Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is rated a "1" because it applied GLP, used appropriate testing procedures, and followed an accepted test guideline.

Data source

Referenceopen allclose all

Title:
Two-generation reproduction studies in rats fed di-isodecyl phthalate
Author:
Hushka LJ, Waterman SJ, Keller LH, Trimmer GW, Freeman JJ, Ambroso JL, Nicolich MJ and McKee RH
Year:
2001
Bibliographic source:
Reproductive Toxicology (15)153-169
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
Cited as Directive 67/548/EEC, Annex V Part B 1988
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Principles of method if other than guideline:
-
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2-benzenedicarboxylic acid, di-C9, C10 and C-11 branched alkyl ester, C10 rich
- Physical state: liquid
- Analytical purity: Assumed 100% pure for purposes of dosing
- Expiration date of the lot/batch: April 1999

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc
- Age at study initiation: (P) 7-8 wks
- Weight at study initiation: (P) Males: 238.2-288.4 g; Females: 148.3-201.5 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: No
- Housing: individually housed during the test period, except during the mating and postpartum periods.
- Diet: Purina Certified rodent Chow (5002 meal), ad libitum
- Water: Automatic watering system, ad libitum
- Acclimation period:16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 1995-07-12 To: 1996-04-07

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The basal diet consisted of Certified Rodent Chow (5002 Meal). The test material was incorporated into the feed and mixed thoroughly to assure homogeneity. The test material diet and mixtures were prepared as fixed concentrations of test material.
Details on mating procedure:
After the 10-week premating exposure period, each P1 male was randomly paired with a P1 female from the same treatment group to produce the F1 generation. The mating period ended when all females were confirmed mated or approximately two weeks had elapsed. The day of confirmation of mating, based on observation of a copulatory plug or sperm in a vaginal rinse, was designated as Gestation day 0 (GD 0) and the date on which parturition was recorded was designated as PND 0.

- M/F ratio per cage: 1:1
- Length of cohabitation: Continuously until mating was confirmed
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Mated females were single housed in clean cages fitted with a stainless steel litter pans and provided with fresh bedding material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of test material-diet blends were checked by the testing laboratory at least once a month in order to ensure continuing accuracy in mixing diets.
Duration of treatment / exposure:
P1 males and females received test material daily for at least 10 weeks prior to mating and during the mating period. Additionally P1 females received test material during the gestation and postpartum periods, until weaning of the F1 (offspring of the P1 generation) offspring on PPD 21. P2 (F1 generation animals chosen to mate) males were dosed from postnatal day (PND) 21 for at least 10 weeks prior to mating, through the mating period for F2 (the offspring of the P2 generation) litters, and until sacrificed. P2 (F1) females were dosed from PND 21 for at least 10 weeks prior to mating, during mating, gestation, postpartum and until they were sacrificed following weaning of the F2 animals on PPD 21.
Frequency of treatment:
continuous (diet)
Details on study schedule:
- Selection of parents for F1 generation when pups were 21 days of age.
Doses / concentrations
Remarks:
Doses / Concentrations:
In the first study 0.2, 0.4, 0.8% were target dietary concentrations, 30/sex/group. In the second study the target concentrations were 0.02%, 0.06%, 0.2%, and 0.4% in diet, 30/sex/group.
Basis:
nominal in diet
No. of animals per sex per dose:
Main Study (P1 Generation)
30/sex/dose for 0.2% and 0.4% concentrations
40/sex/dose for control and 0.8% concentration

Satellite Animals (P1 Generation)
5 males/dose for 0.2% and 0.4% doses
10/sex/dose for control and 0.8% dose

Main Study (P2 Generation)
30/sex/dose

Satellite animals (P2 generation)
5 males/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Doses for this study were selected based on the results of a one-generation reproduction toxicity range-finding study in rats with the test material.

