Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, in accordance with generally accepted scientific standards and described in sufficient details.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
publication
Title:
Determination of the Di-(2-Ethylhexyl) Phthalate NOAEL for Reproductive Development in the Rat: Importance of the Retention of Extra Animals to Adulthood
Author:
Blystone CR, Kissling GE, Bishop JB, Chapin RE, Wolfe GW and Foster PMD
Year:
2010
Bibliographic source:
Tox Sci, 116(2), 640–646

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
3-generation study with satellite groups, only 17 rats/sex/group
Principles of method if other than guideline:
Three-generation study with satellite groups
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylhexylphthalate (CAS No.117-81-7)
- Physical state: liquid
- Analytical purity: 99.8%
- Lot/batch No.: no data
- Stability under test conditions: An 8-day simulated dose study indicated no significant loss of DEHP from approximately 4.12 μg/g DEHP on NIH-07 feed when stored for 8 days at approximately 25°C, 50% relative humidity, with a 12-h light cycle.
- Storage condition of test material: stored at 5°C or lower in sealed containers and protected from light.
- Source: purchased from Aldrich Chemical Company (Milwaukee, WI) and provided by the NTP through Midwest Research Institute(MRI) in Kansas City, Missouri.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Age at study initiation: (F0) 5 weeks old
- Weight at study initiation: (F0) Males: 166-218 g; Females: 124-190 g
- Fasting period before study: no
- Housing: Animals were housed two per cage by sex from receipt to randomization, two per cage (one male and one female) during Task 1, F0, F1, and F2 Cohabitations, one to four per cage during the F1c, F2c, and F3c, holding and rearing phase, and housed individually at all other times. All animals were housed in polycarbonate cages (19"L x 102"W x 8"H) suspended on stainless-steel racks with an Edstrom automatic watering system providing filtered tap water. Racks were equipped with filter paper liners. Polycarbonate caging contained Sani Chip heat treated hardwood laboratory bedding.
- Diet (e.g. ad libitum): Harlan TekladTM NIH-07 Small Animal Feed was available ad libitum in a glass jar with a stainless steel follower (follower was removed from female feeders during lactation periods) and lid, and was used within five months of the milling date (with a few exceptions).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 (68-74 °F)
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF FEED:
For the dose-range finding phase of this study, 20 kg of NIH-07 feed (vehicle) was weighed into aluminum cans for the control group. For all other phases of this study, 0.3 grams was subtracted from the 20 kg of vehicle because the background level of DEHP in the vehicle was determined to be 1.5 ppm. For each phase of this study, it was stored at room temperature and protected from light. Formulations of 0, 5000, and 10,000 ppm were prepared for the dose- range finding study at least every twenty-three days for no more than 35 days of use. For the main study, formulations of 1.5(vehicle only), 10, 30, 100, 300, 1000, and 7500 ppm were prepared weekly for no more than 35 days of use. For each dose level, the appropriate amount of vehicle was weighed into aluminum cans. The proper amount of DEHP was weighed into a glass beaker. Enough 95% ethanol was added to allow DEHP to go into solution. A premix feed was prepared from the previously weighed feed in the aluminum cans. Feed flour was prepared by mixing a portion of the premix feed through a USS 100 mesh stainless steel sieve. A portion of the feed flour was added to a glass beaker and the DEHP solution was poured onto the feed. The beaker was rinsed three times with a small amount of ethanol and poured onto the feed flour. The feed and solution were mixed together with a stainless steel spatula and allowed to air dry in a chemical fume hood until sufficiently dry while being stirred occasionally. Additional feed was added to the beaker, in increasing amounts, while the DEHP and feed was mixed continuously with a stainless steel spatula until no feed/chemical agglomerates remained. A 10-kg portion of the pre-weighed feed was evenly layered into the Patterson-Kelly Blender. Equal amounts of the premix feed was poured into the right and left blender ports and spread evenly.

The premix beaker was rinsed three times with vehicle from the pre-weighed feed. Each rinse was poured evenly into the blender. The remaining pre-weighed feed was evenly poured into the blender. The blender was sealed and the mix was blended for approximately ten minutes with the intensifier bar on, then twenty minutes without the bar, and then another ten minutes with the bar on, for a total of approximately forty minutes. The formulations were stored in sealed aluminum cans and were stable for 35 days at room temperature and protected from light. Contact with plastics was avoided.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): NIH-07 feed
- Storage temperature of food: at room temperature and protected from light

VEHICLE
- Justification for use and choice of vehicle (if other than water): feeding study
- Concentration in vehicle: 1.5(diet only), 10, 30, 100, 300, 1000, and 7500 ppm
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 9 weeks.
The first two litters produced (F1a and F1b) during the cohabitation period had the total number of pups, number of live and dead pups, the number of male and female pups, the total body weight of male and female pups, individual male and female pup weights and AGD measured on PND 1, along with dam and sire weights. All pups were euthanized by sodium pentobarbital overdose on PND 1 and discarded without necropsy. The third litter born (F1c) was reared by the dam until weaning on PND 21.
- Proof of pregnancy: not applicable
- Further matings after two unsuccessful attempts: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two 150-g samples were collected from each batch of each dose level of every formulation upon completion of mixing and stored in heat seal able foil bags and protected from light at approximately -20 to -15°C. At the completion of the first mix, homogeneity samples (150 g each) were collected in duplicate from the right, left, and bottom ports of the Patterson-Kelly blender for the low, mid, and high dose groups. These samples were stored in heat seal able foil bags, protected from light at -20 to -15°C. Samples selected for dose concentration analysis were forwarded on ice packs to MRI.
Archival samples which were not selected for analysis were retained for at least 90 days following preparation then discarded as hazardous waste.
Samples were analyzed by high performance liquid chromatography by MRI. An aliquot of each level of dose formulation and of the control vehicle was analyzed. All dose formulations analyzed were within 90.0-128.3% of the nominal concentration.
Duration of treatment / exposure:
Premating exposure period (males): 81 +/- 10 d
Premating exposure period (females): 81 +/- 10 d
Duration of test: 3 generations in F3 (F3c)
Frequency of treatment:
no data
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 ppm (nominal)
Remarks:
Doses / Concentrations:
1.5 (2 groups), 10, 30, 100, 300, 1000, 7500, 10000 (2 groups) ppm
Basis:
nominal in diet
Dose / conc.:
0.12 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
0.12, 0.78, 2.4, 7.9, 23, 77, 592, and 775 mg/kg/day
Basis:
actual ingested
F0 animals
Dose / conc.:
0.09 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
0.09, 0.48, 1.4, 4.9, 14, 48, 391, and 543 mg/kg/day
Basis:
actual ingested
F1 animals
Dose / conc.:
0.1 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
0.1, 0.47, 1.4, 4.8, 14, 46, 359 mg/kg/day
Basis:
actual ingested
F2 animals
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
30 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
7 500 ppm (nominal)
Dose / conc.:
10 000 ppm (nominal)
Remarks:
2 groups
No. of animals per sex per dose:
(F0), (F1c) and (F2c): 17 males and 17 females per dose were selected for the mating trials. The other animals of each litter were submitted to different examinations.
Control animals:
other: 1.5 ppm DEHP in diet (note that no DEHP was added to the control diet. Approximately 1.5 ppm DEHP was found in the control feed; as a result the control group was indicated as being the 1.5 ppm group)
Details on study design:
- Dose selection rationale:
Sprague-Dawley rats (13 weeks old; 8/sex/group) were given feed containing 0, 5000, or 10,000 ppm DEHP from 7 days prior to mating through the cohabitation period, which extended until necropsy. [The time period of cohabitation was not specified.] DEHP intake was estimated by the study authors at 0, 321.42, and 643.95 mg/kg bw/day. In litters born during the cohabitation period, anogenital distance was measured on PND 1, and growth and mortality were monitored through PND 21. Signs of systemic toxicity included decreased feed and water intake during lactation in females from the 5000 and 10,000 ppm groups and a decrease in body weight gain in females from the 10,000 ppm group. Reproductive effects included a decrease in uterus, cervix, and vagina weights in PND 21 pups from the 5000 and 10,000 ppm groups. At 10,000 ppm, ratio of anogenital distance to pup
weight was increased in female pups, and pup weights were decreased on PND 4 and 21.

