29.9
(11.3 - 47.3)
Supernumerary lumbar vertebra 0.0 1.5 X3.8** 2.1* 0.4
(0.0 - 2.4)
Incomplete ossification of talus; cartilage present 0.3 2.7* 4.2** 2.7 2.1
(0.0 - 4.8)
mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent
* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

As can be seen from the table above., the statistically significantly increased incidences of the listed skeletal variations were either not related to the dose or they were inside the historical control range. Thus, they are not considered to be associated with treatment.


Fetal skeletal unclassified cartilage observations

Some isolated cartilage findings without impact on the respective boney structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the vertebral column, sternum and the ribs and did not show any relation to dosing.

The total of those individual findings resulted in a slightly, but statistically significantly increased affected fetuses per litter-incidence of unclassified cartilage observations in test group 2 (80 mg/kg bw/d). However, as these were individual findings and the incidence is below the historical control mean (HCD: 9.8 mean% [3.1 - 20.5]), these findings were considered to be spontaneous in origin and not treatment-related.

Table: Total unclassified cartilage observations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 11 (5.2) 18 (8.6) 18 (10) 8 (4.3)
Litter incidence N (%) 7 (29) 9 (38) 11 (52) 4 (19)
Affected fetuses/litter Mean% 4.8 8.1 9.6* 3.6
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue malformations

Soft tissue malformations occurred in all test groups including controls (0, 40, 80 or 160 mg/kg bw/d), as listed in Tab. 4.3.3.1.1. Four fetuses of the mid- and high-dose groups had additional external and skeletal malformations. No statistically significant nor toxicologically relevant differences between the groups were noted, and the overall incidences were inside the historical control range.

Table: Individual fetal soft tissue malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 6-06 F small lung, diaphragmatic hernia
19-13 M heart: muscular ventricular septum defect
1 (40 mg/kg bw/d) 43-06 F absent subclavian
2 (80 mg/kg bw/d) 58-04 M a)b) misshapen brain
60-08 M a)b) misshapen brain
61-02 M persistent truncus arteriosus, heart: muscular
ventricular septum defect
3 (160 mg/kg bw/d) 77-01 M malpositioned kidney, short ureter
82-02 F a)b), 82-09 F a)b) small cerebrum
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a) fetus with additional external malformation
b) fetus with additional skeletal malformation

Table: Total soft tissue malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 2 (0.9) 1 (0.5) 3 (1.7) 3 (1.6)
Litter incidence N (%) 2 (8.3) 1 (4.2) 3 (14) 2 (9.5)
Affected fetuses/litter Mean% 0.7 0.4 1.6 1.4
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent


Fetal soft tissue variations

The examinations of the soft tissues revealed a broad variety of soft tissue variations, i.e. dilated cerebral ventricle, malpositioned carotid branch, narrowed pulmonary trunk, dilated aortic arch, heart: enlarged ventricular chamber, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of test groups 0, 1, 2 and 3 (0, 40, 80 or 160 mg/kg bw/d. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. The findings ‘narrowed pulmonary trunk’, ‘dilated aortic arch’ and ‘heart: enlarged ventricular chamber’ occurred exclusively in one fetus of the control group, all the other findings can be found in the historical control data of the test facility at comparable incidences.

Table: Total soft tissue variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 11 (5.2) 6 (2.9) 4 (2.3) 5 (2.7)
Litter incidence N (%) 8 (33) 6 (25) 4 (19) 4 (19)
Affected fetuses/litter Mean% 5.6 3.1 2.1 2.2
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent


Fetal soft tissue unclassified observations

One unclassified soft tissue observation was recorded in ten control, twelve low-dose, two mid-dose and nine high-dose fetuses: a blood coagulum around urinary bladder. This finding is not considered to be treatment-related.

Table: Total soft tissue unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 10 (4.7) 12 (5.7) 2 (1.2) 9 (4.9)
Litter incidence N (%) 4 (17) 6 (25) 1 (4.8) 3 (14)
Affected fetuses/litter Mean% 3.8 6.8 1.0 4.5
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent
Other effects:
no effects observed
Description (incidence and severity):
The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were similar to the control value.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all substance-treated test groups (40, 80 or 160 mg/kg bw/d).

