Bis(2-ethylhexyl) terephthalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility NOAELS were identified as levels ranging from 552 to 893 mg/kg/day for adult male rats, and 697 to 1549 mg/kg/day for adult female rats.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

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.

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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'

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

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.

Dose descriptor:

NOAEL

Remarks:

for neonatal toxicity

Generation:

F1

Effect level:

3 000 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

552 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Relaible, GLP, OECD

Additional information

The potential for di (2-ethylhexyl) terephthalate to cause reproductive toxicity is well understood. In a 13-week oral toxicity study conducted according to US EPA Guideline 799.9310, male and female Sprague-Dawley rats were exposed to 0, 0.1, 0.5 or 1.0% of di (2-ethylhexyl) terephthalate in the diet (equivalent to 561 mg/kg bw/day in males and 617 mg/kg bw/day in females at the 1% dose level). There were no adverse effects on weight of testes or ovaries and gross/microscopic examination of testes, epididymides, male accessory sex glands, ovaries, uterus, vagina, fallopian tubes, and male and female mammary glands revealed no treatment-related effects. In a feeding study of shorter duration, there were no adverse effects on testicular weight or gross/microscopic pathology in male Fischer 344 rats receiving up to 2.5% (equivalent to 2104 mg/kg bw/day) di (2-ethylhexyl) terephthalate in the diet for 21 days. In a 2-year oral study conducted according to EPA OPPTS Guideline 870.4200 in which male and female rats were provided ad libitum access to diets containing up to 12000 ppm (equivalent to 666 and 901 mg/kg bw/day in males and females, respectively) di (2-ethylhexyl) terephthalate, there were no adverse effects on reproductive organs in either sex. In a two-generation reproductive toxicity study conducted in male and female rats according to OECD Guideline 416, there were no adverse effects on sexual function, fertility or reproductive organs in either sex receiving up to 10000 ppm in the diet. All F0 animals were treated for a minimum of 70 days prior to mating until necropsy and F0 offspring chosen for the F1 study received similar treatment. Estrous cyclicity, spermatogenic endpoints (motility, morphology, numbers), and reproductive indices (mating, fertility, copulation and conception) were also unaffected by test substance administration. In a dietary prenatal developmental toxicity study conducted according to OECD Guideline 414, female mice were exposed to test diets containing up to 7000 ppm (equivalent to 1382 mg/kg bw/day) di (2-ethylhexyl) terephthalate during gestation days 0-18. There were no adverse effects on any reproductive parameters including preimplantation loss, number of implantation sites and numbers of corpora lutea. In a similar study in which pregnant Sprague-Dawley rats were exposed to up to 10000 ppm di (2-ethylhexyl) terephthalate in the diet from gestation days 0-20, there were no adverse effects on reproductive organs and no significant differences in numbers of early or late resorptions, corpora lutea, viable fetuses per litter, implantation sites, or pre- or post-implantation loss among any of the groups. Fetal sex ratios were also unaffected. Di (2-ethylhexyl) terephthalate caused no adverse reproductive effects in either sex under conditions used in these studies.

   

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliable without restriction; study conducted according to GLPs and to Redbook 2000 Guideline IV.C.9.b., OPPTS 870.3700, ICH Guideline Section 4.1.3, and OECD Guideline 414.

Qualifier:

according to guideline

Guideline:

other: Redbook 2000 Guideline IV.C.9.b

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ICH Guideline Section 4.1.3

Deviations:

yes

Remarks:

One deviation was noted but it did not impact the quality or integrity of the study.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Test animals:
-Source: Charles River Laboratories, Portage, Michigan
-Sex: female
-Condition at receipt: sexually mature female virgins
-Age at receipt: approximately 80 days old
-Age at study initiation: approximately 12 weeks
-Acclimation period: approximately 6 days
-Housing: Animals after the first 6 days of acclimation were single housed in stainless-steel cages. Cage paper was changed at least three times a week during the study.
-Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test material, ad libitum
-Water: Reverse osmosis-purified drinking water was supplied via an automatic watering system, ad libitum
-Method of animal identification: Each mouse was uniquely identified by a tail tattoo

Environmental Conditions:
-Temperature: 70.4 to 70.6 °F (21.3 - 21.5 °C)
-Humidity: 39.7 - 46% relative humidity
-Photoperiod: 12 hours light/12 hours dark
-Air Exchanges: approximately 10 per hour

Route of administration:

oral: feed

Vehicle:

other: Certified Rodent LabDiet 5002 (PMI Nutrition International, LLC)

Details on exposure:

The control and test diets (1000, 3000 and 7000 ppm) were offered ad libitum and supplied weekly from gestation Days 0-18 to randomly selected groups of 25 pregnant females.

Study Schedule:
-Experimental start date (animal receipt): January 25, 2005
-Experimental start date: February 4, 2005
-Test diet administration: February 4-25, 2005
-Last laparohysterectomy: February 25, 2005
-Experimental termination/completion date: April 18, 2005.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Animals were fed PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test substance. For test diets, an appropriate amount of test substance and diet for each group was weighed, placed into a Hobart mixing bowl, and mixed for 5 minutes. The premix was transferred to a V-blender and mixed for 10 minutes with enough basal diet to form batches of diets containing 1000, 3000 and 7000 ppm. Dose levels were selected based on a previous range-finding study in which doses of 500 to 10000 ppm were well tolerated in pregnant mice.

The test diets were prepared biweekly, placed in labeled high density polyethylene (HDPE) plastic drums with no liners, and stored at room temperature. Diets thus prepared were stable for 15 days, as shown by previous analyses with the test substance. The control diet was weighed biweekly into a plastic bag and placed in a HDPE plastic storage drum with no liner.