- Other:
Each generation was allowed to breed 3 generations (F1a, F1b, F1c; F2a, F2b, F2c; F3a, F3b, F3c). On postnatal day 81+/-10 animals from the 3rd litter of each generation were assigned to mating pairs (N=17).
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
(F0), (F1c): weeks 1, 3 and 5. Males had additional body weights collected biweekly after the completion of the cohabitation period until necropsy. During the lactation period for their F1c litter, F0 females had body weights measured on Post-natal Days 1(sires also), 4, 7, 11, 14, 18, and 21.
(F2c): Body weights were obtained during Weeks 1(body weights only), 2, 4, and 6 for all animals. Additional body weights were collected and feed consumption was measured for the males biweekly (with a few exceptions) after the completion of the cohabitation period until necropsy. During the lactation period for their (F3c) litter, F2 females also had body weights measured on PND 1 (sires also), 4, 7, 11, 14, 18, and 21.

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
- Time schedule:
(F0), (F1c): weeks 1, 3 and 5. Males had additional body weights collected biweekly after the completion of the cohabitation period until necropsy. During the lactation period for their F1c litter, F0 females had feed consumption measured for PND 1-4, 4-7, 7-11, 11-14, 14-18, and 18-21.
(F2c): Feed consumption was measured during Weeks 1(body weights only), 2, 4, and 6 for all animals. During the lactation period for their (F3c) litter, F2 females also had feed consumption measured for PND 1-4, 4-7, 7-11, 11-14, 14-18, and 18-21.
Oestrous cyclicity (parental animals):
(FO), (F1c), (F2c): Upon completion of the lactation phase (at least 4 d after the last weaning litter), vaginal cytology was conducted for 14 days on the dams.
Sperm parameters (parental animals):
- Parameters examined in all male parental generations:
Computer-assisted sperm motion analysis using Cellsoft ™ was conducted, and epididymal sperm density, sperm morphology, and testicular spermatid head count data were measured on all necropsied Group 1, 2, 3, 5, and 7 males. In addition, computer-assisted sperm motion analysis was conducted in Groups 1, 2, 3, 5, 7, and 8 using the Hamilton-Thorne Research Version 10 IVOS Sperm Analysis System (Hamilton-Thorne Research, Beverly, Massachusetts). Computer-assisted sperm motion analysis was not conducted in Groups 4 and 6 since they represented intermediate groups. The right vas deferens was used for sperm motility analysis; the right cauda epididymis was used for sperm density and morphology; and the right testis was frozen (approximately - 80°C) for evaluation of spermatid head counts.
Chromatin analysis was performed using a sample taken from the epididymal sperm suspension prepared for the sperm density and morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
All pups delivered were euthanized by sodium pentobarbital overdose on PND 1 and discarded without necropsy.
For the pups, selection occurred on PND 16. On PND 16, up to five males and two females from each litter, when available, were randomly selected for the cohabitation, the non-mating male phase, or post-natal necropsies.
On PND 21, the selected pups were housed separately from the dam and given diets containing DEHP at the same dose levels as their parents. Pups not
selected were euthanized via carbon dioxide asphyxiation and discarded without necropsy on PND 21.

PARAMETERS EXAMINED
- The following parameters were examined in F1/F2/F3 offspring:
The first two litters produced during the cohabitation period were counted, weighed, and anogenital distance (AGD) and individual pup weights were measured on all pups at PND 1. These litters were euthanized by sodium pentabarbitol overdose and discarded without necropsy on PND 1.
The third litter born was reared, without culling, by the dam until weaning on PND 21. On PND 1, 4, 7, 14, and 21, all pups were counted, weighed, and had AGD measured (PND 1 only). Male pups were examined for retained nipples on PND 12 and 13. On PND 16, up to five males and two females were randomly selected from each litter produced. One female was selected for necropsy on PND 60-74 and the other was selected for the F1 cohabitation.
The males were selected in the following order of priority. First, one male from each litter was assigned to the F1 cohabitation. (If a litter had no males, a male from another litter was selected to make up to 17 males per group.) Second, three males per litter, when available, were then selected to be maintained until approximately two weeks prior to the necropsy of the mating F1 males, at which time they would be necropsied. These males had testicular descent and preputial separation measured and were not cohabited with females. These animals were termed “non-mating males”.
Finally, one additional male was selected for the PND 63-64 necropsy. The animals selected for either PND 63-64 or PND 60-74 necropsies had sexual developmental parameters measured and were then discarded without necropsy. Observations of testicular descent (starting on PND 16), vaginal opening (starting on PND 25), and preputial separation (starting on PND 35) were conducted. On PND 21, all selected animals were housed separately from the dam and remaining pups were euthanized and discarded without necropsy.
Postmortem examinations (parental animals):
SACRIFICE
On SD 175-177 and SD 171, for (F0) animals
OnPND 194-197±12 for non-mating males and selected mating males and on PND 215 – 217±12 for (F1) females.
On PND 241-243±10, selected (F1) non-mating males and on PND 248 – 250 ± 10, selected mating (F2) males and (F2) females.
On PND 63-64 for the (F3) males and PND 60-74 for the (F3) females.

Selected males and females were euthanized by CO2 asphyxiation. Terminal body weights were obtained from all surviving (F0) animals. Animals not scheduled to be necropsied were euthanized and discarded. A complete necropsy was performed on all surviving control animals and 10 treated animals from each dose group (the first 10 animals from Groups 2-8) for each sex. A gross necropsy was performed on the remaining females from each group not selected for a complete necropsy, which included examination of gross abnormalities including missing organs or visible reproductive abnormalities. All necropsies were performed by trained personnel from Pathology Associates International (PAI) under the direct supervision of a Board-certified pathologist. Necropsies included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.

