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REACH

Bis(2-propylheptyl) phthalate

EC number: 258-469-4 | CAS number: 53306-54-0

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Di-(2-propylheptyl) phthalate neither influenced fertility nor reproductive performance in the parental animals in a two generation OECD guideline study. 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.

Link to relevant study records

Reference

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
: 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
: 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

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 test item 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

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 mode of action has been extensively studied with Perchlorate and Phenobarbital for example (Meek et al., 2003 Critical Reviews in Toxicology, 33(6):591–653; Lewandowski et al., 2004 Regulatory Toxicology and Pharmacology 39, 348–362; McClain et al., 1989 Toxicology And Applied Pharmacology 99,216 -228; Fisher et al., 2013 Joural of Environmental Science and Health, Part C, 30:81 -105) and is therefore well understood. It is not relevant to humans as rodents are more sensitive to thyroid effects (summarised in Meek et al., 2003Critical Reviews in Toxicology). Accordingly Bhat et al. (2014), Regulatory Toxicology and Pharmacology 70, 65 -74, who published the comprehensive oral risk assessment undertaken by the US EPA chaired Health Advisory board of NSF International, 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 the German MAK Commission (2015). Furthermore, in humans, activation of peroxisome proliferator-activated receptor α(PPARα) does not lead to increased relative liver weights, oxidative enzyme induction or other responses typically associated with sustained PPARα activation observed in wild-type mice (Corton et al., 2018 Arch Toxicol. 92(1): 83–119). The weight of evidence supports the conclusion that adverse effects related to a PPARα MOA is either “not relevant” or “unlikely to be relevant” in humans (Felter et al., 2018 Regulatory Toxicology and Pharmacology 92, 1 -7).

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.

Supporting evidence for the lack of estrogenic activity for DPHP is coming from Zacharewski et al. (1998) where eight phthalate esters (esterified with C4 – C10 alcohols) were tested in vitro and in vivo and all of them were clearly negative, i.e. absence of estrogenic activity in vivo was demonstrated (Zacharewski et al. (1998), Toxicological Sciences 46, 282-293, see IUCLID section 7.8.3).

As shown by a US EPA lab, there was also no effect on fetal testosterone production, a key parameter in the adverse outcome pathway analysis for phthalates showing toxicity to reproduction, i.e. DPHP did not show any indication for anti-androgenic activity as there was no effect on fetal testicular testosterone production (Furr et al. (2014), Toxicological Sciences 140, 403-424, see IUCLID section 7.8.3).

In summary, available relevant testing data on DPHP do not give any indication for adverse effects on fertility or an endocrine disrupting activity.

Effects on developmental toxicity

Description of key information

OECD 414, rat: NOAEL maternal, embryo toxicity: 200mg/kg bw/d, teratogenicity: 1000mg/kg
similar OECD 414, rat: NOAEL: 1000mg/kg bw/d

OECD 414, rabbit: NOAEL maternal, embryo toxicity: 66 mg/kg bw/d, teratogenicity 127 mg/kg bw/d (highest dose tested)

Link to relevant study records

Referenceopen all close all

Reference 1

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)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Wistar
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Strain: Wistar (CrIGIxBrlHan:WI)
- Age at study initiation: 70 - 84 at delivery
- Weight at study initiation: 146.3 - 188.6 g
- Housing: single, DK III stainless steel wire mesh cages (Becker, Castrop-Rauxel, Germany)
- Diet (e.g. ad libitum): ground Kliba laboratory diet rat/mouse/hamster, Provimi Kliba SA, Kaiseraugts, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: between the supply on day 0 and the first administration on gestational day 6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:: oral: gavage
Vehicle:: olive oil
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in olive 011 Ph.Eur./DAB 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 solutions, an appropriate amount of the test substance was weighed in a graduated beakers (depending on the dose group), topped up with olive oil Ph.Eur./DAB and subsequently thoroughly mixed using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.8, 4, 20%
- Amount of vehicle (if gavage): 5 ml/kg bw
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 olive oil for a period of at least 7 days at room temperature were carried out by HPLC or GC.
Details on mating procedure:: Mating procedure: the animals were mated by the breeder (time-mated) and supplied on day 0 post coitum
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:: day 6 through day 19 of gestation
Frequency of treatment:: daily
Duration of test:: 20 days
Dose / conc.:: 40 mg/kg bw/day (nominal)
Dose / conc.:: 200 mg/kg bw/day (nominal)
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 25
Control animals:: yes, concurrent vehicle
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).
- Cage side observations checked in table were included.

