Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Two generations
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restriction; study was conducted according to GLPs and to OPPTS 870.3800 and OECD Guideline 416.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance: Di-2-Ethylhexyl Terephthalate (DOTP Plasticizer)
-Lot numbers: 9020344 and 0022268
-Physical description: Clear, colorless liquid
-Purity: 97.1%
-Stability of test substance: Stability was verified by the Sponsor prior to use on the study.
-Stability of test substance in feed: Stability of the test substance in the feed was verified by analyzing each dose level immediately after preparation and at the end of a 15 day storage period.
-Homogeneity: Homogeneity of the test substance in feed was verified by analyzing samples from the top, middle, and bottom of each diet formulation prior to use on the study.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
-Strain: Crl:CD®(SD)IGS BR rats
-Source: Charles River Laboratories, Raleigh, NC
-Sex: male and female
-Age at receipt: 42 days
-Age at study initiation: approximately 8 weeks old
-Weight at study initiation: males:223-318 g; females: 150-212 g
-Housing: For the first three days, all rats were housed at least three per cage to acclimate to the automatic watering system. After a minimum of three days, rats were single housed in wire-mesh cages suspended above cage-board. Following positive evidence of mating, females were transferred to plastic maternity cages with nesting material (Bed-O'Cobs®; The Andersons, Industrial Products Division, Maumee, OH 43537). The dams were housed in these cages until weaning on lactation day 21 and the litter, as a whole, were housed in these cages until postnatal day 28. Beginning on PND 28, the F1 pups were individually housed in suspended wire-mesh cages until the start of the mating period.
-Diet: PMI Nutrition International, Inc., Certified Rodent LabDiet® 5002, ad libitum
-Water: reverse osmosis-treated, ad libitum
-Method of animal identification: uniquely numbered Monel® metal eartags
-Method of animal distribution: Computer generated, based on body weight stratification randomized in a block design. On PND 21, 30 F1 pups per sex per group were randomly selected for the next phase of the study.

Environmental Conditions:
-Temperature: 72 ± 4 °F
-Humidity: 30-70%
-Photoperiod:12-hour light/12-hour dark
-Air exchanges: 10 fresh air exchanges per hour

In-Life Study Dates:
-Study Initiation Date: 25 January 2000
-Experimental Start Date: 4 February 2000
-Experimental Completion Date: 12 April 2001

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The F0 animals were offered experimental diets ad libitum beginning on Day 0. The animals were treated continuously throughout the study until necropsy, a total of approximately 16-18 weeks (at least 70 days prior to being mated, up to two weeks during the mating phase,approximately 3 weeks during gestation, and approximately 3 weeks during lactation).

Dose Received:
F0: A concentration of 3000 ppm of the test substance in feed produced dose levels ranging from 133 to 182 mg/kg/day for adult male rats, and 184 to 478 mg/kg/day for adult female rats. A concentration of 6000 ppm of the test substance in feed produced dose levels ranging from 265 to 367 mg/kg/day for adult male rats, and 372 to 940 mg/kg/day for adult female rats. A concentration of 10000 ppm of the test substance in feed produced dose levels ranging from 447 to 614 mg/kg/day for adult male rats, and 595 to 1349 mg/kg/day for adult female rats.