ORGAN WEIGHTS
The following organs were weighed:
liver kidneys (paired), adrenals (paired), right testis, right epididymis, right cauda epididymis, ventral prostate, dorsolateral prostate, ovaries (paired), seminal vesicles with coagulating glands, uterus/vagina/cervix pituitary, brain, gross lesions

HISTOPATHOLOGY
The kidneys, ventral and dorsolateral prostates, seminal vesicles with coagulating glands, pituitary, adrenals, thyroid/parathyroid, uterus, cervix, vagina, brain, liver and gross lesions were preserved in 10% neutral-buffered formalin for histopathological examination. The ovaries were preserved in Bouin's for 24-48 hours and then transferred to 70% ethanol. The left testis and epididymis from all necropsied males were fixed in 2% paraformaldehyde / 3% glutaraldehyde for 3-5 days and then transferred to phosphate buffered saline. The left testis and epididymis from the first 10 males per group will be embedded in glycol methacrylate (GMA), stained with periodic acid-Schiffs’s and hematoxylin and examined microscopically. The kidneys, liver, adreanals, seminal vesicles with coagulating glands, ventral and dorsolateral prostates, and gross lesions from the first ten males and females per group were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by the study pathologist. The pituitary was embedded in paraffin and held for possible future microscopic examination. The uterus/vagina/cervix from all animals was embedded in paraffin, sectioned and stained with hematoxylin and eosin. Both ovaries from each terminally killed female, with a few exceptions, were embedded in paraffin and step sectioned beginning in the middle of each ovary. Each section was approximately 5 microns thick.
Sections were taken at every 100 microns through the ovary for a total of nine sections per ovary. Three sections were mounted in order and stained with hematoxylin and eosin.
Postmortem examinations (offspring):
SACRIFICE
The first two litters produced during the cohabitation period had the total number of pups, number of live and dead pups, the number of male and female pups, the total body weight of male and female pups, individual male and female pup weights and AGD measured on PND 1.

The third litter born (F2c) was reared by the dam until weaning on PND 21. The total number of pups, number of live and dead pups, the number of male and female pups, and total body weight of male and female pups were recorded on PND 1, 4, 7, 14, and 21. On PND 1, individual pup body weights and AGD of all pups were measured. Male pups were observed on PND 12 and 13 for retained nipples.
Statistics:
All comparisons were made between the 1.5 (control) and 10, 30, 100, 300, 1000, and 7500 ppm groups and between the 1.5 (control) and 10,000 ppm groups. Most hypotheses are tested using the nonparametric multiple comparisons procedure of Dunn (1964) or Shirley (1977), as modified by Williams (1986).
If the p-value from Jonckheere’s test was less than 0.01, Shirley's test was used; otherwise, Dunn’s test was applied. For data expressed as a proportion, the Cochran-Armitage test (Armitage, 1971) was used to test for a dose-related trend, and pairwise comparisons were performed using a chi-square test (Conover, 1971).
The ratio of the number of pups born alive to the total number of pups carried to full term was computed for all fertile pairs. The sex ratio, expressed as the proportion of males, was computed for all fertile pairs with at least one live pup. Shirley's or Dunn's test was used to compare dosed groups to controls based on Jonckheere’s test, as described above.
Since the number of pups in a litter may influence the average pup weight, a parametric analysis of covariance (Neter and Wasserman, 1974) was used to test overall equality in average pup weight, after adjustment for average litter size. The covariant used was average litter size, including live and dead pups. Least squares estimates of dose group means adjusted for litter size were computed and tested for overall equality using an F-test. Pairwise comparisons were performed using Dunnett’s test (1955).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(F0)
Clinical signs noted periodically during the (F0) mating phase of the study included abrasions, alopecia, discharge from the eyes, reddened area, urine stains, few feces, ulcers, swelling, paralysis, small stationary tissue mass, hunched posture, paleness, lacrimation, anorexia, squinting, bulging or protruding eyes, opacity of the eye, and thinness. The incidence of these observations throughout the Groups was low (up to 12%), except for abrasions and alopecia (up to 29%). The observations of alopecia and abrasions are commonly seen in the laboratory rat and are most likely caused by contact with the feeder. They were not considered to be treatment-related in incidence or severity.

Mortality occurred in three (F0) males and two (F0) females. One control male was found dead on Study Day (SD) 110. Two males were moribund killed. One 30 ppm male was killed moribund on SD 89 because of severe clinical signs including lethargy, thinness, hematoma on right ear, and paralysis of hind limbs. One 7500 ppm male was killed moribund because of a severe abscess behind left ear on SD 136. Two females (from 10 and 1000 ppm groups) were found dead on SD 89 and SD 121.

(F1)
Clinical signs noted during the (F1) mating phase included abrasions, alopecia, discharge from the eyes, urine stains, anorexia, paleness, swelling, hunched posture, obesity, small and large stationary tissue masses, reddened areas (erythema), lacrimation, malocclusion, papilloma, ulcer, missing anatomy, and thinness. The incidence of these observations throughout the Groups was low (up to 12%), except for alopecia (up to 47%), and urine stains (up to 65%). The observation of alopecia is commonly seen in the laboratory rat and is most likely caused by contact with the feeder. These observations are not considered to be treatment-related in incidence or severity.

Mortality was observed in two mating and four non-mating males and in two control females. One 1000 ppm mating male was moribund killed on PND 170± 10 because of severe tremors and hunched posture. One 300 ppm mating male was found dead on PND 104. One 30 ppm non-mating male was found dead on PND 190 ± 10. One 100 ppm non-mating male was found dead on PND 182±10. Two 7500 ppm non-mating males were found dead on PND 137±10 and PND 189±10. Two control females were found dead on PND 110±10 and PND 111±10. All animals appeared normal prior to their deaths.

(F2)
Clinical signs noted during the (F2) mating phase included abrasions, alopecia, discharge from the eyes, urine stains, anorexia, paleness, swelling, hunched posture, obesity, small and large stationary tissue masses, reddened areas, lacrimation, malocclusion, papilloma, ulcers, missing anatomy, and thinness. The incidence of these observations was low (0-18%), except for alopecia (0-29%), abrasions (0-24%), swelling (0-71%), and urine stains (6-65%), and they were not treatment-related in incidence or severity.

Mortality was observed in three males and three females. One 10 ppm male, one 30 ppm male, and one 7500 ppm male were found dead. All males appeared normal prior to their deaths. One control female was moribund killed because of the clinical observations of thinness and hunched posture. One 30 ppm female which was found dead appeared to have died due to parturition difficulties. One 1000 ppm female was moribund killed due to swelling and paralysis of the hind limbs.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
(F0)
Mean body weights were comparable except for decreases seen in the 10,000 ppm males by approximately 6% during Weeks 21 and 23.