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 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: all animais 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 caiculated from these results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: uterus, ovaries
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: dead fetuses
Fetal examinations:: - External examinations: Yes:
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:: Statistical analysis: Dunnett's test (food consumption, body weight, body weight change, corrected body weight gain, weight of uterus (before opening), weights of fetuses, placentae, corpora lutea, implantations, pre- and post implantation losses, resorptions and live fetuses). KRUSKAL-WALLIS-test (weights of liver, kidneys, spleen). Fisher's exact test (conception rate, mortality of the dams, number of litter with fetal findings). Wilcoxon test (proportion of fetuses with malformations, variations and/or unclassified observation in each litter).
Indices:: - The conception rate (in %) was calculated according to the following formula: number of pregnant animals/number of fertilized animals x 100
- The preimpIantation loss (in %) was calculated according to the following formula: (number of corpora lutea - number of implantations) / number of corpora lutea x 100
- The postimplantation loss (in %) was calculated from the following formula: (number of implantations - number of live fetuses) / number of implantations x 100
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: In total 5 high dose dams showed urine-smeared fur occasionally, predominantly on the last days of the treatment period (days 16 - 20 p.c.).
The clinical finding "urine smeared fur" is a sign of discomfort of the rats and is probably substance-induced. There were no abnormal clinical findings in the other dams of this study.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/d: The mean body weights of the high dose rats were statistically significantly below control values between days 19 - 20 p.c.. On day 20 p.c., the mean body weight of these rats was about 6% below the mean value of the concurrent control females. There was a statistically significant body weight less at 1,000 mg/kg bw/d at initiation of treatment (days 6 - 8 p.c.). This is in-line with the reductions in food intake of these dams during this study phase. Thereafter, weight gains of the high dose dams were generally slightly below corresponding control values without attaining statistical significance. If calculated for the entire treatment period (days 6 - 19 p.c.), however, weight gain of the high dose dams was statistically significantly impaired and about 24% below the respective contral value. A reduction of about 18% occurred at 1,000 mg/kg bw/d, if weight gain was calculated for the total study phase (day 0 - 20 p.c.).
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) was distinctly and statistically significantly lower at 1,000 mg/kg bw/d (about 30% below the concurrent control
value).
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/d: mean food consumption was statistically significantly reduced (up to about 32% below the concurrent control value) on treatment days 6 - 13 p.c.. On the following days of the treatment period, food consumption reached or even exceeded control values. If calculated for the entire treatment phase (days 6 - 19 p.c.), food uptake of the 1,000 mg/kg bw/d dams was still about 11 % below the comparable control value. The transient reductions in food consumption at 1,000 mg/kg bw/d were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing. Therefore, this finding is considered to be substance-induced.
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:: no effects observed
Description (incidence and severity):: UTERUS WEIGHT
1000 mg/kg bw/d: the mean gravid uterus weight of the high dose females, was about 19% below the control value, but the difference did not attain statistical significance due to the high standard deviation. The 3 dams of this group which had no live fetuses at all, but only early resorptions had very low uterus weights, whereas the uterus weights of the other dams of this group remained unaffected.
Gross pathological findings:: no effects observed
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: not examined
Histopathological findings: neoplastic:: not examined
Other effects:: no effects observed
Number of abortions:: not specified
Pre- and post-implantation loss:: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/day: the postimplantation lass value in the top dose was statistically significantly increased (23.1%) and distinctly outside the historical control range (mean value: 6.6%; range: 3.7 - 11.3%). The increased embryo-/fetolethality at 1,000 mg/kg was not equally distributed throughout the dams of this dose group, but was primarily induced by 3 high dose dams which had no live fetuses at all, but only very early resorptions. Thus, the 3 affected dams were only pregnant by stain and had a postimplantation lass of 100% each, whereas the postimplantation loss values of the other 1,000 mg/kg dams were generally similar to control values.
Total litter losses by resorption:: not specified
Early or late resorptions:: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/d: a statistically significant increase in resorption rate (especially early ones).
Dead fetuses:: not specified
Changes in pregnancy duration:: not specified
Description (incidence and severity):: Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:: not specified
Other effects:: no effects observed
Dose descriptor:: NOAEL
Effect level:: 200 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Fetal body weight changes:: no effects observed
Description (incidence and severity):: Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:: not specified
Changes in sex ratio:: no effects observed
Changes in litter size and weights:: not specified
Changes in postnatal survival:: not specified
External malformations:: no effects observed
Description (incidence and severity):: No external malformations or variations were seen in any of the fetuses of test groups, except two unclassified variations which were regarded as isolated and considered to be spontaneous in nature.
Skeletal malformations:: no effects observed
Description (incidence and severity):: Malformations of the skeletons were observed at low incidences in fetuses of the test groups 40 and 1,000 mg/kg bw/d. In total, none out of 115 control fetuses (from 24 litters), 2 out of 105 low dose fetuses (1.9%) in 2 out of 23 litters (8.7%), none out of 114 mid dose fetuses (from 25 litters) and 2 out of 77 high dose fetuses (2.6%) in one of 17 litters (5.9%) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 1.6, 0.0, and 2.4%. The noted skeletal malformations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data
In all groups, signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The mean percentages of affected fetuses/litter with skeletal variations amounted to 96.0, 95.0, 97.4, and 100% at 0; 40; 200 or 1,000 mg/kg bw/d.
However, two skeletal variations, occurred at statistically significantly rates in the high fetuses (1,000 mg/kg bw/d). The increased occurrence of unossified sternebra and supernumerary rib (without cartilage) were clearly above the upper historical-control values. However, the overall rate of skeletal variations at this dose level was not statistically significantly elevated above the concurrent control value (affected fetuses/litter: 96.0, 95.0, 97.4 and 100.0% in all groups).
Also, unclassified cartliage observations, occurred in all groups including the controls. The mean percentages of affected fetuses/litter with these findings amounted to 38.6, 34.6, 47.2, and 63.5%** at 0; 40; 200 or 1,000 mg/kg bw/d (** = p< 0.01). A toxicological relevance for these findings could be excluded.
Visceral malformations:: no effects observed
Description (incidence and severity):: No soft tissue malformations were recorded in any of the fetuses of all groups.
However, soft tissue variations were detected in each group including the control. Uni- or bilateral dilations of the renal pelvis occasionally in association with uni- or bilaterally dilated ureter(s) were found in fetuses of all groups. The mean percentages of affected fetuses/litter with total soft tissue variations were within the historical control range (4.0 - 22.2%) in test groups (8.5% (control), 9.1% (40 mg/kg bw/d) and 14.8% (200 mg/kg bw/d)), but were above the upper historical control value at 1,000 mg/kg bw/d (26.7%; p< 0.05).