F1: A concentration of 3000 ppm of the test substance in feed produced dose levels ranging from 159 to 256 mg/kg/day for adult male rats, and 206 to 516 mg/kg/day for adult female rats. A concentration of 6000 ppm of the test substance in feed produced dose levels ranging from 320 to 523 mg/kg/day for adult male rats, and 423 to 1036 mg/kg/day for adult female rats. A concentration of 10000 ppm of the test substance in feed produced dose levels ranging from 552 to 893 mg/kg/day for adult male rats, and 697 to 1549 mg/kg/day for adult female rats.
Details on mating procedure:
Females were paired 1:1 with males, of the same dose concentration, for a period of up to 14 days and allowed to mate. Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Females that showed no evidence of mating were separated at the end of the mating period and singly housed until necropsy. All females were allowed to deliver naturally and rear their young to weaning (PND 21).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of the test diets was determined using representative batches of diets. Duplicate samples were collected from the top, middle, and bottom of each diet formulation. One set of samples from each diet formulation was analyzed for homogeneity. The remaining set of samples from each formulation was stored at room temperature for 15 days, and stability of the test substance in the diet formulations were assessed at the end of the 15-day storage period. The concentration of the test substance in feed was verified by analyzing the first four bi-weekly diet preparations for all dose levels. Additional analyses were performed on experimental diets once a month.
Duration of treatment / exposure:
All F0 animals were treated for a minimum of 70 days prior to mating until necropsy (approximately 16-18 weeks total). This process was repeated for the chosen offspring of the F0 generation (F1 animals).
Frequency of treatment:
Daily (ad libitum)
Details on study schedule:
The test substance was offered ad libitum in the diet to three groups of 60 Crl:CD®(SD)IGS BR rats (30 rats/sex/group) that comprised each generation. Dose levels selected for the F0 and F1 I generations were 3000, 6000 and 10000 ppm. A concurrent control group of 60 rats (30 rats/sex) received the basal diet, (PMI Nutrition International, Inc.), Certified Rodent LabDiet® 5002, on a comparable regimen. The parental animals received the diets beginning at approximately eight weeks of age for the F0 generation and at postnatal day (PND) 22 for the F1 generation; in both cases, diets were administered for at least 70 days prior to mating and until termination of the generation. All animals were observed twice daily for appearance and behavior; body weights and food consumption were recorded at appropriate intervals. All females were allowed to deliver and rear their pups to lactation day 21. Offspring from the pairing of the F0 animals (30 pups/sex/group) were selected to constitute the F1 generation. Indicators of physical and functional development (balanopreputial separation and vaginal patency) were evaluated for the F1 generation. Necropsies were performed at specified intervals. Designated tissues from F0 and F1 parental animals (10 animals/sex/group from the control and high-dose groups only) were evaluated for histopathological changes. Uteri and vaginas from all females in the control and high-dose groups and uteri and vaginas from all females in the low-and mid-dose groups with uterine weights greater than one gram were examined histopathologically.
Doses / concentrations
Remarks:
Doses / Concentrations:
10,000, 6,000, 3,000, or 0 ppm
Basis:

No. of animals per sex per dose:
30 rats/sex/group/generation
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
Clinical abnormalities: Detailed clinical examinations were recorded weekly for all adult F0 and F1 animals. All animals were observed twice daily for signs of toxicity.

Body Weights: Body weights were recorded weekly for adult F0 and F1 male rats. For adult F0 and F1 female rats, body weights were recorded weekly during the premating phase, during the gestation phase on Days 0, 4, 7, 11, 14, 17, and 20, and during the lactation phase on Days 1, 4, 7, 14, and 21.

Food Consumption: Food consumption was recorded weekly for adult F0 and F1 male rats. For adult F0 and F1 female rats, food consumption was recorded weekly during the premating phase, during the gestation phase on Days 0, 4, 7, 11, 14, 17, and 20, and during the lactation phase on Days 1, 4, 7, 14, and 21. Food consumption was not recorded during the cohabitation of males and females (mating phase).

Reproductive parameters (fertility, mating, days between pairing and coitus, gestation, and parturition) were evaluated for both F0 and F1 generations.
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of estrous for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed or termination of the mating period. Vaginal smears were also prepared and evaluated on the day of necropsy. Average cycle length was calculated for complete estrous cycles.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each F0 and F1 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed and an incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was placed in Dulbecco's phosphate-buffered saline, at approximately 37 ºC with 10 mg/mL bovine serum albumin (BSA). After a 10-minute incubation period, a sample of sperm was loaded into a 100-μm cannula for determination of sperm motility. All cannulas and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 ºC) using the Hamilton-Thorne HTM-IVOS Version 10 computer-assisted sperm analysis (CASA) system. Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported. Sperm morphology was evaluated by light microscopy and abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.

The left testis and epididymis from all F0 males from all test substance-exposed groups were weighed, stored frozen, homogenized, and evaluated for determination of homogenization-resistant spermatid count and calculation of sperm production rate using the Hamilton-Thorne CASA system. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye that stained DNA that is present in the head of the sperm. Each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the HTM-IVOS analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or 20 fields were counted for each sample. The sperm production rate was calculated.

Sperm Indices:

-Percent Motile (or Progressively Motile) Sperm = Number of Motile (or Progressively Motile) Sperm/Total Number of Sperm Counted X 100


-Sperm Production Rate = Number of Sperm per Gram of Testis/6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Litter observations:
All pups were individually identified by application of tattoo markings on the digits on PND 0. Litter parameters (mean live litter sizes, number of pups born, percentage of males per litter at birth, and postnatal survival) were evaluated for both F0 and F1 generations.

Parameters Assessed for F1 and F2 litters (pups):

Clinical abnormalities: Each litter was examined daily for mortality and clinical abnormalities; findings were recorded as developmental variations or malformations. Detailed observations were collected on PND 1, 4, 7, 14, and 21. Animals were sexed on postnatal days 0, 4, and 21. Litters were culled to 8 pups (4 male and 4 female, when possible) on postnatal day 4. F1 pups selected for the next phase of the study were also evaluated for balanopreputial separation and vaginal perforation, starting on postnatal day 35 and 25, respectively.