The feed consumption values (g/kg body weight /day) were increased in the 100 (males only), 7500 (males only), and 10,000 ppm animals by 5.6 - 10.4% during Week 5, in the 10,000 ppm males by 9.2% during Week 17, in the 10, 30, 100, 300, 1000, 7500, and 10,000 ppm males during Week 19 by 1.2 - 12.6%s, during Week 21 in the 100, 7500, and 10,000 ppm males by 6 - 11%, during Week 23 in the 7500 ppm males by 16.6% and in the 10,000 ppm males during Week 23 by 11.2%.

Feed consumption (g/animal/day) was decreased by 9.2% in the 1000 ppm females during Week 1 and increased in the 10,000 ppm males by 4.6 - 6.5% during Weeks 5 and 17.
Dam feed consumption (g/kg body weight/day and g/animal/day) during many of the intervals throughout lactation was decreased by 10 - 20%.

Dam weights were decreased by almost 8% at the time of littering for the F1a, F1b, and F1c litters, while the sire weights were decreased by about 8% at the time of the F1c litter. Dam weights remained decreased by 10-20% on PND 4 - 21. Dam feed consumption (g/animal/day) was decreased by 12-29% at all intervals except PND 1-4. Feed consumption on a g/kg body weight/day basis was only decreased at days 4-7 (approximately 11%).

(F1)
Mean adult body weights were decreased in the 10,000 ppm males during Weeks 1, 3, 5, 11, 13, 16, 17, 19, 21, and 22 (mating males only) by approximately 16 -19%. During Weeks 1, 3, 5, 8, 10, 17, and 22, the body weights of the 10,000 ppm females were decreased by approximately 15 - 24%. The 7500 ppm male body weights were also decreased by 9.2 - 12.8% during Weeks 13 (non-mating only), 15 (non-mating only), and 17 (mating only).

Mean feed consumption (g/animal/day) was decreased in the 10,000 ppm males 8.3 - 12.0% during Weeks 17, 19, and 21 and in the 10,000 ppm cohabited pairs by 9.9 - 19.4% during Weeks 1, 3, and 5. Non-mating males group housed feed consumption (g/animal/day) was decreased by 9.1% during Week 1 and by 8.6% during Week 17 in the 10,000 ppm animals and was increased by 4.6% in the 300 ppm males during Week 3, by 5.3% in the 100 ppm males during Week 5, and by approximately 8% in the 100 and 300 ppm males during Week 15. Mean feed consumption (g/kg body weight/day) was increased in the 7500 and 10,000 ppm males during Weeks 11, 13 (7500 ppm only), 15, 17, and 19 by 6.5 - 16.4% and in the 10,000 ppm cohabited pairs by 9.1% during Week 1. Non-mating males group housed feed consumption (g/kg body weight/day) was increased by 7.3% in the 7500 ppm animals during Week 1, by 6.1%, 4.2%, 7.2%, and 17.2% during Week 3, in the 100, 300, 7500, and 10,000 ppm males, respectively, during Week 5 in the 100 ppm (4.7%), 7500 ppm (10.4%), and 10,000 ppm (22.4%) males. An increase of 17.0% was also seen during Week 11 in the 10,000 ppm males. Increases of 11.8% and 41.3% were also seen during Week 13 in the 7500 and 10,000 ppm males. During Week 15, increases of 2.8 - 11.7% were seen in the 100, 300, 1000, and 7500 ppm males. During Week 17, increases of 5.4 - 8.5% were seen in the 100, 300, and 10,000 males and during Week 19 in the 1000 ppm males by 4.0%. In the 10,000 ppm non-mating males, increases were seen during Weeks 17, 19, and 21 of 14.6 - 20.2%.

(F2)
Mean body weights of the mating males were decreased 7.4% and 8.2% during Weeks 2 and 4 at the 30 ppm dose level. In the 100 ppm males, mean body weights were decreased 6.5% - 8.5% during Weeks 2, 4, 6, 13, 17, and 19. During Weeks 1, 2, 4, 6, 11, 13, 15, 17, 19, and 20 (required for F2 Crossover Mating Trial), decreases were seen in 7500 ppm mating males mean body weights of 12.1% - 15.3%. Decreases of 12.8% - 18.7% were also seen in the mean body weights of 7500 ppm non-mating males during Weeks 1, 2, 4, 6, 11, 13, 15, 17, 19, 21, and 23. Mean body weights of the 7500 ppm females were decreased by 8.7%, 9.3%, and 17.7% during Weeks 1, 2, and 6, respectively.

Mean male feed consumption (g/animal/day) was decreased during Week 15 by 8.2%, 9.7%, 8.8%, and 9.4% at the 100, 300, 1000, and 7500 ppm dose levels, respectively. Mean feed consumption (g/kg body weight/day) was increased during Week 1 by 9.1% in the 7500 ppm males. During Week 13, feed consumption was increased by 17.2%, 12.8%, 8.3%, and 22.2% in the 100, 300, 1000, and 7500 ppm males, respectively. During Week 17, increases of 5.7%, 5.0%, 5.5%, and 10.5% were seen in 30, 100, 1000, and 7500 ppm males, respectively. An increase of 8.5% was seen in the 7500 ppm males during Week 19. Cohabited pairs group housed feed consumption (g/kg body weights/day) was increased by 12.5% during Week 2 and by 10.8% during Week 6 in the 7500 pairs.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
(F0) The average test article intake was 0.12, 0.78, 2.37, 7.91, 23.30, 77.45, 592.30 and 774.65 mg/kg/day in the (F0) animals.
(F1) The average test article intake was 0.09, 0.48, 1.40, 4.86, 14.38, 48.42, 391.36 and 542.98 mg/kg/day in the (F1) animals.
(F2) The average test article intake was 0.1, 0.47, 1.44, 4.79, 13.97, 46.34 and 358.65 mg/kg/day in the (F2) animals.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
(F0) Vaginal cytology was performed on (F0) dams for 14 days after weaning of the (F1c) litters. There was significant evidence that the 30, 300, 1000, and 7500 ppm females differed from the control females in the relative amount of time spent in oestrous stages, however no changes were revealed in the number of females with regular cycles, cycle length, number of cycles, and in number of cycling females across the dose groups as compared to the control females.

(F1) Vaginal cytology was performed on (F1) dams for 14 days after weaning of the (F2c) litters. There was no evidence of any differences between the dosed and control groups in cycle length, in number of cycles, or in relative length of time spent in oestrous stages.