Prior to use, samples (approximately 100 g each) of each diet (including the control) were taken from the top, middle and bottom strata for homogeneity/ concentration analyses. A GC/FID area % purity determination was also performed. Samples of at least 10 g were collected prior to and after treatment and were analyzed for structure of the test substance. All prepared diets were homogeneous (samples from all strata were within 100-102% of target concentrations) and contained the amount of test substance desired (ranged from 96-106% of target concentrations of 1000, 3000 and 7000 ppm). The substance used in the study was at least 97.6% pure at the end of the study (compared to a purity of 99.79% on the Certificate of Analysis). A GC-MS analysis showed the substance to be authentic di (2-ethylhexyl) terephthalate. No contaminants were present in the feed or water at concentrations sufficient to affect the outcome of the study.

Details on mating procedure:

Females in good health that weighed at least 20 g were paired for mating in the home cage of a resident, sexually mature Crl:CD1(ICR) male from the same source. Each mating pair was examined daily. Following positive evidence of mating (vaginal copulatory plug), the females were returned to their individual cages. The day on which evidence of mating was identified was designated as Gestation Day 0. Actual body weight values ranged from 25.8 to 34.2 g on Gestation Day 0.

Duration of treatment / exposure:

Gestation Days 0-18

Frequency of treatment:

continuous

Duration of test:

18 days

No. of animals per sex per dose:

25 pregnant females per dose

Control animals:

yes, concurrent no treatment

Maternal examinations:

-Clinical observations: All mice were observed twice daily for moribundity and mortality. Individual, detailed clinical observations were recorded daily from Gestation Days 0-18. All significant findings were recorded.
-Body Weight: All animals were weighed daily from Gestation Days 0-18
-Food Consumption: All animals were weighed daily from Gestation Days 0-18
-Mean Compound Comsumption: Mean compound consumption (in mg/kg/day) for each group was determined by dividing the concentration of test material in the diet (in mg/kg) by the g/kg/day food consumption value for each interval.

Ovaries and uterine content:

All surviving mice were euthanized on Gestation Day 18 by CO2 inhalation. The thoracic, abdominal and pelvic cavities were opened and examined, and any abnormalities were recorded. Liver weights were recorded. The uterus and ovaries were exposed and excised. The numbers of corpora lutea on each ovary were recorded. All implantation sites (including resorptions) were numbered in consecutive order (beginning with the left distal to the left proximal uterine horn), noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues exhibiting gross changes were retained in 10% neutral-buffered formalin for possible future examination. Uteri with no evidence of implantation were placed in 10% ammonium sulfide solution for detection of early implantation loss.

One female in the high dose group delivered on Gestation Day 18. This animal was euthanized and necropsied. The number and location of implantation sites, corpora lutea and viable pups were recorded and included in the mean fetal data. Viable pups from this female were euthanized and examined viscerally and skeletally. A control female was euthanized on Gestation Day 6 in extremis. The uterus had no macroscopic evidence of implantation and was placed in 10% ammonium sulfide. Any grossly affected tissues from this animal were retained in 10% neutral-buffered formalin and subsequently examined.

Fetal examinations:

At necropsy each fetus was weighed, sexed, examined macroscopically for any external findings, and euthanized by an intrathoracic injection of sodium pentobarbital (if necessary). The external examination included (but was not limited to) an examination of the eyes, palate and external orifices. The crown-rump length, weight, and sex of nonviable fetuses (not autolyzed) were determined. Crown-rump measurements and degrees of autolysis were recorded for late resorptions (if present). Each viable fetus was examined viscerally (included examination of the heart and major blood vessels). Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one half of the fetuses in each litter were placed in Bouin's fixative and subsequently examined using the Wilson sectioning technique. The heads from the rest of the animals were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation, each fetus was macerated in KOH and stained with Alizarin Red S and Alcian Blue. External, visceral and skeletal findings were recorded as developmental variations (changes representing slight deviations from normal that were considered to have no effect on animal health or body conformity) or malformations (anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Fetal data were presented according to the numbers of fetuses and litters available for examination in each group, and the number of affected fetuses per litter on a proportional basis.

Statistics:

Analyses were conducted using two-tailed tests (except where noted) at significance levels of p < 0.05 and 0.01, comparing each test group to the control group. Data were presented as mean ± SD. Mean maternal body weight (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, fetal body weights (separately and by sex combined) and liver weights were analyzed using a one-way analysis of variance (ANOVA). If the ANOVA revealed a significant intergroup variance, Dunnett's test was used to compare test groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post implantation loss and fetal sex distribution) and total malformations and developmental variations (external, visceral, skeletal and combined) and of each malformation or variation were analyzed using the Kruskal- Wallis nonparametric ANOVA test to determine intergroup variances. If the variance was significant, the Mann- Whitney U-test was used to compare test to control data.

Indices:

Intrauterine data were summarized using two methods of calculation. An example of each method of calculation follows:

1. Group Mean Litter Basis:
No. Dead Fetuses, Postimplantation Loss/Litter = [Resorptions (Early/Late)/Group] / [No. Gravid Females/Group]

2. Proportional Litter Basis:
Summation Per Group (%) = [∑ Postimplantation Loss/Litter (%)] / [No. Litters/Group]

Where:
Postimplantation Loss/Litter (%) = [No. Dead Fetuses, Resorptions (Early/Late)/Litter] / [No. Implantation Sites/Litter] x 100



The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = [∑ Viable Fetuses Affected/Litter (%)] / [No. Litters/Group]

Where:
Viable Fetuses Affected/Litter (%) = [No. Viable Fetuses Affected/Litter] / [No. Viable Fetuses/Litter] x 100

Historical control data:

Historical Control data from the testing laboratory was provided as part of the study report consisting of 22 developmental toxicity/teratogenicity studies.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal data with dose level:
Clinical observations and survival: All animals survived to study termination, with the exception of one high dose animal that was euthanized after delivery on Gestation Day 18 and one control animal that was euthanized in extremis on Gestation Day 6. This animal had facial lacerations and was hypoactive, cool to the touch, and exhibited tremors. At necropsy, this animal was found to have a fractured nasal bone and was nongravid. Clinical findings were limited to single animals and/or did not occur in a dose-related manner.