Body Weights: Individual body weights were collected on PND 1, 4, 7, 14, and 21.
Postmortem examinations (parental animals):
All surviving F0 adults were euthanized following the selection of the F1 generation. All surviving F1 adults were euthanized following weaning of the F2 pups. A complete necropsy examination was conducted on all parental animals that were found dead or euthanized either in extremis or at study termination. All animals were euthanized by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera. For F0 and F1 females, the number of former implantation sites was recorded. The following organs from all F0 and F1 parental animals euthanized at scheduled termination were weighed: adrenals, brain, epididymides (total and cauda), kidneys, liver, ovaries, pituitary, prostate, seminal vesicles with coagulating glands (with accessory fluids), spleen, testes, thymus, and uterus (with cervix and oviducts). The following tissues and organs were collected and saved in 10% neutral-buffered formalin: adrenals, aorta, bone with marrow (sternebrae), brain (forebrain, midbrain, hindbrain), cervix, coagulating gland, eyes with optic nerve (2), esophagus, stomach, duodenum, jejunum, ileum, cecum, rectum, colon, heart, kidneys, liver (sections of two lobes), lungs (including bronchi, fixed by inflation with fixative), lymph node (mesenteric), ovaries and oviducts, pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary gland (submaxillary mandibular), seminal vesicles, skeletal muscle (vastus medialis), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus, thyroids (with parathyroids, if present), trachea, urinary bladder, uterus with vagina, and all gross lesions.

Microscopic evaluations were performed on the following tissues for F0 and F1 parental animals (10/sex/group) from the control and high dose groups and for all parental animals found dead or euthanized in extremis: adrenal glands, brain, cervix, coagulating gland, epididymis (right): caput, corpus, and cauda, kidneys, liver, ovaries, oviducts, pituitary, prostate, seminal vesicles, spleen, testis (right), thymus, urinary bladder, uterus, vagina, vas deferens, and all gross (internal) lesions.

The ovaries from 10 F1 females in the control and 10000 ppm groups were prepared to obtain primordial follicle and corpora lutea counts. The ovaries were fixed, trimmed and embedded in paraffin blocks, five sections, approximately 100 microns apart, were taken from the inner third of each ovary. The sections were then placed onto clean glass microscopic slides and stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Intact F1 and F2 pups that died were necropsied and pups with external abnormalities were processed for skeletal evaluation and on postnatal day 4 non selected F1 pups were euthanized and necropsied On postnatal day 21, the remaining F1 pups that were not selected for the next phase of the study and all F2 pups were euthanized and necropsied. Brain, spleen and thymus gland weights were recorded for one randomly selected pup/sex/litter. The brain, spleen and thymus from these animals were retained in 10% neutral-buffered formalin for possible future histopathologic examination.
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5%, comparing each test substance-exposed group to the control group by sex. Reproductive toxicity data obtained from nongravid animals were excluded from statistical analyses following the mating period. Where applicable the litter was used as the experimental unit. Parental mating, fertility, copulation and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Mean body weights, body weight changes and food consumption (including food utilization), estrous cycle lengths, pre-coital intervals, gestation and lactation body weights and body weight changes, offspring weights, age of acquisition of vaginal perforation and balanopreputial separation, absolute and relative organ weights, numbers of primordial follicles and corpora lutea, sperm production weight and sperm numbers were analyzed by One-way ANOVA with Dunnett’s test. Sperm motility, morphologically normal sperm, and litter proportions of pup viability were analyzed using the Kruskal-Wallis test with the Mann-Whitney U test. All histopathology data was analyzed using Fisher’s Exact Test. Offspring weights were analyzed by Analysis of Covariance (with litter size as the covariate), and Student’s T-test.
Reproductive indices:
Mating, fertility, copulation and conception indices were calculated as follows:

-Male (Female) Mating Index (%) = Number of Males (Females) with evidence of Mating (or Females Confirmed pregnant)/Total number of Males (Females) Used for Mating X 100

-Male Fertility Index (%) = Number of Males Siring a Litter/Total Number of Males used for Mating X 100

-Male Copulation Index (%) = Number of Males Siring a Litter/Number of Males with Evidence of Mating (or Females confirmed pregnant) X 100

-Female Fertility Index (%) = Number of Females with Confirmed Pregnancy/Total Number of Females used for Mating X 100

-Female Conception Index (%) = Number of Females with Confirmed Pregnancy/Number of Females with Evidence of Mating (or Females Confirmed Pregnant) X 100
Offspring viability indices:
Litter parameters were defined as follows:

-Mean Live Litter Size = Total Viable Pups on PND 0/Number of Litters with Viable Pups on PND 0

-Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (%Per litter) = ∑(Viable Pups Per Litter on PND 0 or PND 4/Number of Pups Born Per Litter)/ Number of Litters per group X 100

-Postnatal Survival for All Other Intervals (% per litter) = ∑(Viable Pups per Litter at End of Interval N/Viable Pups per Litter at Start of Interval N)/Number of Litters per Group X 100
n=PND 0-1, 1-4 (Pre-selection), 4 (Post-selection)-7, 7-14, 14-21 or 4 (Post-selection)-21

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

Mortality:
F0: Treatment-related deaths were observed for three female rats from the 10000 ppm group approximately one week after the F1 pups were weaned. Although one male rat from the 6000 ppm group was euthanized in extremis during Week 11, and one male rat from the control group died during Week 12, these deaths were not considered treatment-related. All other F0 animals survived to scheduled necropsy.