(F2) Vaginal cytology was performed on (F2) dams for 14 days after weaning of the (F3c) litters. There was no evidence of any differences between the dosed and control groups in cycle length, in number of cycles, or in relative length of time spent in oestrous stages.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
(F0)
Computer-assisted sperm motion analysis (using Cell-Soft) revealed a decrease in velocity (μm/sec) by 11.3% in the 10,000 ppm males. The remaining sperm motion parameters were comparable to the controls. Using the Hamilton-Thorne Integrated Visual Optic System, a decrease of 12.8% in track speed was revealed along with a 15.6% decrease in the lateral amplitude in the 10,000 ppm animals. Beat frequency, straightness, linearity, motile percentage, and progressive and path velocities of the treated groups were comparable to the controls. No changes were seen in epididymal sperm density, sperm morphology and number of sperm per cauda epididymis or spermatids per mg testis. The total number of spermatids per testis was decreased by 30.6% in the 10,000 ppm males, but was increased by 16.5% in the 10 ppm males. As a result of technical difficulties, the epididymal sperm data for the 10,000 ppm males was not obtained.

(F1)
Computer-assisted sperm motion analysis using Hamilton Thorne Integrated Visual Optic System revealed no treatment-related changes in any of the sperm motion parameters. Samples from the 10,000 ppm males did not contain a sufficient quantity of sperm for analysis using this system. No spermatids were present in the testes of the 10,000 ppm males. Only a small quantity of sperm was seen in the cauda of only two of the 10,000 ppm males. No sperm was present in the cauda of the remaining 10,000 ppm males. A 55.6% decrease was seen in the spermatid per mg testis in the 7500 ppm males. Total spermatid per testes was also decreased by 68.7% in the 7500 ppm males. Total sperm per cauda was decreased in the 7500 ppm males by 61.1%. No changes were seen in sperm morphology or number of sperm per cauda epididymis.

(F2)
Computer-assisted sperm motion analysis using Hamilton Thorne Integrated Visual Optic System revealed a 25.2% decrease in the motile percentage of the 7500 ppm males. No other treatment-related changes in any of the remaining sperm motion parameters were seen. Decreases were seen in epididymal sperm density (63.8%), the total number of sperm per cauda (72.3%), spermatids per mg testis (57.0%), and the total number of spermatids per testis (73.8%) of the 7500 ppm males. Abnormal sperm morphology was also seen in the 100, 300, 1000, and 7500 ppm males.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
(F0)
Numerous treatment-related changes were observed in the reproductive data of the 10,000 ppm cohabited pairs in the (F0) Mating Trial. There was an increase in the number of cumulative days to deliver the (F1a) litter, but not the (F1b) or (F1c).

(F1)
Decreases were seen in the pregnancy index for 10,000 ppm females in the (F1) Mating Trial. No litters were produced by the 10,000 ppm pairs during the (F1) Mating Trial. Because of this effect, this dose level was not continued after the (F1) crossover Mating Trial and the (F1) necropsy.

(F2)
Pregnancy index was increased in the 10 ppm animals during the (F3c) litter (16/17 compared to 11/17).
The following decreases were observed in the reproductive data during the (F2) cohabitation period:
- Pregnancy index during the (F3a) (9/17 compared to 17/17) and (F3b) (8/17 compared to 17/17) litters in the 7500 ppm animals
- Average litters per pair by 44.0% in the 7500 ppm animals


ORGAN WEIGHTS (PARENTAL ANIMALS)
(F0) At the higher doses, increases were observed in terminal body weights and the weights of liver, kidney, and male sex accessory organs.

(F1) The (F1) adult necropsy revealed decreased terminal body weights, increased liver weights, decreased male sex accessory organ weights generally at the 7500 and 10,000 ppm doses. There was no apparent difference between mating and non-mating males at 7500 and 10,000 ppm.

(F2) The (F2) adult necropsy revealed decreased terminal body weights, increased liver weights, decreased male sex accessory organ weights generally at the 7500 and 10,000 ppm doses.


HISTOPATHOLOGY (PARENTAL ANIMALS)
(F0)
Treatment-related findings were found at 10,000 ppm in the testis, epididymis, liver, and adrenal glands and at 7500 ppm in the liver only.
In the testis, there was minimal to marked atrophy of the seminiferous tubules characterized by loss of germ cells and “Sertoli cell only” tubules over varying proportions of the testis section. This change observed only at 10,000 ppm and was correlated with “small testis” observed grossly in the most severe cases. In one male, this change was accompanied by minimal retention of mature spermatids in the seminiferous tubule. There were secondary changes in the corresponding epididymis at 10,000 ppm consisting of sloughed epithelial cells/residual bodies (3/10 males) and aspermia (1/10 male).

There was a slight increase in the incidence and severity of cortical vacuolation in the adrenal glands of the 10,000 ppm males (6/10 at 10,000 ppm versus 1/10 in the controls). The liver was characterized by adaptive changes consisting of hepatocellular hypertrophy. This change was minimal to mild and was observed at 7500 ppm (10/10 males, 9/10 females) and 10,000 ppm (9/10 males, 10/10 females).

All other microscopic changes were those commonly found in laboratory rats and were unrelated to treatment. Incidental gross findings seen at necropsy included pitted right kidney, dilated right kidney, liver nodule, small right adrenal gland, small ventral prostate, small right and left testes, and a discolored preputial gland. Incidental gross findings in the females at necropsy included left and right pitted kidneys, dilated and pitted right kidney, dilated urinary bladder, torsion in the mesentery fat, alopecia, and an adhesion to the right accessory lobe of the liver. These findings were spread throughout the dose groups and the incidence and severity were not related to dose in both sexes.

(F1)
Treatment-related changes were found at the 7500 and 10,000 ppm dose levels in the testis, epididymis, kidneys, adrenal gland, and liver.
Findings were also noted at 1000 ppm in the kidneys, liver, and adrenal glands.
Changes in the testis included changes identical to those described in the (F0) males (i.e. minimal to marked atrophy of seminiferous tubules with occasional failure of sperm release). These changes were found in all 7500 and 10,000 ppm males. Minimal atrophy of seminiferous tubules was also observed in 1/10 male at 100 and 300 ppm. There were secondary changes in the corresponding epididymis at 7500 ppm consisting of sloughed cells/residual bodies (6/10 males) and aspermia (4/10 males) and at 10,000 ppm consisting of aspermia (9/10 males). The results from the NIEHS PWG observed Sertoli cell vacuolation in the control group as well as in the 1000 ppm and 7500 ppm males. It was not observed in the 10,000 ppm animals with diffuse seminiferous tubule atrophy. In the 7500 ppm males, Sertoli cell vacuolation was observed in seminiferous tubules without atrophy. This vacuolation was similar to that observed in the control group males.