Body weight: Maternal body weight, body weight gain, net body weight, net body weight gains and gravid uterine weights were not affected by treatment with any dose. The only significant change was reduced body weight gain during Gestation Days 12-13 in the 3000 ppm group (2.0 ± 0.63 g in treated vs. 2.5 ± 0.43 g in control), which was not seen at 7000 ppm.
Food consumption: There was no effect of test material on food consumption.

Gross pathology, incidence and severity: There were no internal findings in the high dose animal that delivered on Gestation Day 18. All females were internally normal, with the exception of one female in the high dose group that had a white area on the liver. The numbers of females that were nongravid in the control, 1000, 3000 and 7000 ppm groups were 3, 4, 4 and 1, respectively.

Organ weight changes: Mean absolute liver weights of animals in the 3000 (3.0053 ± 0.35957) and 7000 (3.2516 ± 0.29470) ppm groups were 8.4% (p < 0.05) and 17.3% (p < 0.01) higher than control.

Reproductive parameters: The numbers of nongravid females in the control, 1000, 3000 and 7000 ppm groups were 3/25, 4/25, 4/25 and 1/25, respectively (no significant difference). Preimplantation loss, number of implantation sites and numbers of corpora lutea were not affected by any dose of test material. The only significant change was a higher mean litter proportion of preimplantation loss in the 3000 ppm group (6.7 ± 8.18% in treated vs. 3.0 ± 9.21% in control). Since this was not observed in the 7000 ppm group (preimplantation loss was 2.7 ± 5.87%), it was not considered to be related to test material.

Dose descriptor:

NOEL

Effect level:

1 000 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal data with dose level:
Litter size, weight and sex ratio: There was no effect of treatment on live litter size, fetal body weight or sex ratios.

Total numbers of malformations/variations: The numbers of fetuses (litters) available for examination were 265(21), 276(21), 246(21) and 308(24) in the control, 1000, 3000 and 7000 ppm groups, respectively. Malformations (all of which were considered to be spontaneous) were observed in 4(3), 1(1), 8(4) and 3(3) fetuses (litters) in the respective groups. There was no effect of test material on the fetal incidence or mean litter percent with malformations.

Grossly visible abnormalities: No external developmental variations were reported in any group. External malformations occurred in the control and 3000 ppm groups only. One fetus in the control group had a meningocele. Four fetuses from one litter in the 3000 ppm group had tarsal flexure and cleft palate, and one fetus each in two different litters from this group had tarsal flexure or cleft palate. Although the litter proportions of these malformations in this group (2.0% and 2.2%, respectively) were higher than study (0% for both malformations) and historical controls (0.6% and 0.7%, respectively), they were not observed in the 7000 ppm group and were primarily clustered in one litter. Therefore, they were not considered to be related to treatment.

Visceral malformations and variations: The only visceral malformation noted was an absent kidney and ureter in one fetus in the 3000 ppm group. Visceral variations were noted in single fetuses in the 3000 (retroesophageal right subclavian artery; no brachiocephalic trunk) and 7000 ppm (hemorrhagic iris) groups. None of these changes were considered to be related to administration of test material.

Skeletal malformations and variations: Skeletal malformations were noted in three controls (fused sternebrae in two animals and severely malaligned sternebrae in another), one low dose (fused and/or malpositioned costal cartilage), one mid dose (severely malaligned sternebrae) and three high dose fetuses (severely malaligned sternebrae, fused ribs and fused and/or malpositioned costal cartilage). There were no differences in the incidences of these malformations in treated animals vs. controls when evaluated on a percent litter basis. Variations were noted in all groups (including controls) and consisted of 14th rudimentary rib(s), 14th full rib(s), 7th sternebra, accessory skull bones, malaligned sternebrae (slight or moderate), and 7th cervical rib(s). Vertebral centra unossified, extra site of ossification anterior to sternebra No. 1, extra site of ossification anterior to cervical arch No. 2, 25 presacral vertebrae and 27 presacral vertebrae were found in one, two or three fetuses in the 1000, 3000 or 7000 ppm groups. There were no significant differences in the incidences of any skeletal variations between treated and control animals when evaluated by incidence or on a percent litter basis and there was no dose-response relationship.

Dose descriptor:

NOEL

Effect level:

7 000 ppm

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a teratology study in which groups of pregnant CD-1 mice were exposed to 0, 1000, 3000, and 7000 ppm di (2-ethylhexyl) terephthalate ad libitum via the diet from gestation days 0-18, the only evidence of maternal toxicity was higher mean absolute liver weights noted at the two highest concentrations. Intrauterine growth and survival was unaffected at all dose levels and there was no evidence of teratogenicity or fetotoxicity, even at maternally toxic doses. The no-observed-effect-level (NOEL) for maternal toxicity was 1000 ppm (197 mg/kg bw/day) and the NOEL for developmental toxicity was 7000 ppm (1382 mg/kg bw/day) under conditions of the study.

Based on the results of this study, di (2-ethylhexyl) terephthalate is not classified for “Developmental or Reproductive Toxicity” according to GHS.