The dose levels tested in the rangefinding study (with only a 3 week pre-mating period) were 0.25%, 0.50%, 0.75% and 1.0%. Signs of toxicity were apparent at dose levels of 0.75% and 1.0%, and observed in both the parental animals and offspring. Signs of toxicity in the parental animals included decreased doby weight, suppression of body weight gain, and/or decreased food consumption. In the 0.5% dose group, adverse findings were limited primarily to decreases in food consumption compared to controls in the females during the postpartum period. Overt signs of toxicity observed in the offspring were limited to growth retardation in males and females at 0.75% and 1.0%. There also was slight evidence of growth retardation at 0.5%. The postnatal day 0 and 4 offspring mean body weights were outside the historical control range of this laboratory (although not statistically significantly less than controls) indicating possible growth retardation.

Based on these results, 0.8% was selected as the high dose for the definitive two-generation reproduction toxicity study in rats with the test material. This dose was anticipated to produce signs of toxicity, primarily lower body weights, in the parental males but also in the females during gestation and postpartum. Additionally, this dose was considered low enough to allow for sufficient survivorship in the F1 generation. A low dose of 0.2% was selected because it was expected to be a level without effect, particularly in the F2 generation. Finally, 0.4% was selected as the mid dose.

Examinations

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


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: On the first day of dosing (day 0) and at least weekly therafter until sacrifice. Females: Prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21, and on PPD0, 4, 7, 10, 14, and 21. An exam was also given to each P1 male and female on its day of sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: Males: prior to P1 selection, on the first day of dosing, and at least weekly thereafter until sacrifice. Females: Prior to P1/P2 selection, on the first day of dosing and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21 and on PPD 0, 4, 7, 10, 14, and 21, and/or at least weekly until sacrifice. Body weight also was measured on the day of sacrifice for all P1 males and females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured concurrently with body weight after Day 0, except during mating or during the postweaning period of the F1 litters.
Oestrous cyclicity (parental animals):
The estrous cycle of each P1 female was evaluated daily by vaginal smears beginning three weeks prior to mating (day 49) and continuing until the end of cohabitation. The vaginal smears were stained with Wright's stain before being evaluated. Estrous cycles were not evaluated on the day positive vaginal smears for either sperm or plugs were present.
Postmortem examinations (parental animals):
Complete gross postmortem examinations were conducted on all animals in the study. Selected organs including liver, kidneys (paired), testes (individual), prostate, seminal vesicles, right epididymis (total and cauda), ovaries (individual), uterus, and brain from all parental animals that survived to scheduled termination were removed and weighed. The pituitary, testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries, mammary gland, oviducts, thymus, adrenals, coagulating gland, kidney, liver and gross lesions from all parental animals in the control and 0.8% dose groups were examined microscopically. In addition, the reproductive organs of all animals in the 0.2% and 0.4% dose groups that had abnormal sperm, estrous cycles, or failed to produce viable litters were examined. The testes of teh P1 and F1 males were preserved in Bouin's solution. All other tissues were fixed in 10% neutral formalin, The tissues were processed, embedded in paraffin, sectioned at 5um and stained with hematoxylin and eosin. Five sections of each ovary from females in the control and high dose were examined for oocyte evaluation.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 0.8 other: %
Sex:
male/female
Basis for effect level:
other: Reproductive NOAEL is the highest dose tested = 0.8% Lowest estimated dose for 0.8% = 427mg/kg/bw/d (P1 males, premating) Highest estimted dose for 0.8% = 1,424 mg/kg/bw/d (P2 females, post partum)

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
> 0.06 other: %
Sex:
male/female
Basis for effect level:
other: lowest estimated dose for 0.06% = 33mg/kg/bw/d