Two fetuses of the control, one fetus of the low-dose, three fetuses of the mid- dose and four fetuses of the high-dose group had more than one malformation or were multiple-malformed across the different examination areas. For a better overview, all those malformations were listed the table below

Table: Fetuses with more than on malformation
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 6-06 F small lung, diaphragmatic hernia
8-06 M short tail, absent caudal vertebra
1 (40 mg/kg bw/d) 34-06 F spina bifida, splayed lumbar arch
2 (80 mg/kg bw/d) 58-04 M short snout, misshapen brain, multiple skeletal mal-formations (dwarfism)
60-08 M multiple external malformations (meningoencephalo-cele, misshapen upper jaw, absent nostrils, skin tag and anophthalmia), misshapen brain, severely mal-formed skull bones
61-02 M persistent truncus arteriosus, heart: muscular ventricu-lar septum defect
3 (160 mg/kg bw/d) 77-01 M malpositioned kidney, short ureter
82-02 F microglossia, cleft palate, small cerebrum, severely malformed skull bones
82-09 F cleft palate, small cerebrum, severely malformed skull bones
87-03 M multiple skeletal malformations in the area of sternum, cervical and thoracic vertebrae, ribs and scapula
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Other malformations, such as umbilical hernia, absent subclavian, misshapen lumbar vertebra and fused rib were scattered observations in individual fetuses of test groups 0, 1 or 3. They were not dose-related and all of them can be found in the historical control data at comparable or higher frequency.

There was no statistically significant difference in the distribution of total malformations about the groups, and the incidences in the treated groups were either below or close to the historical control means.

No ontogenetic pattern is recognizable for the individual malformations or in the configuration of malformations in multiple-malformed fetuses, nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.

Overall, an association of all these findings to the treatment is not assumed.

Table: Total fetal malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 4 (1.9) 4 (1.9) 3 (1.7) 5 (2.7)
Litter incidence N (%) 3 (13) 4 (17) 3 (14) 4 (19)
Affected fetuses/litter Mean% 1.3 2.1 1.6 2.6
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

A spontaneous origin is assumed for the external variations, soft tissue varia-tions and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls.

If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at a comparable frequency.

Table: Total fetal variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 40 mg/kg bw/d 80 mg/kg bw/d 160 mg/kg bw/d
Litter N 24 24 21 21
Fetuses N 213 209 173 184
Fetal incidence N (%) 194 (91) 195 (93) 152 (88) 171 (93)
Litter incidence N (%) 24 (100) 24 (100) 21 (100) 21 (100)
Affected fetuses/litter Mean% 91.5 93.2 88.3 93.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

No unclassified external observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0-3. The distribution and type of these findings do not suggest any relation to treatment.

Thus, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (160 mg/kg bw/d).
Dose descriptor:
NOAEL
Effect level:
127 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: various findings without relevance
Developmental effects observed:
no
Lowest effective dose / conc.:
127 mg/kg bw/day (actual dose received)
Treatment related:
no
Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Palatinol 10-P as homogeneous inclusion in the diet to pregnant New Zealand White rab-bits from implantation to the expected day of parturition (GD 6-29) caused evidence of maternal toxicity at the high-dose level, such as reduced food consumption, carcass weight and corrected (net) body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is effectively 66 mg/kg bw/d (target dose 80 mg/kg bw/d).

Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 127 mg/kg bw/d (target dose 160 mg/kg bw/d).
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification in chapter 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Species:
rabbit
Dose descriptor:
NOAEL
Effect level:
66 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
127 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Species:
rat
Quality of whole database:
Two prenatal developmental toxicity study on rats with the test item according to OECD/EU guideline and in compliance with GLP regulations are available. The studies are considered reliable without restrictions.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity of 2-Propylhexan-1-ol (99.8%) was examined in a reliable GLP-study (according to OECD 414), where 25 Wistar rats (per dose) were administered an aqueous emulsion of 50, 200 and 600 mg/kg bw 2-Propylheptan-1-ol orally via gavage. The administration was daily from day 6 through day 19 post coitum (BASF, 2004). On gestation day 20, the dams were sacrificed and ovaries and uterine content as well as fetuses were examined.

At 600 mg/kg bw signs of maternal toxicity like clinically salivation and discomfort, reduced food consumption, reduced absolute and net body weight gain and increased water consumption were recorded. Clinical pathology revealed slight changes such as mild thrombocytopenia and marginal changes in electrolyte levels in the peripheral blood of the high dose dams. Organ weight determination revealed an increased absolute and relative liver weight. Also, at mid dose level (200 mg/kg bw) were still some signs of maternal toxicity such as salivation, reduced absolute and net maternal body weight gain as well as a slightly increased relative liver weight recorded. The increase in the absolute and relative liver weight (200 and 600 mg/kg bw) is likely to be associated to a peroxisome proliferation and is therefore not relevant for humans. The oral administration of 2-Propylheptan-1-ol to dams at all 3 dose levels had no influence on gestational parameters. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or values calculated for pre- and postimplantation losses were unaffected by the treatment. There was a statistically significant reduction of mean fetal body weight at the high dose level (600 mg/kg bw) of about 11% below the corresponding control value. This finding is assessed to be secondary to the distinct maternal toxicity that has been observed in the appropriate dams. The mean fetal body weights in low and mid dose (50 and 200 mg/kg bw) were not influenced by the test substance. No toxicologically relevant influence of the test substance on mean placental weights was noted. The external, soft tissue and/or skeletal examinations of the fetuses revealed no malformations which might be related to the test substance and no specific malformation pattern was found. A slight increase of the overall incidence of skeletal variations was noted at high dose level (600 mg/kg bw), but also these findings are considered as secondary to maternal toxicity and not relevant in terms of developmental toxicity.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg body weight/day. The developmental NOAEL is 200 mg/kg body weight/day. No indications for a teratogenic potential of the test compound were noted.