Executive summary:

In a teratological evaluation, groups of 25 pregnant CD-1 mice were exposed to di (2-ethylhexyl) terephthalate via the diet at concentrations of 0, 1000 (197 mg/kg bw/day), 3000 (592 mg/kg bw/day) or 7000 (1382 mg/kg bw/day) ppm from gestation days 0-18. Dams were sacrificed on Gestation Day 18 and fetuses were individually weighed and examined for external, skeletal and visceral malformations and variations. The two highest concentrations were maternally toxic, as manifested by higher mean absolute liver weights. There was no evidence of teratogenicity or fetotoxicity at any dose concentration. Under conditions of the study, the NOEL for maternal toxicity was 1000 ppm (197 mg/kg bw/day) and the NOEL for developmental toxicity was 7000 ppm (1382 mg/kg bw/day).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 weeks

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Valid without restrictions; study conducted according to GLPs, EPA OPPTS 870.3700, and OECD Guideline 414.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

the deviations did not impact the quality or integrity of the study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

the deviations did not impact the quality or integrity of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Test animals:
-Source: Crl:CD®(SD)IGS BR rats Charles River Laboratories, Raleigh, NC
-Sex: mature, virgin females
-Age at receipt: approximately 10 weeks
-Age at study initiation: approximately 12 weeks
-Acclimation period: 14 days
-Weight at study initiation: 241-298 g on gestation day 0
-Housing: singly housed in suspended stainless-steel wire-mesh cages
-Diet: PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (meal), ad libitum
-Water: Reverse osmosis-treated water, ad libitum
-Method of animal identification: uniquely identified by a Monel® metal eartag
-Method of animal distribution: Mated females were assigned to groups, after evidence of mating, using a computer program based on stratification of the gestation day 0 body weights into a block design.

Environmental Conditions:
-Temperature: 71 ± 5 °F
-Humidity: 30 - 70% relative humidity, recorded daily
-Photoperiod: 12 hours light/12 hours dark
-Air Exchanges: 10 fresh air exchanges per hour

In-Life Study Dates and date of animal receipt:
-Study Initiation Dates: 19 June 2001
-Experimental Start Date: 3 July 2001
-Experimental Completion Date: 1 November 2001

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The female rats were offered certified PMI Rodent Meal containing 0, 0.3, 0.6, or 1.0% of the test substance beginning on day 0 and continuing until day 20 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in feed was previously determined by analyzing diets ranging from 0.3 to 1.0% in concentration for 15 days (Report WIL-387001).

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.

The concentration of the test substance in feed was verified by analyzing each bi-weekly diet preparation for all dose levels. Diets were stored at room temperature.

Details on mating procedure:

Each female rat was paired with an untreated, sexually mature male rat for breeding. Females were approximately 12 weeks old when paired. Mating was confirmed by the presence of a copulatory plug or sperm in a vaginal smear. The day mating was confirmed was recorded as gestation day 0.

Duration of treatment / exposure:

Days 0 to 20 of gestation

Frequency of treatment:

Daily (ad libitum)

Duration of test:

Approximately 3 weeks

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent no treatment

Maternal examinations:

Clinical Observations: Clinical examinations were performed on each animal once daily, and moribundity/mortality checks were performed twice daily.

Body Weights: Individual body weights were measured daily, from gestation day 0-20.

Feed Consumption: Individual food consumption was measured daily, from gestation day 0-20.

Laprohysterectomy: All surviving dams were euthanized by carbon dioxide on day 20 of gestation. The abdominal, thoracic and pelvic cavities were opened and the organs examined.

Organ Weights: Liver (Dams only)

Ovaries and uterine content:

Examination of Uterine Content: Gravid uterine weights, the number of corpora lutea, and the number of implantation sites were measured at necropsy. Uteri with no macroscopic evidence of nidation were excised, opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski.

Fetal examinations:

Examination of Fetuses: The number of viable and nonviable fetuses and the number of early and late resorptions were recorded at necropsy. In addition, each viable fetus was examined, weighed, sexed, tagged, and euthanized (if necessary) by intrathoracic injection of sodium pentobarbital. Nonviable fetuses with minimal autolysis were also examined, weighed, sexed, and tagged, and the crown-rump length was measured. For nonviable fetuses with advanced autolysis, the crown-rump length was measured and the degree of autolysis recorded.

Necropsy of Fetuses: The thoracic and abdominal cavities of the fetuses were opened and examined. The sex of the pups was verified by internal examination. Fetal kidneys were graded for renal papillae development. The heads from approximately one-half of the fetuses in each litter were removed and fixed in Bouin's solution for further examination. The remaining heads were examined by a mid-coronal slice. All carcasses (including nonviable fetuses when possible) were then eviscerated, fixed in ethyl alcohol, and stained with an appropriate dye for subsequent skeletal examination.

Statistics:

The following computerized statistical models were used to analyze the data: One-way analysis of variance (ANOVA) with Dunnett's test; and Kruskal-Wallis test with Mann-Whitney U test. All analyses were conducted using two-tailed tests for minimum significance levels of 5% and 1% comparing each test substance-treated group to control. Data are presented as the mean with the standard deviation (S.D.).

Indices:

Intrauterine data were summarized in the following manner:
1) Group Mean Litter Basis:
Postimplantation Loss/Litter= [No. Dead Fetuses, Resorptions (Early/Late)/Group] ÷ No. Gravid Females/Group

2) Proportional Litter Basis:
Summation per Group (%)= ∑ Postimplantation Loss/Litter (%) (^a) ÷ No. of Litters/Group

Where a = [(No. Dead Fetuses, Resorptions (Early/Late-)/Litter) ÷ (No. Implantation Sites/Litter)] X 100.

The fetal developmental findings were summarized by presenting the incidence of a given finding and by considering the litter as the basic unit for comparison, and by calculating the number of affected fetuses in a litter on a proportional basis based on the following:

Summation per Group (%)= ∑ Viable Fetuses Affected/Litter (%) (a) ÷ No. of Litters/Group

Where a = [(No. Viable Fetuses Affected/Litter) ÷ (No. Viable Fetuses/Litter)] X 100

Historical control data:

Historical control data were provided by the testing facility as part of the final report. Data from this study were compared to historical control to determine relevance of study findings. At the time the study was finalized, the historical control database contained 665 laprohysterectomies, 9643 fetuses examined externally, and 9642 fetuses examined viscerally.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: No mortality was observed during the study.

Clinical Abnormalities: There were no treatment-related clinical abnormalities noted during the study.

Body Weights: All mean body weights were comparable among groups throughout the study. Mean body weight changes were significantly lower for the 10000 ppm group between Days 16-20. As a result, the mean net body weight and net body weight change were also significantly lower for the 10000 ppm group when compared with the control group.