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Parental toxicity: In the P1 generation, there were statistically significant increases in the mean absolute and relative liver weights of the 0.4% dose males 12% and 14%, respectively) and 0.4% dose females (12% and 13%,respectively)  compared with controls.  These increases were consistent with findings in the previously conducted two-generation reproductive study (Exxon Biomedical Sciences, 1997d), and related to the known capability of DIDP to cause peroxisome proliferation.  There were statistically significant increases in mean absolute and relative kidney weights of the 0.4% males (14% and 18%, respectively).  These increases were also consistent with findings in the previously conducted two-generation reproductive study (ExxonBiomedical Sciences, 1997d).  There also was a statistically significant increase in the 0.4% female mean relative kidney weight (6%) compared with the controls.  In the P2 generation, there were statistically significant increases in the mean absolute and relative liver weights of the 0.4% males(13% and 14%,respectively), 0.4% females (23% and 20%, respectively), and 0.2% females (17% and 9%, respectively)compared with controls. In the kidneys, there were statistically significant increases in mean absolute and relative weights of the 0.4% dose males (20% and 19%, respectively), and the 0.2% males (10% and 7%,respectively) compared with controls.  There was a statistically significant increase in mean absolute kidney weight in the 0.2% females (13%) compared with controls.  There were no treatment-related deaths.  There were no gross postmortem observations judged to be related directly to treatment with the test material.  The majority of P1 and P2 animals throughout the groups were free of observable abnormalities at postmortem examination.  In the females, there was an apparent dose-related increase in thick and/or discolored stomachs.  This stomach irritation was attributed to ingestion of bedding materials since it was observed only in females and observed in all groups, including controls.  Notable postmortem observations in the P2 animals surviving to termination were limited to an increased incidence (8/29) of dilated renal pelves in the 0.4% dose males compared with controls.  Dilated renal pelves also were observed in the other treated groups (2-5/30),but the incidence generally was similar to controls (3/30).  Dilated renal pelves also were noted in several females.  There were no statistically significant differences in the mean body weight between the treated and control males or females during the P1 or P2 generation, including the gestation and postpartum intervals.

    

Offspring toxicity: There were no biologically significant differences in F1 survivorship between the treated and control offspring and all survival indices were within the historical control range for this laboratory.  Statistically significant differences were limited to an increase in the live birth index of the 0.06% and 0.4% dose groups compared with controls.  These increases were not considered biologically important. In the F2 generation, there was a dose-related decrease in the Day 1 and Day 4 survival indices, with statistically significant decreases being observed in the 0.2% dose group (4% and 10%, respectively) and0.4% dose group (6% and 13%, respectively) compared with controls.  These values were outside the historical control range of this laboratory and were considered treatment related.  These results were consistent with the decreased F2 survivorship in the previous two-generation study.  There were no statistically significant differences between the control and treated animals for post-implantation loss.  The live birth index for the 0.2% dose group was higher than the historical control and this was not considered biologically important.  There were statistically significant increases in Day 14 and viability at weaning indices of the 0.02% and 0.06% dose groups compared with controls. These increases were not considered biologically important.  The Day 21 survival indices of the 0.2% and 0.4% dose groups were marginally outside the historical control range for this laboratory, but not statistically significantly different from the control.  No biological importance was assigned to these observations (cf. Table 4.31).  In the F1 offspring, there were no statistically significant differences in mean body weights between treated and control animals of either sex up to PND 21 nor statistically significant differences in mean body weight or mean food consumption between treated and control offspring of either sex during the two-week post weaning measurements.  In the F2offspring, there were statistically significant lower mean body weights in the 0.4% males on PND 14, the 0.4%females on PND 14 and 21 and the 0.2% females on PND 14 compared with controls.  Although, these weights were within the historical control range of the laboratory, these may have been a treatment-related effect.  There also was a statistically significant increase in the 0.02% male mean body weight on PND 21.  This increase was not considered biologically important. Mean post weaning body weights were significantly decreased compared to controls in the 0.4% dose males during PNDs 28 and 35, and in the 0.2% dose males at PND35 only.  At PNDs 42, 49, and 56, an apparent recovery occurred in the 0.2% and 0.4% treated males and their mean body weights were no longer statistically different from controls.  There were no statistically significant differences in post weaning body weights between treated and control females on PND28.  There were no treatment-related clinical signs observed in the F1 or F2 offspring of any group and the majority of offspring in all groups were free of observable abnormalities from PND 0-21 and during the post weaning periods.  However, there was an increased incidence of cannibalization of F2 pups at PND 1 or 2,not sporadically but a few P2 females in the 0.2 and 0.4% groups were specifically concerned (for instance in the 0.4% treated group one female cannibalized all its litter, e.g. 10 pups/10).  In general, there were no gross postmortem observations in the F1 or F2 offspring judged to be related to treatment with the test material.  The majority of animals selected for necropsy were free of observable abnormalities at the scheduled terminal sacrifice on PND 21.  The majority of animals that died prior to weaning (GD 22 - PND 21) also were free of observable abnormalities.  In the F1 generation, there were no statistically significant differences in mean absolute or relative organ weights (kidney or liver) between treated and control animals of either sex.  In the F2 generation, there were no statistically significant differences in mean absolute or relative organ weights(kidney or liver) between treated and control animals of either sex with the exception of the 0.4% dose group female mean relative liver weight.  There was a statistically significant increase in the mean relative liver weight of the 0.4% dose group females compared with controls.  In the absence of a similar trend in the respective absolute liver weight, this single difference was considered the result of the lower mean bodyweights of the 0.4% females at study termination and not treatment-related.  There were no statistically significant differences in F1 or F2 offspring mean PND 0 anogenital distance between treated and control animals of either sex.  Nipple retention was similar between treated and control offspring of both sexes: the majority of females in all groups had six nipples retained on PND 13/14, while all males in all groups had zero.  In the F1 animals, there were no statistically significant differences in age or weight at preputial separation between treated and control male offspring.  There were no statistically significant differences in age or weight at vaginal patency between treated and control female offspring.  In the F2 animals, there was a statistically significant delay in preputial separation for the 0.4% males when compared to the control male offspring.  This delay was small (1.2 days) and preputial separation was still included within the historical data of CD-rats(Bates et al., in Developmental Toxicology Handbook, 1997).  There were no statistically significant differences in the mean body weight at which preputial separation occurred between treated and control male offspring.  There were no statistically significant differences for age of vaginal patency between treated and control female offspring.  However, there was a statistically significant decrease in the mean body weight at the time that the 0.4% females achieved vaginal patency compared with the control female offspring.  This decrease was small (6%) and not considered biologically significant.