There are data from a further developmental toxicity study in rats according to OECD 414 available. This study was conducted as a screening test with a limited number of animals. 8 – 10 Wistar rats (per dose) were administered 158, 790 and 1580 mg/kg bw 2-Propylheptan-1-ol orally via gavage. The administration was daily from day 6 through day 15 post coitum (BASF, 1995). On gestation day 20, the dams were sacrificed and ovaries and uterine content as well as fetuses were examined.

Based on the results where impairments in food consumption and body weight gain, salivation after the daily treatment and reduced gravid uterus weights were reported at 790 mg/kg bw/day, the NOAEL for maternal toxicity is 158 mg/kg body weight/day. Additionally, increased liver weight was reported at 790 mg/kg/bw/day. This, however, was due to peroxisomal proliferation and is therefore not relevant for humans. Also reduced fetal body weights and increased rates of various skeletal and total variations were seen at 790 mg/kg bw. According to this results the NOAEL for embryo- and fetotoxicity and for teratogenicity was determined at 158 mg/kg bw, but the observed fetal variations were commonly noted in fetal morphology and were assessed as indications for fetal growth retardations and/or maternal stress, but not as selective effects on the fetal organism. No indications for teratogenicity occurred.

There is no developmental toxicity / teratogenicity study in a second species available for 2 -propylheptan-1 -ol, but a read-across can be made from Di-(2-propylheptyl) phthalate (CAS 53306 -54 -0):

Di-(2-propylheptyl) phthalate was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD guideline 414. The test substance was administered as a homogeneous addition to the food to groups of 25 inseminated female New Zealand White rabbits in target doses of 40, 80 and 160 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 29. The effective doses (= test substance intake) were 33, 66 and 127 mg/kg bw/d. The control group, consisting of 25 females, was kept on basal diet only. At terminal sacrifice on GD 29, 22-24 females per group had implantation sites. Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings. The following test substance-related adverse effects/findings were noted: In test group 3 (effective dose 127 mg/kg bw/d, target dose 160 mg/kg bw/d) the dams showed reduced mean food consumption (24% below control)during the treatment period (GD 6-29) and a reduced mean carcass weight (-4%) and corrected (net) body weight gain (-359.9 g vs.202.8 g in control). No adverse findings were detected in dams of test group 2 and 1 (effective dose 66 and 33 mg/kg bw/d). No test substance-related adverse effects were observed in the fetuses in any of the test groups. Under the conditions of this prenatal developmental toxicity study, the oral administration of di-(2-propylheptyl) phthalate as homogeneous inclusion in the diet to pregnant New Zealand White rabbits from implantation to the expected day of parturition (GD 6-29) caused evidence of maternal toxicity at the high-dose level, such as reduced food consumption, carcass weight and corrected (net) body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is effectively 66 mg/kg bw/d (target dose80 mg/kg bw/d). Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 127 mg/kg bw/d (target dose 160 mg/kg bw/d).

Toxicity to reproduction: other studies

Additional information

In a reliable 90 day subchronic GLP study with Fischer 344 rats, according to OECD guideline 408, groups of 10 male and 10 female animals received dose levels of 30, 150 or 600 mg/kg bw 2-Propylheptan-1-ol (99.5%) per gavage for three months (BASF, 1996). This study was no specific fertility study but treatment was long enough to cover sperm maturation (no mating performed). After a treatment period of 3 months covering the time of sperm maturation there was no effect on the reproductive organs examined by weight determinations and histopathological examinations. For substance-related effects not considering reproduction see 7.5.1 Repeated dose toxicity: oral.

Justification for classification or non-classification

The available data concerning reproduction and developmental toxicity/teratogenicity are reliable and suitable for classification purposes under Regulation 1272/2008.

Though slight signs of a retardation of prenatal development (lower fetal weights, delay of ossification) were observed, they were considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity. As a result, the substance is not considered to be classified for reproductive toxicity under Regulation (EC) 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Additional information