F1: Treatment-related deaths were observed for a total of seven female rats from the 10000 ppm group: four female rats died approximately one week after the F2 pups were weaned, and the remaining three female rats were euthanized, in extremis, during Week 36. Although one male rat from the 10000 ppm group was euthanized in extremis during Week 28, it was due to a mechanical injury to the face and not considered treatment-related. All other F1 animals survived to scheduled necropsy.


Body Weights:
F0:
Males: Mean body weights were comparable among groups for male rats throughout treatment. Sporadic increases and decreases in mean weekly body weight gains were observed during the premating period for male rats from the 10000 ppm group. These differences from control values were frequently statistically significant, but did not demonstrate a consistent pattern or a dose-related trend. Similar increases and decreases in body weight gain were noted infrequently in the 3000 and 6000 ppm groups, again without any pattern or dose-related trends. A consistent, significant decrease (approximately 11%) in mean cumulative body weight gain was noted for male rats from the 10000 ppm group for intervals (Weeks) 0-5, 0-7, 0-8, and 0-10, a change considered to represent a treatment-related effect. No changes in cumulative weight gain were noted in the 3000 or 6000 ppm male groups.

Females: Mean body weights were comparable for female rats prior to breeding. A reduction in mean body weights was observed for female rats from the 10000 ppm group throughout gestation, but the difference was significant only on gestation Days 4 (-6.3%) and 20 (-5.8%). Mean body weights were also significantly reduced (by 6.6 - 11.8%) for this group throughout lactation (Days 1-21). Sporadic increases and decreases in mean weekly body weight gains were observed during the premating period for female rats from the 10000 ppm group. These differences from control values were frequently statistically significant, but did not demonstrate a consistent pattern or a dose-related trend. Similar increases and decreases in body weight gain were noted infrequently in the 3000 and 6000 ppm groups, again without any pattern or dose-related trends. Mean body weight gains were significantly reduced for female rats from the 10000 ppm group between gestation Days 0-4 (-53%), 17-20 (-20.9%), and when the entire period was evaluated (Days 0-20)(-12.7%). Mean body weight gains were significantly higher than control for the 10000 ppm group during lactation Days 14-21 and for the 3000 ppm groups from lactation Days 1-21. These changes were not considered to be related to test material. A similar trend as males was noted in the cumulative weight gain parameters for the 10000 ppm female group, although these differences from the control group were rarely statistically significant. No changes in cumulative weight gain were noted in the 3000 or 6000 ppm female groups.

No other changes in mean body weight, body weight gains, or cumulative body weight gains were observed for any F0 group during the study.

F1:
Males: Reduced mean body weights (-10.1 to -16.1%) were noted in the 10000 ppm male group from the time of initial exposure to the test diet (approximately PND 10) through necropsy. The reduced mean body weight was the result of a reduced rate of body weight gain (-9.4 to -75% weekly) during the initial exposure period until approximately twelve weeks postweaning. Cumulative weight gains in the 10000 ppm male group were also reduced (-13.4%) due to the reduction in weight gain.

Mean body weights in the 6000 ppm male group were reduced (-5.7 to-6.9%) from week 26 - termination (week 37). Mean body weight gain in the 6000 ppm male group was reduced in the first week postweaning (-12.1%) and sporadically thereafter. The reductions in body weight gain resulted in reduced mean body weights and cumulative weight gains in the 6000 ppm male group sporadically during the premating period. The males in the 3000 ppm group had infrequent increases and decreases in body weight parameters (relative to the control groups) but did not demonstrate any consistent, dose-related patterns indicating that these differences were not related to treatment.

Females: Mean female body weights, rate of weight gain, and cumulative weight gain in the 10000 and 6000 ppm female groups were frequently increased or decreased relative to the control group during the premating period. In the absence of any consistent pattern, these changes were not considered treatment related. The females in the 3000 ppm group had infrequent increases and decreases in body weight parameters (relative to the control groups) during the premating period but did not demonstrate any consistent, dose-related patterns indicating that these differences were not related to treatment.