Satellite studies: Cross fostering: In the cross fostering satellite study, offspring born to high-dose dams and cross-fostered to control dams on PND 0 exhibited body weights which were not different from main study control offspring throughout the postnatal phase. Conversely, the mean body weights of the offspring cross-fostered to the high-dose dams were statistically significant lower (up to 19%) than the main study control offspring of both sexes on PND 14 and 21. This indicates that DIDP may be transferred through the milk but at a low level, evidenced by a low decrease of body weight; a statistical level of significance was obtained when lactation exposure effects and direct toxicity via feed (solid food is absorbed by pups from PND 14) were combined. Following weaning, these animals remained on control or high-dose diet corresponding with their cross fostering treatment, for the second generation premating phase. The mean body weight of the offspring cross-fostered with high-dose dams continued to be statistically significant lower (9-11% males; 7-10% females) than the mean body weight of the offspring cross-fostered with control dams during premating. In parent equivalents (adult rats stemming from cross-fostered pups), during the premating period there were statistically significant increases in the mean absolute and relative kidney weights of the pups cross fostered with high-dose dams (an increase of respectively 16% and 30% in males, and respectively 9% and 22% in females) compared with pups cross-fostered with control dams. The same trend was observed for liver weights: the mean absolute and relative liver weight of the cross-fostered high-dose group was increased compared with the cross-fostered control group (an increase of respectively 11% and 23% in males and 22% and 35% in females). Pertaining to reproductive organ weight changes in males, mean absolute right and left testis weight of the cross-fostered high-dose group were statistically significantly decreased compared with the cross-fostered control group. Since, relative right and left testis and epididymis weights were increased, this effects is probably due to lower body weight. In females there was an increase of the uterus, right and left ovary weights, only statistically significant when expressed relative to body weights. In absence of histopathology, it could not be determined if changes in tissue structure and function occurred.