Food Consumption: All mean food consumption values were comparable among groups throughout the study.

Organ Weights: Mean liver weights were significantly higher for the 10000 ppm group when compared with the control group.

There were no significant differences in mean gravid uterine weights among any of the groups.

Gestation Length: There were no premature births noted during the study.

Reproductive Parameters: There were no significant differences in the numbers of: early or late resorptions, corpora lutea, viable fetuses per litter, implantation sites, or pre- or post implantation loss among any of the groups. Fetal sex ratios and fetal body weights were unaffected.

Dose descriptor:

NOAEL

Effect level:

6 000 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Mortality: There were no significant differences in the number of live or dead fetuses among any of the groups.

Body Weights: There were no significant differences in mean fetal body weights among any of the groups.

External Malformations: There were no statistically significant differences in the percentage of external malformations or external variations in litters from any of the groups.

Visceral Malformations: There were no statistically significant differences in the percentage of visceral malformations or visceral variations in litters from any of the groups.

Skeletal Malformations: There were no statistically significant differences in the percentage of skeletal malformations in litters from any of the groups. There was an increased occurrence of 14th rudimentary ribs observed in the 10000 ppm group that was considered test substance-related but this was not considered an adverse effect. The overall percentage of skeletal variations per litter was comparable among groups.

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a prenatal developmental toxicity study in which groups of pregnant Sprague-Dawley rats were exposed to 0, 3000, 6000, and 10000 ppm di (2-ethylhexyl) terephthalate ad libitum in the diet from gestation days 0-20, signs of maternal toxicity were restricted to higher liver weights and reduced body weight and body weight gain at the highest concentration, 10000 ppm. There was no evidence of teratogenicity or fetotoxicity, even at maternally toxic doses. The no-observed-adverse-effect-level (NOAEL) for maternal toxicity was 6000 ppm (458 mg/kg bw/day) and the NOAEL for developmental toxicity was 10000 ppm (747 mg/kg bw/day) under conditions of the study.

Based on the results of this study, di (2-ethylhexyl) terephthalate is not classified for “Developmental or Reproductive Toxicity” according to GHS.

Executive summary:

In a prenatal developmental toxicity evaluation, groups of 25 pregnant rats were exposed to di (2-ethylhexyl) terephthalate per os via the diet at concentrations of 0, 3000 (226 mg/kg bw/day), 6000 (458 mg/kg bw/day) or 10000 (747 mg/kg bw/day) ppm from gestation days 0-20. Dams were sacrificed on gestation day 20 and fetuses were individually weighed and examined for external, skeletal and visceral malformations and variations. The highest concentration was maternally toxic, as manifested by higher mean absolute liver weights and reduced body weights. There was no evidence of teratogenicity or fetotoxicity at any dose concentration. There was an increased occurrence of 14th rudimentary ribs observed in the 10000 ppm group that was considered test substance-related, but this was not considered an adverse effect. Under conditions of the study, the NOAEL for maternal toxicity was 6000 ppm (458 mg/kg bw/day) and the NOAEL for developmental toxicity was 10000 ppm (747 mg/kg bw/day).

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The analyses of the test item (= test substance) were carried out at Analytical North America, BASF Corporation, Wyandotte, Michigan 48192 and/or Tarrytown, New York 10591.
Homogeneity: given (vis.)
Storage conditions: Room temperature

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

Sexually mature, virgin New Zealand White rabbits (Crl:KBL(NZW)) were supplied at 13-18 weeks of age by Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France. Only animals free from clinical signs of disease were used for the investigations.

HOUSING AND DIET
During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (cage volume in mm 653*653*95). Underneath the cages, waste trays were fixed containing dust-free wooden bedding (the present supplier is documented in the raw data). For enrichment, wooden gnawing blocks were added (Typ KNH E-041, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria). The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 20±2°C and a range of relative humidity of 30-70%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study. The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h). Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant. The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test substance was administered daily as a homogeneous addition to the food with daily concentration adjustment on the basis of body weight and food consumption measurements (GD 6-29). The test substance preparations were prepared once before the start of the study and were stored at room temperature. The concentration of Bis(2-ethylhexyl)terephthalate in the diet was calculated with the following formula:
Food consumption: 90 g, 120 g or 150 g per day*
Mean body weight: 3500 g*
* = based on historical control data

(BW x D/FC)= ppm

BW = mean body weight [g]
D = Dose [mg/kg body weight]
FC = daily food consumption [g]
ppm = dietary concentration of Bis(2-ethylhexyl)terephthalate

For each concentration, the calculated amounts of the test substance were weighed out once before the start of the study, and put in appropriate boxes at the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The boxes with the test substance were sent to Provimi Kliba SA, Kaiseraugst, Swizerland. Provimi Kliba SA mixed the respective amounts of food in a “Diosna 2 – Mischer” for 2 x 15 minutes. The mixtures of test substance and food was pelleted in a pelleting machine using distilled water, thereafter put in a pellet dryer to reach ≤ 11% analytical moisture and stored at 13 – 17°C. More details on the preparation of the mixtures by Provimi Kliba SA, Kaiseraugst, Swizerland, can be found in the raw data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the delivery of medicated diet and at the end of the administration period) for verification of the concentrations. The samples taken for the concentration control analyses at the beginning of the study (delivery of medicated diet) were also used to verify the homogeneous distribution of the test item in the diet in samples of all concentrations, as well as to verify the stability ot the test item in diet after storage of the medicated diet until the end of the treatment period. From each formulations, three samples were taken from the preparation vessel (one from the top, middle and bottom).