In the kidneys, treatment-related changes were found in the medulla and consisted of dilation of the tubules and mineralization, occasionally associated with chronic pyelonephritis. These changes were found at 1000 ppm in 1/10 females, at 7500 ppm in 3/10 males and 5/10 females, and at 10,000 ppm in 5/10 males and 3/10 females. In the adrenal gland, there was a slight increase in the incidence and severity of cortex vacuolation in the males at 7500 ppm (4/10 males versus 2/10 controls) and at 10,000 ppm (5/10 males versus 1/10 controls). The liver was characterized by adaptive changes consisting of hepatocellular hypertrophy. This change was minimal at 1000 ppm (5/10 males) and minimal to mild at 7500 ppm (10/10 males and 10/10 females) and 10,000 ppm (6/10 and 9/10 females). All other microscopic changes were those commonly found in laboratory rats and were unrelated to treatment.

Incidental gross findings in the (F1) females at necropsy included dilated left horn of the uterus, cyst on left ovary, enlarged pituitary, enlarged spleen, mass in abdomen near enlarged mammary gland, discolored right kidney, and a mass in the serosa. These were spread throughout the dose groups and the incidence and severity were not related to dose. The following were incidental gross findings in the (F1) males: deformity of both kidneys, pitted kidney surface, cyst on kidney, small kidney, dilated pelvis of kidney, small seminal vesicles, hypoplasia of the seminal vesicles, discolored and small ventral prostate, small dorslateral prostate, opaque and thick ménages, thick cranial bones, skin ulcer on right forelimb, right lobe missing from seminal vesicles, and a stomach nodule. Aplasia was seen in the following organs: right testes, right epididymis, caudal epididymis, and adrenals. Most of these were spread throughout the dose groups and the incidence and severity were not related to dose with the exception of the following gross findings which were considered to be a dose-related response: small testis in 100% of the 10,000 ppm mating and non-mating animals, in 70% of the 7500 mating males, and 4% of the 300 ppm non-mating males, small epididymis in 100% of the 10,000 ppm non-mating males and 2% of the 300 ppm non-mating males.

(F2)
Treatment-related changes were found at 7500 ppm in the testis, epididymis, kidneys, adrenal gland, and liver and at 1000 ppm in the liver. Atrophy of the seminiferous tubules, present in 10/10 males at 7500 ppm, was correlated to the gross observation of atrophy. There was failure of sperm release in 1/10 male. Secondary changes were present in the corresponding epididymis including aspermia, oligospermia, and residual bodies/sloughed epithelial cells.

Changes in the medulla of the kidneys consisted of mineralization, tubular dilation and were occasionally associated with chronic pyelonephritis. These changes were observed at 7500 ppm in 4/10 males and 5/10 females. The liver was characterized by adaptive changes consisting of hepatocellular hypertrophy. This change was minimal at 1000 ppm and minimal to mild at 7500 ppm (10/10 males and 10/10 females).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Effects not related to reproductive toxicity in adult animals
Effect level:
ca. 300 other: ppm (nominal) (equivalent to approximately 23 mg DEHP/kg bw/day in the F0 animals, and 14 mg DEHP/kg bw/day in the F1 and F2 animals)
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: see 'Remark'
Remarks on result:
other: Generation: F0, F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
1 000 other: ppm (nominal) (equivalent to approximately 77 mg DEHP/kg bw/day in the F0 animals, and 48 and 46 mg DEHP/kg bw/day in the F1 and F2 animals respectively)
Sex:
male/female
Basis for effect level:
other: based on impaired fertility and litter parameters noted at 7,500 ppm and above, and decreased various sperm end-points noted at 7500 (F1-, F2-, F3 males) and 10,000 ppm (F0-, F1 males)
Remarks on result:
other: Generation: F0, F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
100 other: ppm (nominal) (equivalent to approximately 8 mg DEHP/kg bw/day in the F0 animals and 4.9 and 4.8 mg DEHP/kg bw/day in the F1 and F2 animals, respectively)
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

VIABILITY AND BODY WEIGHT (OFFSPRING)
(F1)
The changes observed in the reproductive data of the 7500 ppm cohabited pairs in the (F0) Mating Trial, included a decrease of approximately 20% in the number of live male pups when all litters were combined, the number of live pups combined from all litters, and the number of live pups in the (F1a) litter. Also, male anogenital distance in the (F1a) and (F1b) litters was decreased by 6.6 - 8.5%.

Pup weight and number were also decreased at the 10,000 ppm level. The number of live pups and male pups during the (F1a) litter was decreased by 21% and 26%, respectively. The proportion of pups born alive throughout the (F0) generation was decreased by 4%. Male, female, and combined weight, both unadjusted and adjusted for litter size, was decreased by 8-13% in the (F1a), (F1b) and (F1c) and combined (F1) litters. (F1c) pup weights were decreased 14-30% during lactation (PND 4, 7, 14, and 21).

No mortality was observed.


(F2)
Many treatment-related changes were seen in the reproductive data of the 7500 ppm cohabited pairs during the (F1) Mating Trial. Male and total pup survival throughout the lactation period decreased by 19-20%. Also, the proportion of pups born alive during the (F2a) litter decreased by 9%. Anogenital distance of the 7500 ppm males was decreased during the (F2a) and (F2c) litters by 13-18%.

Pup weights were decreased throughout the (F1) Mating Trial at the 7500 ppm level. Decreases of approximately 11% were noted in live total and female unadjusted and adjusted pup weights and in adjusted male pup weights combined across all (F2) litters. The live male, female, and combined pup weight on PND 1, adjusted and unadjusted for litter size, was decreased by 12.7 to 16.6% for the (F2c) litter. The live F2c male and female pup weight was decreased by 20-30% on PND 4, 7, 11, 18, and 21. In addition, dam feed consumption (g/animal/day) was decreased on PND 7-11, by 25%.

Mortality was seen in four males. Two control males were found dead. One animal was thin, hunched, pale, and cold to the touch prior to its death and the other appeared normal prior to its death. One 30 ppm male was found dead and was seen to have languid behaviour and hunched posture prior to its death. One 7500 ppm male was found dead and appeared normal prior to its death.


CLINICAL SIGNS (OFFSPRING)
(F1)
Clinical signs noted during the study included abrasions, discharge from the eyes, anorexia, languid behaviour, dyspnoea, hypothermia, ulcer, hunched posture, thinness, few faeces, swelling, and alopecia. The incidence of these observations was low throughout the Groups (0-6%) and they were not treatment-related in incidence or severity.

(F2)
Clinical signs noted during the study were abrasions, alopecia, and discharge from the eyes. The incidence of these observations was low (0-6%) and they were not treatment-related in incidence or severity.

(F3)
Clinical signs noted during the study included abrasions, alopecia, and discharge from the eyes. The incidence of these observations was low (0-12%), and they were not treatment-related in incidence or severity.


SEXUAL MATURATION (OFFSPRING)
(F1)
Anogenital distance (AGD) of the male pups was decreased by 13.6 - 15.1% in the (F1a), (F1b) and (F1c) litters. The female AGD/pup weight ratio was decreased by 10.5 - 16.7% in the (F1a), (F1b) and (F1c) litters. Other significant findings in the reproductive data were not considered to be treatment-related since they did not occur in a dose-related manner.