No so-called unclassified soft tissue observation (like blood inhibition of kidneys) was recorded in any of the fetuses.
Other effects:: no effects observed
Details on embryotoxic / teratogenic effects:: Details on embryotoxic / teratogenic effects:
WEIGHT
The mean fetal body weights in test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.

SEX DISTRIBUTION
The sex distribution of the fetuses in the test groups 1 - 3 was comparable with that of the control fetuses. The differences observed in comparison to the control were without any biological relevance.

EXTERNAL EXAMINATION
No external malformations or variations were seen in any of the fetuses of test groups, except two unclassified variations which were regarded as isolated and considered to be spontaneous in nature.

SOFT TISSUE EXAMINATION
No soft tissue malformations were recorded in any of the fetuses of all groups.
However, soft tissue variations were detected in each group including the control. Uni- or bilateral dilations of the renal pelvis occasionally in association with uni- or bilaterally dilated ureter(s) were found in fetuses of all groups. The mean percentages of affected fetuses/litter with total soft tissue variations were within the historical control range (4.0 - 22.2%) in test groups (8.5% (control), 9.1% (40 mg/kg bw/d) and 14.8% (200 mg/kg bw/d)), but were above the upper historical control value at 1,000 mg/kg bw/d (26.7%; p< 0.05).
No so-called unclassified soft tissue observation (like blood inhibition of kidneys) was recorded in any of the fetuses.

SKELETAL EXAMINATION
Malformations of the skeletons were observed at low incidences in fetuses of the test groups 40 and 1,000 mg/kg bw/d. In total, none out of 115 control fetuses (from 24 litters), 2 out of 105 low dose fetuses (1.9%) in 2 out of 23 litters (8.7%), none out of 114 mid dose fetuses (from 25 litters) and 2 out of 77 high dose fetuses (2.6%) in one of 17 litters (5.9%) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 1.6, 0.0, and 2.4%. The noted skeletal malformations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data
In all groups, signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The mean percentages of affected fetuses/litter with skeletal variations amounted to 96.0, 95.0, 97.4, and 100% at 0; 40; 200 or 1,000 mg/kg bw/d.
However, two skeletal variations, occurred at statistically significantly rates in the high fetuses (1,000 mg/kg bw/d). The increased occurrence of unossified sternebra and supernumerary rib (without cartilage) were clearly above the upper historical-control values. However, the overall rate of skeletal variations at this dose level was not statistically significantly elevated above the concurrent control value (affected fetuses/litter: 96.0, 95.0, 97.4 and 100.0% in all groups).
Also, unclassified cartliage observations, occurred in all groups including the controls. The mean percentages of affected fetuses/litter with these findings amounted to 38.6, 34.6, 47.2, and 63.5%** at 0; 40; 200 or 1,000 mg/kg bw/d (** = p< 0.01). A toxicological relevance tor these findings could be excluded.
Dose descriptor:: NOAEL
Effect level:: 200 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: embryotoxicity
Dose descriptor:: NOAEL
Effect level:: 200 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: fetotoxicity
Dose descriptor:: NOAEL
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: teratogenicity
Abnormalities:: not specified
Developmental effects observed:: not specified