The female rats from the 6000 and 10000 ppm groups had decreased mean body weights (-5.8 to -8.1% and -9 to -18.2%, respectively) during gestation and lactation. Mean body weights during gestation and lactation were unaffected in the 3000 ppm group. The mean body weight gains during the entire gestation interval were significantly lower than control in the 3000, 6000 and 10000 ppm females (by 10.2%, 13.2% and 19.9%, respectively). However, there were no significant differences in body weight gains over each 3 day interval in any of these groups. Mean body weight gains during lactation were highly variable in the control and treated groups; however, there was no consistent, dose-related pattern that could be attributed to treatment. The reduced mean body weights in the 6000 and 10000 ppm groups during gestation and lactation were considered due to the decreased body weights in these animals prior to breeding.

No other changes in mean body weight, body weight gains, or cumulative body weight gains were observed for any F1 group during the study.

Food Consumption:
F0: Mean weekly food consumption (g/animal/day) values were comparable among groups for male and female rats throughout the premating treatment period and for the male animals during the postmating period. For female rats from the 10000 ppm group, reduced food consumption values were noted during gestation and lactation. Mean food consumption parameters were not affected in the 3000 ppm or 6000 ppm groups during the gestation and lactation periods.

F1: Mean weekly food consumption values (g/animal/day) were reduced for male rats in the 10000 ppm group but were unaffected in the remaining male and female groups. When evaluated on a g/kg/day basis, the male and female rats in the 10000 ppm groups had increased food consumption, due to the reduced body weights for the animals in those groups. There were no other treatment related changes in food consumption (g/kg/day) in the remaining male treatment groups or in any of the female treatment groups. Weekly food efficiency values did not indicate any treatment- related trends, with slight increases and decreases being found in all three treatment groups. Food consumption (g/animal/day) and food efficiency values were reduced in the 10000 ppm group during gestation. Effects observed in food consumption (g/kg/day) values in the 10000 ppm group during gestation were due to reduced body weights in this group. Sporadic increases and decreases in food consumption and food efficiency values noted in the 3000 and 6000 ppm groups did not demonstrate any consistent pattern or dose-related trend and were not considered related to treatment.

During lactation, mean food consumption (g/animal/day and g/kg/day) and food efficiency values were reduced for female rats from the 10000 ppm group. Mean food consumption values were also reduced for female rats from the 6000 ppm group on lactation Days 7-14, the period of greatest milk production and pup growth. No other changes in mean food consumption were noted for the remaining time periods for the 6000 ppm group or for any periods for the 3000 ppm group during lactation.

Organ Weights:
F0: Organ weight changes were limited to lower mean absolute pituitary gland weights for male rats from the 10000 ppm group (0.0157 g in treated vs. 0.0181 g in control), and higher mean relative (to body weight) liver weights for female rats from the 10000 (4.469 g/100 g in treated vs. 3.970 g/100 g in control) and 6000 (4.288 g in treated vs. 3.970 g/100 g in control) ppm groups. The lower mean absolute pituitary weight was due to the reduced body weights of the 10000 ppm male animals at necropsy (mean of 620 grams vs. a control value of 651 grams) as demonstrated by the lack of difference in pituitary weights when corrected for body mass (relative to body weight) basis. The higher mean relative (to body weight) liver weights for the female rats from the 6000 ppm and 1000 ppm groups were considered related to treatment.

F1:
Males: Mean terminal body weights were reduced in male and female rats from the 10000 and 6000 ppm groups. As a consequence of the reduced terminal body weights, several absolute organ weights were reduced the male and female rats from the 10000 ppm group. For the male rats from the 10000 ppm group this included lower mean absolute liver (-14.7%), kidney (-11.5%), spleen (-13.4%), testes (-7.9 and -10.4%) and adrenal gland (-14.6%) weights. These organ weight changes disappeared when evaluated on a relative (to body weight) basis. Reduced absolute spleen weights were also noted in the 3000 and 6000 ppm males (-12.4 % and -9.3 %, respectively). Relative weight of the spleen was decreased in 3000 ppm males but not in males treated with higher doses. Since the magnitude of the weight reductions in the spleens were not dose-dependent and there were no histopathological changes, reduced spleen weights do not appear to be related to treatment. Higher mean relative (to body weight) brain and right epididymis weights were found in the 10000 ppm male group. The increased relative (to body weight) brain weight in the 10000 ppm male group is a common finding in animals as brain growth is spared during periods of reduced rate of weight gain.