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial
insemination.
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary
lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before
insemination. The ejaculate samples used for the artificial insemination were obtained from
male New Zealand White rabbits of the same breed as the females. Each female was inseminated
with the sperm of a defined male donor as documented in the raw data. The male donors
were kept under conditions (air conditioning, diet, water) comparable to those of the females
participating in this study.
During the acclimatization period the animals were assigned to the different test groups according
to a randomization plan (NIJENHUIS and WILF) and on the basis of their body
weights.
The day of insemination was designated as GD 0 (beginning of the study) and the following
day as GD 1.
Based on the pregnant animals the body weight on GD 0 varied between 3119 – 4322 g.

Duration of treatment / exposure:

The test substance was administered to the animals as a homogenous addition to the food from GD 6-29. The animals of the control group were kept on basal diet.

Frequency of treatment:

continously via feed

Duration of test:

GD 6-29

Dose / conc.:

44 mg/kg bw/day (actual dose received)

Remarks:

1167 ppm (assumed daily food intake of 150 grams); 1458 ppm (assumed daily food intake of 120 grams); 1944 ppm (assumed daily food intake of 90 grams)

Dose / conc.:

135 mg/kg bw/day (actual dose received)

Remarks:

3500 / 4375 / 5833 ppm (assumed daily food intake as specified before)

Dose / conc.:

312 mg/kg bw/day (actual dose received)

Remarks:

10500 / 13125 / 17500 ppm (assumed daily food intake as specified before)

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

On GD 29, the females were sacrificed in randomized order and examined. The fetuses were removed from the uterus and investigated.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
A check was made twice a day on working days or once a day on Saturdays, Sundays or on
public holidays (GD 0-29).

DETAILED CLINICAL OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).

BODY WEIGHT: Yes
All animals were weighed on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The consumption of food was recorded daily during GD 0-29.

The intake of test substance was calculated from the amount of food consumed and expressed in milligram per kilogram body weight per day.
The calculation of the group values/day was carried out according to the following formula:

ITx= (FCx x C)/BWy

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
On GD 29, the surviving does were sacrificed in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal) and later, fetuses were removed from the uterus. All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined. After the does had been sacrificed, they were necropsied and were assessed by gross pathology.

OTHER:

Ovaries and uterine content:

The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

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

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by oral administration (one drop into the oral opening) of pentobarbital (Narcoren®; dose: approx.. 0.05 – 0.1 mL/fetus).

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

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

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

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

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

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

Statistics:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means (DUNNETT C W (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096-1121; DUNNETT C W (1964): New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482-491)

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

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians (Nijenhuis A, and Wilf H S (1978): Combinatorial Algorithms. Academic Press New York, 32-33; Hettmansperger T P (1984): Statistical Inference based on Ranks. John Wiley & Sons New York, 132-142)

Indices:

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

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

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

Historical control data:

13 Studies between Jan 2009 and Jun 2013 (gavage)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female of the control and the high-dose group, each, had blood in bedding towards the end of the gestation period (No. 3 on GD 29 and No. 95 on GD 28). In total, reduced defecation was observed in three control, twelve low-dose, five mid-dose and nineteen high-dose females (0, 44, 135 and 312 mg/kg bw/d). No defecation was observed in one female of test group 1.
There were no abnormal clinical findings in the other does in the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no substance-related or spontaneous mortalities in any female of all test groups (0, 44, 135 or 312 mg/kg bw/d).
One high-dose female (No. 98 – 312 mg/kg bw/d) was sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study (historical control data). Thus, this was considered to be a spontaneous incident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights (BW) and the average body weight gain (BWC) of the high-dose rabbits (312 mg/kg bw/d) were statistically significantly reduced on GD 12-22 (BW) and on GD 6-12 (BWC), which is considered to be treatment-related. During the treatment period (GD 6-29), these does gained on average 43% less body weight than the concurrent control animals. The mean body weights and the average body weight gain of the low- and mid-dose groups (44 or 135 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. However, there were some statistically significantly reduced body weight gain values in the low-dose group during GD 6-8, GD 12-14 and GD 28-29.

Corrected (net) body weight gain
Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all test groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group the mean food consumption of the does in the substancetreated groups was reduced during major parts of the treatment period, attaining statistical significance on GD 6-19 (test group 3 - 312 mg/kg bw/d), on GD 6-8 (test group 2 - 135 mg/kg bw/d) and on GD 6-9 and GD 10-17 (test group 1 - 44 mg/kg bw/d). During the treatment period (GD 6-29), the high-dose rabbits consumed 24% less, the mid-dose rabbits 8% less and the low-dose rabbits 13% less food.

The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was determined during the administration period (GD 6-29). The overall approximate values are shown in the table below:
Test group 1 Test group 2 Test group 3
44 mg/kg bw/d 135 mg/kg bw/d 312 mg/kg bw/d
GD 6 to 29 43.8 135.2 312.4
GD = gestation day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean gravid uterus weights of the rabbits of test groups 2 and 3 (135 and 312 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance. However, the mean gravid uterus weight of the does in test group 1 (44 mg/kg bw/d) was statistically significantly reduced in comparison to the control group (about 18% below controls), which is likely to be a subsequent effect of a lower number of implants in this test group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No necropsy findings which could be attributed to the test substance were seen in any doe. A number of spontaneous findings were noted in individual does of test groups 0, 1 and 2 (0, 44 and 135 mg/kg bw/d). These gross findings were:
• Watery feces in females No. 14, 38, 83.
• Absence of left uterine horn in control doe No. 27.
• A stomach filled with very dry, hard feed in low-dose doe No. 38.
• A bilobed gallbladder in low-dose doe No. 53.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• females Nos. 1, 2, 24, 25 – not pregnant
Test group 1 (44 mg/kg bw/d):
• females Nos. 32, 43, 47 – not pregnant
Test group 2 (135 mg/kg bw/d):
• females Nos. 70, 79 – not pregnant
Test group 3 (312 mg/kg bw/d):
• female No. 98 – sacrificed after abortion
• females Nos. 104, 113, 117, 119 – not pregnant
Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The conception rate was 87% in the control and the high-dose groups (0 and 312 mg/kg bw/d), 90% in the low-dose group (44 mg/kg bw/d) and 93% in the mid-dose group (135 mg/kg bw/d). Thus, a sufficient number of pregnant females was available at terminal sacrifice for the purpose of the study. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age (historical control data). However, in the low-dose group (44 mg/kg bw/d) the number of implantation sites was statistically significantly reduced. This was caused by an unusually high preimplantation loss in two does of this test group (No. 53 [77.8%] and No. 55 [72.7%]). This is assessed as incidental finding.