Reference 2

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 18-22°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 (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 administration via the diet was chosen because similar materials are known to cause stomach ulcerations related with dry and hard feed, cease of food intake, abortions and lethality after bolus gavage in rabbits. The local irritation of gastric mucosa and all subsequent effects are circumvented by the administration via diet.

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 (new name: Granovit AG), Kaiseraugst, Swizerland. Provimi Kliba 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 first concentration control analysis at the delivery of food were also used to verify the homogeneity of the samples of all concentrations. The concentrations measured at the delivery of food and the end of administration period provided also information on the stability of the substance preparation, as the same preparation was used throughout the entire study period. 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:: 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

The high dose was selected based on signs of toxicity noted at a dose level of 300 and 1000 mg/kg bw/d in a previously conducted test study and in a maternal toxicity range-finding study which preceded this definitive prenatal developmental toxicity study.

In the test study 5 non-pregnant female NZW rabbits per group were diet exposed to the test compound over 3 weeks, at target dose levels of 100, 300 and 1000 mg/kg bw/d. The 1000 mg/kg bw/d dose cause an immediated disruption of food consumption in all females (reduction by 96%), during the first treatment week the animals consumed about 20 g food per day or less. Afterwards a partial recovery was noted, at the end of the test the animals reached about 50% of their normal food consumption. At 300 mg/kg bw/d a significant decrease of food consumption (by 30-40%) was noted during the first week of treatment, followed by a gradual recovery until the end of the test. The 100 mg/kg bw/d dose was comparable to the control. Similar effects were noted for body weights/body weight gain. At 1000 mg/kg bw/d the rabbit showed a significant weight loss, which did not recover. At 300 mg/kg bw/d the rabbit gained less weight than the control. No effect was noted for 100 mg/kg bw/d. Clinical chemistry revealed some mild signs of metabolic disorder (decreased alanine aminotransferase and alkaline phosphatase; increased creatinine, cholesterol, triglycerides, bilirubin) at 1000 mg/kg bw/d.
In the maternal toxicity range‐finding study, 5 presumed pregnant NZW rabbits were administered the test substance via diet from gestational day (GD) 6 through GD 28, at doses of 75, 150 and 300 mg/kg bw/d.
The 300 mg/kg bw/d dose caused in the pregnant rabbits a significant reduction of food consumption in all females, in mid-pregnancy about 70-85% below control. Towards the end of the treatment recovery was noted. At 150 mg/kg bw/d still a decrease of food consumption was noted, in mid-pregnancy about 40-70% below control, followed by a gradual recovery until the end of treatment. Food consumption at the 75 mg/kg bw/d dose was slightly below control, but not very distinct. Similar effects were noted for body weights/body weight gain. At 300 mg/kg bw/d the rabbits showed a weight loss, which did not recover. At 150 mg/kg bw/d the rabbit gained less weight than the control but recovered towards the end of treatment. No effect was noted for 75 mg/kg bw/d. Clinical pathology, organ weights and macroscopic pathology revealed no relevant findings.

The selected high dose for the definitive study (160 mg/kg bw/d) represented a dose which caused a moderate reduction of food consumption in pregnant female rabbits. Considering the specific characteristic of rabbit gastrointestinal physiology and the consequences of their disruption for the maintenance of pregnancy in rabbits this procedure meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 86/609/EEC on animal protection.

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. Individual animals with no or very low food consumption over several consecutive days were permanently kept on the lowest test item concentration of the respective dose group, to ensure the maximum possible food intake of these animals.

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 - 30.5]), 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).

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 200 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Two prenatal developmental toxicity study 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

Developmental toxicity of di-(2-propylheptyl) phthalate was analyzed in a Prenatal Developmental Toxicity Study performed under GLP according to guidelines OECD 414 and U.S. EPA Health Effects Test Guidelines OPPTS 870.3700 (BASF, 2003). Groups of 25 mated female Wistar rats received doses of 0, 40, 200 and 1000 mg/kg bw/d in olive oil by stomach tube on day 6 through day 19 post coitum (p.c.). No signs of substance-induced maternal toxicity occurred at the low and the mid dose level. However, maternal toxicity was substantiated by clinical signs of discomfort of 5 rats and statistically significant reductions in food consumption, impairments in body weight and body weight gain and reductions in corrected body weight gain at 1000 mg/kg bw. Furthermore, the mean gravid uterus weight was distinctly affected.