Females: For female rats from the 10000 ppm group, reduced organ weights due to decreased body mass included lower mean absolute kidney and uterus weights (-7.6% and -22.1%, respectively). These organ weight changes disappeared when evaluated on a relative (to body weight) basis. Increased mean relative (to body weight) brain, liver, spleen, and thymus gland weights were found in the 10000 ppm female group. The increased relative brain weight is expected for the reason stated previously for the F0 animals. The increased relative liver weight (4.434 g/100 g in treated vs. 4.120 g/100 g in control) is considered a treatment related effect in the 10000 ppm female group. The increased relative spleen and thymus weights were considered to have resulted from the reduced terminal body weights in the 10000 ppm female group. Absolute organ weight changes observed in female animals from the 6000 ppm group consisted of lower mean uterine weights (-17.4%) for the female rats. The uterine weight change disappeared when evaluated on a relative (to body weight) basis as the terminal body weights of the 6000 ppm female groups were reduced relative to control values. Increased mean relative (to body weight) brain, liver, spleen, and thymus gland weights were reported for female rats from the 6000 ppm group. The increased relative liver weight (4.410 g/100 g in treated vs. 4.120 g/100 g in control) was considered related to treatment while the increases in relative brain, spleen, and thymus weights were related to the decrease in terminal body mass of the animals. Organ weight changes observed in female animals from the 3000 ppm group were limited to lower mean absolute and relative (to body weight) uterine weights (-20.9% and -20.0%, respectively). The lack of a dose-dependent change in the uterine weight suggests that the effect was not related to treatment. Several control female rats had unusually high uterine weights and upon microscopic examination were found to be in stages of estrus (estrus or proestrus) that increased uterine weights due to physiological phenomena. The female animals from the treated groups that were in either estrus or proestrus had similar uterine weights to the control animals in those stages of estrus. Therefore, the apparent changes in uterine weight were due to the stage of estrus for the animals selected for organ weight determinations, rather than a true effect of the test substance. No other significant differences in organ weights were detected among any group.

Histopathology: There were no test substance-related microscopic lesions in any of the tissues examined for the adult F0 and F1 rats.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for reproductive toxicity
Effect level:
10 000 ppm
Sex:
male/female
Basis for effect level:
other: No reproductive toxicity was observed in this study; gonadal function, estrous cyclicity, mating behavior, conception, gestation and parturition, and spermatogenic endpoints, were unaffected by test substance administration.
Dose descriptor:
NOAEL
Remarks:
for parental toxicity
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Body Weights:
F1 (weanlings):
The mean body weights of the pups in the control and three treated groups were affected by the number of pups born per litter, as litters with a larger number of pups have reduced mean pup body weights and litters with a fewer number of pups have increased mean pup body weights. The number of pups born per litter was 13.2, 14.5, 14.5, and 13.6 pups/litter for the control, 3000 ppm, 6000 ppm, and 10000 ppm groups, respectively. In order to correct for the effect of differing litter size on mean pup body weight, the mean pup body weights from PND 1 and PND 4 were analyzed using litter size as a covariate. Although this is an imperfect analysis (i.e. the pups in larger litters do not instantaneously gain weight on PND 4 after culling), it represents an attempt to correct for differences in litter size before the culling procedure.

Following the covariate analysis, the mean male and female pup body weights were reduced for the 6000 ppm and 10000 ppm groups throughout weaning (PND 1-21). However, the concurrent control group had unusually large pups (probably due to the small litter size) and exceeded the laboratory historical control maximum mean value by 0.5 grams. In addition, the mean body weights from the F2 control litters matched those from the 3000 ppm and 6000 ppm groups during the first week of postnatal life. The male and female pups from the 6000 ppm group had reduced mean body weights and rate of weight gain from PND 7-14 when compared to both the F1 and F2 control values. This period of reduced weight gain in the 6000 ppm pups coincides with the reduced food consumption by the 6000 ppm dams during this period of lactation. The reduced weight gain in the 6000 ppm male and female pups was considered a treatment related effect. The rate of weight gain in the male and female pups from the 6000 ppm group from PND 14-21 was reduced when compared to the F1 control values; however, the values from the 6000 ppm group from PND 14-21 exactly matched the F2 control values from the same time period. Therefore, the reduced rate of weight gain in the 6000 ppm from PND 14-21 was not considered treatment related. The reduced mean pup body weight throughout lactation in the 10000 ppm was considered treatment related and may be related to the reduced food consumption noted in the dams from this group during gestation and lactation. The slightly reduced mean pup body weight from the 3000 ppm male pups on PND 4 and female pups on PND 7 matched both the historical control values and the F2 control litter values and therefore were not considered treatment related. The rate of weight gain for the 3000 ppm male and female pups throughout lactation matched or exceeded those found for the F2 control group during these same periods.

No other differences in mean body weight or body weight gains were observed among any group.