Switched diet phase:  In the switched diet phase, weaning from high-dose animals was given control diet, while weaning from control animals was given high-dose diet. The high-dose offspring of both sexes switched to control diet displayed signs of recovery in body weight immediately after weaning and displayed normal growth patterns. However a trend toward lower body weight similar to the main study high-dose males was observed after day 42. The control offspring of both sexes switched to high-dose diet displayed slight reduction of body weight gain as the study progressed, similar to the main study high-dose animals during the P1 premating interval. In addition, in the switched diet high-dose P2 equivalents (adult rats stemming from the switched diet pups) there were statistically significant increases of the absolute and relative liver weights (an increase of respectively 36% and 34% in males, 31 and 39% in females) and kidney weights (an increase of 27% in males, respectively 15% and 23% in females), right and left testis and epididymis weights compared with the switched diet control P2 equivalents. The increase of testicular weight observed in males might be related to transient hypothyroidism in early phases of development. Results from the cross-fostering and switched diet satellite groups indicate that lactation exposure may participate to toxicity of DIDP (EU RAR on DIDP (2006)).

Summary: No-Observed-Adverse-Effect Levels (NOAELs) for fertility was 0.8%, and for offspring survival was 0.06% (F2 gen, PND 1 -4).  There were no important or dose-dependent clinical signs of toxicity in either the P1 males or females in any dose group.  There were no treatment-related deaths or gross postmortem observations judged to have been related directly to treatment with the test material in any P1 or F1 animals. Increased liver and kidney weights were found at all dose levels in male and female adults.  There were no differences observed in mating, male or female fertility, gestational index, or length of gestation.  Furthermore, there were no differences in mean littersize or sex ratio.  The percentage of live offspring was significantly decreased in the 0.8% dose group and below the laboratory historical control range (HCR).  In addition, reduced offspring survival was observed at postnatal days 1 and 4 in the F2 generation (but not in the F1 generation) at levels of 0.2% DIDP and greater.  There was a statistically significant increase in age of vaginal patency in the 0.4 and 0.8% dose groups, but overall the differences were small (approximately 2 days) and with in the range of unknown biologic relevance.  No clinical signs of toxicity or gross postmortem observations were noted in the offspring. There were no differences in viability at weaning during the postnatal period.  There were no differences in F1 offspring developmental landmarks, anogenital distance, nipple retention, preputial separation, or vaginal patency.  There were no differences in testicular weights in either parents (P1) or offspring (F1) and there were no pathological changes in the testes.  Further, there were no differences in offspring body weight during the postnatal period to PND 35. Results from the cross-fostering and switched diet satellite groups indicate that lactation exposure may participate to toxicity of DIDP (EU RAR on DIDP (2006)).

Applicant's summary and conclusion

Conclusions:
There were no statistically significant differences in male mating, male fertility, female fertility, female fecundity, or female gestational indices between treated and control animals in the P1 or P2 generation. Mean days of gestation and mean litter size and of the treated and control groups
were similar. There were no statistically significant differences in the mean sex ratio of the treated offspring compared with controls.
Parental toxicity: In both studies, liver and kidney effects were observed in the P1 generation. Increased liver weights and associated hepatocellular hypertrophy were observed at dietary concentrations of 0.4% and greater in both studies. These dietary concentrations also produced kidney effects that were associated with alpha 2u microglobulin toxicity, a male rat specific effect and thus not relevant to humans. In the first study, minor effects on the liver were observed at 0.2% (103 – 203 mg/kg/day). In the second study, no hepatic effects were recorded at this concentration (114 – 225 mg/kg/day). As there is a range in intake levels, it is likely this dietary concentration results in ingestion of DIDP at or near the NOAEL for systemic effects from repeated doing. Up to the highest dose tested no overt signs of reproductive toxicity were reported and no effect was observed on fertility parameters. For offspring toxicity, a decrease in survival indices (day 1 and day 4) in F2 generation leads to a NOAEL of 0.06% (33 mg/kg bw/d, lowest estimated dose for 0.06% DIDP in diet). No effect was observed on developmental landmarks assessed at any dose tested.