Concerning the gestational parameters there was a high rate of resorptions at the top dose, which led to an elevated postimplantation loss value. These differences are due to 3 dams with very early resorptions of all embryos. Data for the remaining animals did not differ from controls. The body weight loss of the high dose rats at initiation of treatment with reductions in food consumption mirrors this direct adverse effect on the dams. It can therefore be neglected at that gestational stage.

There occurred no substance-induced effects on the gestational parameters at 40 or 200 mg/kg body weight/day. The mean placental and fetal body weights were not affected by treatment, but were similar to or even identical with concurrent control values. Marginal effects on fetal morphology, but no clear indications of substance induced teratogenicity occurred exclusively at the high dose level. The rates for soft tissue, skeletal and total variations were slightly, but statistically significantly increased. Based on these effects, the NOAEL for maternal and embryo toxicity was 200 mg/kg, while the NOAEL for teratogenicity was 1000mg/kg in this study.

In another unpublished report (BASF 1995) a prenatal screening study was performed under GLP according to guidelines OECD 414, EU Method B.31 and US EPA OTS 798.4900. Groups of 9 - 10 pregnant female Wistar rats were administered with di-(2-propylheptyl) phthalate at doses of 40, 200 and 1000 mg/kg bw/d in olive oil on day 6 through day 15 post coitum. As results, no signs of maternal toxicity and no signs of developmental toxicity up to and including a dose of 1,000 mg/kg bw/d were found. Also, there were no indications of teratogenic effects which could be causally related to the administration of di-(2-propylheptyl) phthalate. Thus, the no observed adverse effect level (NOAEL) for the maternal and the fetal organism is 1,000 mg/kg body weight/day for this prenatal toxicity screening study in Wistar rats.

Furthermore, 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:

Test group 3 (effective dose 127 mg/kg bw/d, target dose 160 mg/kg bw/d): Dams:

·Reduced mean food consumption (24% below control)during the treatment period (GD 6-29)

·Reduced mean carcass weight (-4%) and corrected (net) body weight gain (-359.9 g vs. ‑202.8 g in control)

Fetuses

·No test substance-related adverse effects

Test group 2 and 1 (effective dose 66 and 33 mg/kg bw/d):

·No test substance-related adverse effects

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 testsubstance 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).

Justification for classification or non-classification

Due to the results obtained in the Two-Generation Reproduction Toxicity Study and in the Prenatal Developmental Toxicity Studies, all performed according to OECD guidelines, no classification according to GHS criteria is necessary.

Additional information

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		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%**

		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%**

Dose (mg/kg bw/d)	0	40	200	1000
Mated females	25	25	25	25
Pregnant females	24	23	25	20
Mortality of dams	0	0	0	0
Pregnant on C-section	24	23	25	20
Clinical symptoms:	-	-	-	+
Food consumption day 6-19 p.c. g	16.9	17.2	17.6	15.1
Mean body weight day 0 (g)	169.2	170.7	173.2	171.9
Mean body weight day 6 (g)	196.9	197.1	201.7	198.3
Mean body weight day19 (g)	265.2	264.4	271.1	250.0*
Mean body weight gain (days 6-19; (g))	68.4	67.4	69.4	51.7**
Uterus weight (g)	49.4	47	48.7	40.2

Dose (mg/kg bw/d)	0	40	200	1000
Corpora lutea (mean)	10.1	9.8	10.5	10.6
Implantation sites (mean)	9.6	9	9.4	9.4
Post-implantation loss (mean %)	6.2	4.2	6.4	23.1**
Dead fetuses/litter (mean %)	0	0	0	0
Resorption sites/litter (mean)	0.5	0.4	0.6	2.2**
Mean No. of live fetuses/litter	9	8.6	8.7	8.6
Mean fetal weights males (g)	3.6	3.6	3.7	3.6
Mean fetal weights females (g)	3.4	3.5	3.5	3.4
% of fetuses with external malformations	0	0.5	0	0
% of fetuses with visceral malformations	0	0	0	0
% of fetuses with skeletal malformations	0	1.9	0	2.6
% of fetuses with external variations	0	0	0	0
% of fetuses with visceral variations	8.8	8.7	13	23
% of fetuses with skeletal variations	96	96	97	100

Registration Dossier

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Diss Factsheets

Bis(2-propylheptyl) phthalate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

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