F2:
As was noted for the F1 pups, the mean body weights of the pups can be affected by the number of pups born per litter, as litters with a larger number of pups have reduced mean pup body weights and litters with a fewer number of pups have increased mean pup body weights. In the F2 generation, the number of pups born per litter was 14.3, 14.3, 14.5, and 14.2 pups/litter for the control, 3000 ppm, 6000 ppm, and 10000 ppm groups, respectively. In order to allow comparison with the F1 generation data, the F2 mean pup body weights were corrected for the effect of differing litter size on PND 1 and PND 4 by analyzing the mean pup body weight using litter size as a covariate. Although this is an imperfect analysis (i.e. the pups in larger litters do not instantaneously gain weight on PND 4 after culling), it represents an attempt to correct for differences in litter size prior to the culling procedure.

The mean male and female pup body weights from the 10000 ppm group were reduced throughout lactation. The mean male and female pup body weights from the 6000 ppm group were reduced on PND 14 and 21. The rate of weight gain was also reduced in the male and female pups from the 10000 ppm group starting on PND 4 and continuing until weaning, a period of lactation where the dams had reduced food consumption. The male and female pups from the 6000 ppm group had reduced body weight gains from PND 7-14, a period of lactation where the dams from the 6000 ppm group had reduced food consumption. The rate of weight gain in the 6000 ppm pups was unaffected from PND 14-21, a period where the dams had food consumption similar to the control values. The mean pup body and rate of weight gain for the male and female pups from the 3000 ppm group were unaffected by treatment. A slight decrease in rate of weight gain from PND 4-7 in the male pups from the 3000 ppm group was not considered related to treatment due to the slight nature of the effect and the lack of effect in the 6000 ppm group during this period of lactation.

No other differences in mean body weight or body weight gains were observed among any group.

Food Consumption: Not Applicable

Developmental Landmarks:
F1 (weanlings):
The mean age of the F1 male pups at acquisition of balanopreputial separation was comparable between the control, 3000 ppm, and 6000 ppm group animals. The age of acquisition of balanopreputial separation was delayed by approximately 2 days in the 10000 ppm group, due to the reduced growth rate (see above). All pups were observed to have separation by PND 51. Mean body weights were comparable between the male pups from the control, 3000 ppm, 6000 ppm, and 10000 ppm groups (due to the delay in the 10000 ppm group). The mean age of acquisition of vaginal perforation was comparable between the control and treated groups, although the 3000 ppm group had a mean value of one day less than the control value. The mean body weight of the female pups at the acquisition of vaginal perforation was reduced in all of the treatment groups due to the reduced rate of weight gain in the 6000 ppm and 10000 ppm groups (see above) and due to the earlier age of the 3000 ppm group animals at acquisition.

Organ Weights:
F1 (weanlings):
The mean terminal body weights of the F1 weanling affected the organ weights of the animals. The terminal body weights of the 3000, 6000, and 10000 ppm male weanlings were 95%, 91%, and 76% of the corresponding male control value. The terminal body weights of the 3000, 6000, and 10000 ppm female weanlings were 96%, 88%, and 79% of the corresponding female control value. For male and female pups from the 10000 ppm group, organ weight changes consisted of lower mean absolute brain, spleen, and thymus gland weights, and higher mean relative (to body weight) brain weights. Male pups from this group also had lower mean relative (to body weight) spleen weights. The female pups from the 6000 ppm group had lower mean absolute thymus gland weights due to reduced body weights as this difference from control values disappeared when corrected for body weight differences (the mean relative thymus weight was unaffected).

No other organ weights changes were detected.

F2:
The mean terminal body weights of the F2 weanling affected the organ weights of the animals. The terminal body weights of the 3000, 6000, and 10000 ppm male weanlings were 95%, 91%, and 77% of the corresponding male control value. The terminal body weights of the 3000, 6000, and 10000 ppm female weanlings were 96%, 92%, and 79% of the corresponding female control value. For male and female pups from the 10000 ppm group, organ weight changes consisted of lower mean absolute brain, spleen, and thymus gland weights, and higher mean relative (to body weight) brain weights. Female pups from this group also had lower mean relative (to body weight) spleen and thymus weights. The male pups from the 6000 ppm group had lower mean absolute thymus gland weights due to reduced body weights as this difference from control values disappeared when corrected for body weight differences (the mean relative thymus weight was unaffected).

No other organ weights changes were detected.

Histopathology: Microscopic examinations were not performed on organs from pups.

Reproductive parameters: Reproductive parameters (fertility, mating, days between pairing and coitus, gestation, parturition, and estrous cycling) were unaffected during the F0 and F1 generations.

Litter parameters: The F1 and F2 mean live litter sizes, number of pups born, percentage of males per litter at birth, and postnatal survival were unaffected by the test substance.