The occurrence of retained nipples was unchanged by DEHP exposure. However, sexual developmental parameters were all delayed in the 7500 and 10,000 ppm animals compared to the controls. Vaginal opening was delayed by approximately 3 days in the 7500 ppm females and by 8 days in the 10,000 ppm females. Testicular decent was delayed in the 7500 and 10,000 ppm males by approximately 3 days and 6 days, respectively. In the 7500 and 10,000 ppm males, preputial separation was delayed by approximately 3.5 days and 11 days, respectively.

(F2)
The occurrence of retained nipples was comparable across the dose levels. Delays were seen in all sexual developmental parameters measured. Vaginal opening was delayed approximately 6 days in the 7500 ppm females. Testicular descent was delayed approximately 1-2 days in the 30, 100, 300, and 1000 ppm males and was delayed approximately 3 days in the 7500 ppm males. Preputial separation was delayed approximately 1-2 days throughout out the 10, 30, 100, 300, and 1000 ppm males. Preputial separation in the 7500 ppm males was delayed approximately 7 days.

(F3)
The following decreases were observed in the reproductive data during the (F2) cohabitation period:
- Average dam body weights at delivery of the (F3a) litter by 15.5% in the 7500 ppm females.
- Average sire body weight at delivery of the (F3a) litter by 5.5 - 18.8% in the 30, 100, 300, and 7500 ppm males and by 15.5% at delivery of the (F3b) litter in the 7500 ppm animals
- Average live female pup weight (adjusted for litter size) by 7.5% during the (F3a) litter
- Male anogenital distance by 12.9% in the 7500 ppm pups in the (F3a) litter.
- Male anogenital distance /pup weight ratio by 7% during the (F3b) litter.
Pregnancy index was increased in the 10 ppm animals during the (F3c) litter (16/17 compared to 11/17). Also, the number of live female pups per litter was increased 41.5% in the 100 ppm animals during the (F3a) litter. An increase of 30.4% was seen in the lactating dam feed consumption (g/animal/day) during PND 1-4 in the 100 ppm animals.

An 11% increase in the occurrence of retained nipples was seen in the 7500 ppm male pups on both PND 12 and 13. Delays were seen in the sexual developmental parameters of the 7500 ppm animals: testicular descent was delayed 2.5 days, preputial separation was delayed 5.1 days, and vaginal opening was delayed 6 days.


HISTOPATHOLOGY (OFFSPRING)
Mean terminal body weights at PND 63 - 64 for the males and PND 60-74 for the females were comparable for each group at the same sex. A dose-related increase in the absolute and relative liver weights was seen in the 1000 ppm (21% and 17%, respectively) and 7500 ppm (51% and 63%, respectively) males. The relative liver weight in the 7500 ppm females was also increased by 36%. Absolute and relative right testis weights were decreased by 42-48% in the 7500 ppm males. Decreases in absolute dorso-lateral prostate weight (41%) and relative epididymis weights (35%) were also seen in the 7500 ppm males.

All measured sperm parameters exhibited significant treatment-related changes in the 7500 ppm adult offspring. Epididymal sperm density and total sperm per epididymis were decreased by 94 - 95%. The total number of spermatids per testes was decreased by 79%, while total spermatids per mg of tests were decreased by 67%.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Effects not related to reproductive toxicity in adult animals
Generation:
F1
Effect level:
ca. 300 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Generation:
F1
Effect level:
1 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: impaired fertility and litter parameters
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
100 ppm (nominal)
Sex:
male
Basis for effect level:
other: based on decreased absolute and/or relative testis weights noted at 7,500 (F1, F2 and F3 males) and 10,000 ppm (F0 and F1 males),

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F1 Crossover Mating Trial (10,000 ppm)

Body weights of Crossover nulliparous naive animals were comparable to the controls. Alopecia was the only clinical sign seen in the naive animals during the Crossover Mating Trial. The incidence of this observation was low (6%).

The following changes were observed in reproductive performance during the Crossover Mating Trial of the 10,000 ppm females with the naive males compared to the control females with the naive males:

- Increases in the pregnancy (11/17 compared to 4/15) and fertility (11/16 compared to 4/15).

- Decrease in the live male pup weight (adjusted for litter size) by 13.6%.

- Decrease in the anogenital distance by 16.9%.

- Increase in the ratio of female AGD/pup weight by 17.6%.

- Decrease in the dam body weight at delivery by 25.6%.

The following changes were observed in reproductive performance during the Crossover Mating Trial of the 10,000 ppm males with naive females compared to the control males with the naive females:

- Decreases in the mating (0/17 compared to 17/17) and pregnancy (0/17 compared to 11/17) indexes.

- 97.9% decrease in the number of implantations.

F2 Crossover Mating Trial (7500 ppm)

Body weights of Crossover naive animals were comparable to the controls. Clinical signs of the naive animals seen during the crossover Mating Trial included alopecia, discharge, and an abrasion. The incidence of these observations was low (6%) and they were not considered treatment-related.

The following changes were observed in reproductive performance during the Crossover Mating Trial of the 7500 ppm females with the naive males compared to the control females with the naive males:

- Decreases in the body weights of male, female, and total pups by 12.1 - 15.8%.

- Decreases in the body weights of male, female, and total pups when adjusted for litter size by 8.2 - 12.3%.

- Decrease in the male anogenital distance by 11.5%.

- Decrease in the average dam body weight at delivery by 17.3%.

The following changes were observed in reproductive performance during the Crossover Mating Trial of the 7500 ppm males with the naive females compared to the control males with the naive females:

- Decreases in the pregnancy (8/17 compared to 15/17) and fertility (8/14 compared to 15/17) indexes.

- Decrease in the number of female pups per litter by 31.0%.

- Increase in the male live pup weight by 9.2%.

- Decrease in the number of implantation sites by 54.5%.

Applicant's summary and conclusion

Conclusions:
The estimated NOAEL for the developmental effects is estimated at 100 ppm, in the 3–5 mg/kg bw/day range.
Executive summary:

To assess the potential reproductive effects over multiple generations, Diethylhexylphthalate (DEHP) was administered in the diet at concentrations of 1.5 (control), 10, 30, 100, 300, 1,000, 7500, and 10,000 ppm to groups of 17 male and 17 female Sprague-Dawley rats. The control dose level was set at 1.5 ppm as this was the amount of DEHP found in the control feed. Animals in the F0 generation began exposure as adults and were bred to produce the F1 generation (F1a, 1b, 1c), the F1 adults were bred to produce the F2 generation (F2a, 2b, 2c), and the F2 adults were bred to produce the F3 generation (F3a, 3b, 3c). Additional non-mating males (up to three per litter) were selected from the F1c, F2c, and F3c litters, and were maintained following similar procedures as those for mating males, except they were not cohabited with females.