Dose descriptor:

NOAEL

Effect level:

135 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 (44, 135 and 312 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (44, 135 and 312 mg/kg bw/d) was comparable with the sex ratio of the control fetuses. Any observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One external malformation (umbilical hernia) was recorded for a control fetus (0 mg/kg bw/d). Furthermore, one external malformation (acaudate) occurred in two high-dose fetuses of the same litter (312 mg/kg bw/d). These individual external findings are considered to be incidental.
One external variation, i.e. paw hyperflexion, was recorded in one fetus, each, of test group 0, 1 and 3 (0, 44 and 312 mg/kg bw/d). This finding was not considered biologically relevant. It can be found in the historical control data.
One unclassified external observation, i.e. polyhydramnios, was recorded in one litter of the control group. Furthermore, discolored amniotic fluid occurred in one mid-dose and two highdose fetuses. These findings are not considered biologically relevant.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were recorded for three fetuses, each, in test groups 0, 1 and 2 (0, 44 and 135 mg/kg bw/d), and in five fetuses of test group 3 (312 mg/kg bw/d) (see table below). No statistically significant differences between the test substance-treated groups and the control were noted and no dose-response relationship was observed.
Test group Finding
0 (0 mg/kg bw/d) malpositioned and bipartite sternebra
lumbar hemivertebra
extra ossification site above tuberculum ventrale
1 (44 mg/kg bw/d) short rib, branched rib
misshapen thoracic vertebra, fused rib
additional vertebral arch and corresponding rib
2 (135 mg/kg bw/d) lumbar hemivertebra, misshapen lumbar vertebra
cleft sternum
misshapen thoracic vertebra
3 (312 mg/kg bw/d) fused rib
misshapen caudal vertebra
misshapen interparietal
misshapen lumbar vertebra

For test groups 0-3, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The individual skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. Although the rates of affected fetuses per litter were statistically significantly increased in the test substance-treated groups (44, 135 or 312 mg/kg bw/d), these incidences were well within the historical control range. Furthermore, the numbers affected fetuses per treatment group or of affected litters was either below control or quite similar to control. Thus, no association to the treatment is assumed for this apparent increase.
Nearly all of the increased incidences of skeletal variations were either not related to dose or they were inside the historical control range. The only exception was a higher rate of unossified talus (cartilage present), which marginally exceeded the historical range. Incomplete or non-ossifications represent slight delays of ossification which did not affect morphology, as the underlying cartilage template was completely intact.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups 0-3. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Four soft tissue malformations were recorded for one control (absent subclavian), one mid-dose (absent subclavian) and four highdose fetuses (absent subclavian, cardiomegaly, diaphragmatic hernia, absent gallbladder). No statistically significant differences between the groups were noted.
The examination of the soft tissues revealed malpositioned carotid branches in individual fetuses of all test groups including the control group (0, 44, 135 and 312 mg/kg bw/d). An absent lung lobe (Lobus inferior medialis), was noted in all substance-treated groups and showed a slightly but statistically significantly increased incidence in test group 3 (312 mg/kg bw/d). However, this is a quite common finding in this rabbit strain and was well covered by the historical control range of the test facility. Other variations occurred in individual offspring such as dilated cerebral ventricle, dilated aorta, narrowed pulmonary trunk in one fetus of test group 2, dilated renal pelvis in one other fetus of the same group, and cystic brain dilatation in one litter (two fetuses affected) of test group 3. The incidences of these variations were neither statistically significantly different from control nor do they present themselves as a specific pattern. Thus, they are not considered biologically relevant. Most of them can be found in the historical control data. All these sporadic findings sum up to a statistically significantly increased total incidence of soft tissue variations in test groups 2 and 3 (135 and 312 mg/kg bw/d). However, the mid-dose incidence closely matches the historical mean and the high-dose incidence is still covered by the historical control range, while the control incidence is unusually low. Thus, no relationship to the treatment is assumed for this apparent increase.
Some unclassified soft tissue observations were recorded: atelectasis in one fetus of test group 1 (44 mg/kg bw/d), a distended stomach in two fetuses in one litter of test group 2 (135 mg/kg bw/d) and a blood coagulum around urinary bladder in one fetus, each, of test group 2 (135 mg/kg bw/d) and test group 3 (312 mg/kg bw/d). These findings were not considered biologically relevant.

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 (44, 135 and 312 mg/kg bw/d) were not influenced by the test substance administration and were similar to the control value.

Details on embryotoxic / teratogenic effects:

There were noted external, soft tissue and skeletal malformations in four control, three low-dose, four mid-dose and nine high-dose fetuses. The distribution of total malformations about the groups was not related to dose. Six fetuses were multiple-malformed. Two high-dose fetuses of one litter (male fetus No. 105-02, female fetus No. 105-10 – 312 mg/kg bw/d) showed externally acaudate, respectively, which proved to be misshapen caudal vertebrae during skeletal examination. Female control fetus No. 22-01 had an umbilical hernia and an extra ossification site above tuberculum ventrale. For female fetus No. 45-12 (44 mg/kg bw/d) two malformations affecting the ribs (i.e. short and branched ribs) were recorded, while the findings in male fetus No. 51-03 (44 mg/kg bw/d) consisted of misshapen thoracic vertebra and fused ribs. Furthermore, female fetus No. 72-12 (135 mg/kg bw/d) had two malformations affecting the lumbar vertebrae (i.e. lumbar hemivertebra and misshapen lumbar vertebra). No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. Further malformations, such as absent subclavian, cardiomegaly, diaphragmatic hernia, absent gallbladder, misshapen interparietal, additional vertebral arch and corresponding rib, malpositioned and bipartite sternebra and cleft sternum were scattered observations in individual fetuses of all test groups. They were not dose-related and most of them can be found in the historical control data at comparable or higher incidences, thus, a relation to the treatment is not assumed. A spontaneous origin is assumed for the external variation which was observed in one fetus, each, of test group 0, 1 and 3 (0, 44 and 312 mg/kg bw/d). Soft tissue variations occurred in all test groups including control. The total incidence of soft tissue variations was statistically significantly increased in test groups 2 and 3 (135 and 312 mg/kg bw/d), while it was comparable to the controls in the low-dose group (44 mg/kg bw/d). Also, a range of skeletal variations occurred in all test groups including control. Generally, these skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at a comparable or higher frequency. The only exception was a higher rate of unossified talus (cartilage present). Incomplete or non-ossifications represent slight delays of ossification which did not affect morphology, as the underlying cartilage template was completely intact. As can be seen from the historical background data, increased incidences of such incomplete or nonossifications of the affected skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this NZW rabbit strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. They are neither considered to be evidence of developmental toxicity on their own nor to be adverse.
Although the rates of affected fetuses per litter were statistically significantly increased in the substance-treated groups (44, 135 or 312 mg/kg bw/d), the overall incidences of all classified fetal variations were well within the historical control range. Furthermore, the numbers of affected fetuses per treatment group was below control, the number of affected litters was quite similar to control. Thus, no association to the treatment is assumed. A spontaneous origin is also assumed for unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3. The distribution and type of these findings do not suggest any relation to treatment. Thus, the oral administration of Bis(2-ethylhexyl)terephthalate to pregnant New Zealand White rabbits during pregnancy caused no toxicologically relevant adverse effects on fetal morphology at all dose levels tested (44, 135 or 312 mg/kg body weight/day). Particularly, no indications for test substance-induced teratogenicity occurred.

Dose descriptor:

NOAEL

Effect level:

312 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Bis(2-ethylhexyl)terephthalate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-29) at a dose of 312 mg/kg bw/d caused evidence of maternal but no toxicologically relevant developmental toxicity. The no observed adverse effect level (NOAEL) for maternal toxicity is 135 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 312 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 382 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

Reliable, GLP, OECD

Additional information

The potential for di (2-ethylhexyl) terephthalate to cause developmental toxicity is well understood. In a two-generation reproductive toxicity study conducted according to OECD Guideline 416, male and female Sprague-Dawley rats were allowed ad libitum access to diets containing up to 10000 ppm di (2-ethylhexyl) terephthalate. Age of acquisition of balanopreputial separation and vaginal patency were unaffected in the F1 and F2 offspring. 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.  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. In a dietary prenatal developmental toxicity study conducted according to OECD Guideline 414, female mice were exposed to test diets containing up to 7000 ppm (equivalent to 1382 mg/kg bw/day) di (2-ethylhexyl) terephthalate during gestation days 0-18. There was no adverse effect on live litter size, fetal body weight or sex ratio and there was no increase in the incidence of external, skeletal or visceral malformations or variations even at maternally toxic doses. In a similar study in which pregnant Sprague-Dawley rats were exposed to up to 10000 ppm di (2-ethylhexyl) terephthalate in the diet from gestation days 0-20, intrauterine growth and survival and incidences of fetal external, visceral or skeletal malformations were unaffected by test substance administration at any dose level. An increase in the occurrence of rudimentary 14^thribs observed in the 10000 ppm exposure group was not considered an adverse effect. In a supporting study in which di (2-ethylhexyl) terephthalate was administered in corn oil via oral gavage to pregnant female Sprague-Dawley rats at a dose level of 0.75 g/kg bw/day from gestation day 14 to postnatal day 3, there were no adverse effects in male offspring on any endpoints specific to androgen disruption including anogenital distance, onset of puberty, serum testosterone levels, retained nipples, cleft phallus, blind vaginal pouch, hypospadias, undescended testes, histopathology of the testes, and sperm analysis. Di (2-ethylhexyl) terephthalate is not a teratogen and is not selectively toxic to the developing fetus. Minor developmental toxicity, such as weight reduction, was observed but only in the presence of maternal toxicity.

   

Justification for classification or non-classification

There were no adverse effects on reproductive organs in either sex of rat exposed ad libitum to 12000 ppm (equivalent to 666 and 901 mg/kg bw/day in males and females, respectively) di (2-ethylhexyl) terephthalate in the diet for up to two years and there were no adverse effects on any reproductive organs or reproductive parameters in a 2-generation reproductive toxicity study in which males and females from both generations were exposed to up to 10000 ppm in the diet for a minimum of 70 days prior to mating until necropsy. Di (2-ethylhexyl) terephthalate was not previously classified under Directive 67/548/EEC, i.e., Annex I of the Dangerous Substances Directive for reproductive toxicity. Based on a weight-of-the-evidence assessment, di (2-ethylhexyl) terephthalate is not classified for “Reproductive Toxicity” according to the UN Globally Harmonized System of Classification and Labeling (GHS) or the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) no. 1272/2008. 

  

Di (2-ehtylhexyl) terephthalate is not teratogenic. In several guideline studies, there was no increase in external, visceral or skeletal malformations at any dose level tested. While slight fetotoxicity in the form of reductions in body weight was observed, this was generally in the presence of maternal toxicity. Di (2-ethylhexyl) terephthalate was not previously classified under Directive 67/548/EEC, i.e., Annex I of the Dangerous Substances Directive for developmental toxicity. Based on a weight-of-the-evidence assessment, di (2-ethylhexyl) terephthalate is not classified for “Developmental Toxicity” according to the UN Globally Harmonized System of Classification and Labeling (GHS) or the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) no. 1272/2008.