Spermatogenic Endpoints: There were no toxicologically significant differences noted for any sperm parameter measured for any treated group from the F0 and F1 generations.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
for neonatal toxicity
Generation:
F1
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no adverse effects on mating performance, fertility, or reproductive organs in a 2-generation study in which 30 rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate ad libitum in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm. Gonadal function, estrous cyclicity, mating behavior, conception, gestation and parturition, and spermatogenic endpoints (motility, morphology, numbers) were unaffected by test substance administration. At the highest concentration (10000 ppm) tested, mortality was observed in both the F0 and F1 female parental generations following weaning of their pups. There were no treatment-related deaths in F0 or F1 males. Reductions in mean body weight gains and/or body weights were observed in one or both sexes at various times in the study in both the F0 and F1 generations receiving 6000 or 10000 ppm diets. Mean maternal body weights and body weight gains were reduced for F0 and F1 females in the 10000 ppm groups throughout gestation and mean maternal body weights were also reduced throughout lactation. Mean maternal food consumption was also reduced in these groups. Increases in mean absolute (F0 females) and relative (F0 and F1) liver weights were observed in the 6000 and 10000 ppm groups but these effects occurred in the absence of corresponding gross or microscopic changes. Mean F1 offspring weights and weight gains were reduced for both sexes in the 6000 and 10000 ppm groups throughout the pre-weaning period. In the F2 offspring, reduced offspring weight gains were observed in the 6000 and 10000 ppm groups during lactation. Based on an absence of reproductive effects in either generation of parental animals, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was considered to be 10000 ppm. Based on general signs of toxicity observed in both generations of parental animals, the NOAEL for parental toxicity was considered to be 3000 ppm. Although some developmental toxicity (lower body and organ weights) was observed in the offspring of both generations at 6000 and 10000 ppm, these generally only occurred in the presence of maternal toxicity and the NOAEL for neonatal toxicity was considered to be 3000 ppm.

These results indicate that di (2-ethylhexyl) terephthalate is not selectively toxic to the developing fetus and it is not classified for “Developmental or Reproductive Toxicity” according to GHS.
Executive summary:

In a two generation reproductive toxicity study, thirty Crl:CD®(SD)IGS BR rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm. The parental animals received the diets beginning at approximately eight weeks of age for the F0 generation and at postnatal day (PND) 22 for the F1 generation; animals consumed diets for at least 70 days prior to mating and until termination of the generation. Clinical observations, body weights, and food consumption were recorded at specific intervals during the study. All females were allowed to deliver and rear their pups to weaning, and 30 F1 pups/sex/group were selected to constitute the F1 generation. Indicators of physical and functional development (Balanopreputial separation and vaginal patency) were evaluated for the F1 generation. Gross necropsies were performed on all animals, 10 rats/sex/group randomly selected had tissue and organ histopathology performed and 1 pup/sex/group randomly selected had organ weight analysis performed. Reproductive parameters (fertility, mating, days between pairing and coitus, gestation, parturition, and estrous cycling) were evaluated for both F0 and F1 generations.

    

Reproductive parameters were unaffected by test substance administration at concentrations of 3000, 6000 and 10000 ppm during the F0 and F1 generations. Mean live litter sizes, numbers of pups born, percentages of males per litter at birth and postnatal survival were unaffected by parental consumption of the test substance. Mean F1 and F2 male and female offspring weights and weight gains in the 6000 and 10000 ppm group were, however, reduced throughout the pre-weaning period for the F1 offspring and beginning on PND 4 and 7 for the F2 offspring in the 6000 and 10000 ppm groups, respectively. Reductions in mean absolute and relative spleen weights were also observed for F1 and F2 male offspring and reductions in mean absolute and relative spleen and thymus weights were observed for F2 female offspring in the 10000 ppm group. Based on these results, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was considered to be 10000 ppm and the NOAEL for neonatal toxicity was considered to be 3000 ppm.

    

In the parental generations, ten females (3 in F0 and 7 in F1) in the 10000 ppm group either died or were euthanized in extremis. These unscheduled deaths were considered to be test substance-related. No test substance-related deaths were observed in either generation of male rats.  Mean weekly body weights were reduced for both males and females in the 10000 ppm group and for F1 males in the 6000 ppm group. Mean maternal body weights and body weight gains were reduced for F0 and F1 females in the 10000 ppm group throughout gestation, as were mean maternal body weights during lactation. Mean weekly food consumption was slightly reduced in F1 males in the 10000 ppm group throughout the generation and in the 6000 ppm group in the week following weaning. Food consumption was reduced in the F0 and F1 10000 ppm females during gestation and lactation, and food efficiency was reduced in the F0 and F1 10000 ppm females during gestation. Although increases in absolute (F0 females) and relative (F0 and F1 females) liver weights were observed in the 10000 ppm groups, these were not considered adverse findings since there were no corresponding gross or microscopic changes observed at necropsy. Based on these results, the no-observed-adverse-effect level (NOAEL) for parental toxicity was considered to be 3000 ppm.