The 10,000 ppm animals only completed the F1 generation and were terminated due to the inability to produce any F2 generation animals. Parameters evaluated over the course of the study included body weights, feed consumption, clinical observations, reproductive performance, anogenital distance, pup survival, sexual development, estrous cyclicity, sperm endpoints, gross pathology, organ weights, and limited/selected histopathology.

Based on measured feed consumption, mg/kg daily doses were calculated to be 0.12, 0.78, 2.4, 7.9, 23, 77, 592, and 775 mg/kg/day in the F0 animals; 0.09, 0.48, 1.4, 4.9, 14, 48, 391, and 543 mg/kg/day in the F1 animals; and 0.1, 0.47, 1.4, 4.8, 14, 46, 359 mg/kg/day in the F2 animals.

Reductions in terminal body weights were noted at 7500 ppm in the F1 and F2 males (10% and 14%, respectively), and at 10,000 ppm in the F0 and F1 males (6% and 21%, respectively) and females (12% and 19%, respectively). Feed consumption was generally comparable in all groups in all generations on a g/animal/day basis, but was increased at 7500 and 10,000 ppm on a g/kg body weight/day basis. Clinical signs were generally comparable among all groups in all generations.

Reproductive effects were noted in the 7500 ppm and 10,000 ppm groups. The total number of males per litter was decreased at 10,000 ppm in the F1a litter and at 7500 ppm across all F1 litters combined (F1a + 1b + 1c). The total number of F1a pups per litter was decreased at 7500 and 10,000 ppm. The total number of pups per litter across all F1 (F1a + F1b + F1c) litters combined was also decreased at 7500 ppm. At 10,000 ppm, male and female pup weights, unadjusted and /or adjusted for litter size, were decreased in the F1a and F1b litters on PND 1 and in the F1c litters on PND 1, 4, 7, 14, and 21. Male anogenital distance (AGD) was decreased at 10,000 ppm in the F1a, F1b, and F1c pups and at 7500 ppm in the F1a and F1b pups. Testes descent, vaginal opening, and preputial separation were delayed at 10,000 ppm and 7500 ppm in the F1c pups. None of the F1 mating pairs produced offspring at 10,000 ppm. Male and female pup weights, unadjusted and adjusted for litter size, were decreased at 7500 ppm in the F2c litter and combined F2a, b, c litters. Male and female pup weights were decreased at 7500 ppm throughout the lactation period (PND 1-21) of the F2c pups. Male anogenital distance (AGD) was decreased at 7500 ppm in the F2a and F2c pups. There was also a decrease in the pregnancy index for the F2 mating pairs (45%) at 7500 ppm. Male anogenital distance (AGD) was decreased at 7500 ppm in the F3a pups. Testes descent, vaginal opening, and preputial separation were delayed at 7500 ppm in the F3c pups.

Retained nipples were observed in the F3c male pups at 7500 ppm. No changes were noted in the female AGD throughout all the Mating Trials.

Crossover matings were conducted using the 7500 and 10,000 ppm males and females. At 7500 and 10,000 ppm, when treated males were crossed with nulliparous naive females, there were decreased numbers of implantation sites, and decreased indices of mating, pregnancy, and fertility. At 7500 and 10,000 ppm, when treated females were crossed with naive males there was a decrease in AGD in the male pups. Also at 7500 ppm, male, female, and  combined pup weights were decreased, both when unadjusted and adjusted for litter size.

At terminal necropsies, various sperm end-points were found to be decreased at 7500 ppm in the F1, F2, and F3 males and at 10,000 ppm in the F0 and F1 males. Density (sperm/mg cauda), sperm/cauda, spermatids/testis, and spermatids/mg testes were decreased at 7500 ppm in the F1, F2, and F3 males. Spermatids/testis were decreased at 10,000 ppm in the F0 males and no sperm or spermatids were noted in the F1 males.

Organ weight changes were seen in the liver, kidney, and male accessory sex organs. The absolute and/or relative liver weights were increased at 1,000 ppm in the F1 males at 7500 ppm in the F0, F1, and F2 males and at 10,000 ppm in the F0 males. Absolute and relative liver weights were also increased at 7500 ppm in the females in all generations; in some cases these increases in liver weights extended to the 300 and 1,000 ppm groups. Absolute and/or relative kidney weights were increased at 7500 ppm in the F0 and F2 males, and F1 females and at 10,000 ppm in the F0 males, and F0 and F1 females. The absolute and/or relative cauda, epididymis, and testis weights were decreased at 7500 ppm in the F1, F2, and F3 males and at 10,000 ppm in the F0 and/or F1 males.

Gross necropsy observations of possible significance were observed in the F1 and F2 males. In the non-mating males selected from the F1 and F2 male pups, aplastic testes and epididymis, and small testes, seminal vesicles, and prostates were noted in 1-3 animals at 300 ppm. While the incidence of these findings is low, they are consistent with the syndrome of effects seen with other phthalate-induced male reproductive toxicity. The number of animals with small testes is greater than that of TherImmune historical control. The toxicological significance of male reproductive abnormalities seen at 300 ppm may be questionable because the incidence is low and no other reproductive effects were noted. These effects, however, are consistent with phthalate-induced male reproductive toxicity and represent sampling of only a small number of animals (1 male/litter); therefore, they are considered as potentially treatment-related.

Treatment-related histopathologic abnormalities were noted at 7500 and 10,000 ppm in the testes, epididymis, liver, adrenal, and kidney in the F0, F1, and F2 animals and at 1,000 ppm in the liver of F1 and F2 animals. In the testes, minimal to marked atrophy of the seminiferous tubules characterized by loss of germ cells and the presence of Sertoli cell-only tubules, as well as occasional failure of sperm release, were noted at 10,000 ppm in the F1 males and at 7500 ppm in the F2 males. The epididymis was observed to have sloughed epithelial cells and residual bodies at 7500 and 10,000 ppm in the F0 and F1 males and also in the F2 males at 7500 ppm. Minimal to mild hepatocellular hypertrophy was noted at 10,000 ppm in the F0 animals, at 7500 ppm in the F0, F1, and F2 animals, and at 1,000 ppm in the F1 and F2 animals. Dilation of the tubules and mineralization occasionally associated with chronic pyclonephritis was observed at 1,000 ppm in the F2 animals, at 7500 ppm in the F1 and F2 animals, and at 10,000 ppm in the F1 animals. Cortex vacuolization of the adrenals was noted at 7500 ppm in the F1 animals, and at 10,000 ppm in the F0 and F1 animals.

The data obtained in this study indicate that DEHP is a reproductive toxicant at 7500 and 10,000 ppm with the presence of toxicity in the liver, kidneys, and adrenals. Other than the hepatocellular toxicity at 1,000 ppm, there was no general toxicity observed at dose levels below 1,000 ppm. There was no reproductive toxicity observed at doses lower than 7500 ppm except for a possible increase of small testes and prostates which may represent an increased incidence of developmental abnormalities in the male reproductive organs at 300 and/or 1000 ppm.