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Toxicity to reproduction

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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: High quality study conducted in accordance with recognised guidelines and to GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002
Reference Type:
publication
Title:
Reproductive toxicity evaluation of dietary Butyl Benzyl Phthalate (BBP) in rats.
Author:
Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Brine DR, Barter RA and Butala JH
Year:
2004
Bibliographic source:
Reprod. Toxicol. 18, 241-264.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Santicizer 160
- Physical state: clear liquid
- Analytical purity: 98.5%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Purity test date:
- Lot/batch No.: DL1120N
- Expiration date of the lot/batch:
- Stability under test conditions: 12 days
- Storage condition of test material: frozen
- Other:

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: (P) ~7 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 221.5-264.0 g; Females: 157.3-195.2 g; (F1) Males: ~100-120 g; Females: ~80-110 g
- Fasting period before study: no data
- Housing: individually housed in solid-bottom polycarbonate cages with stainless steel wire lids
- Diet (e.g. ad libitum): Rodent chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Every 4-6 weeks
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: frozen
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Until successful mating, or until the end of the 2-week mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dietary formulations were analysed prior to use, and BBP concentrations were 90.9-107% of target concentrations.
Duration of treatment / exposure:
The males and females of the parental (F0) generation were exposed to BBP in the diet for 10 weeks prior to mating and during the 2-week mating period. The males were then killed, while the females continued with the same treatment throughout gestation, lactation and weaning, until sacrifice on postnatal day (PD) 21 (a total of about 17 weeks).

The F1 offspring were reduced to 10 pups/litter on PD 4, and were reared by their dams to PD 21. (They would therefore have been exposed to BBP both indirectly during gestation and lactation and directly from the feed during weaning.) On PD 21, at least one male and one female per litter was randomly selected to produce the second (F2) generation, and continued under the same regime as their parents.
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 10-12 weeks after being selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 750, 3750 and 11,250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 50, 250 and 750 mg/kg bw/day
Basis:
other: nominal ingested
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Concentrations were selected to provide target doses of 50, 250 and 750 mg/kg bw/day. The top dose of BBP was selected as it was reported to produce effects on male offspring in a study by Gray et al. (2000); the low dose was selected as it was reported as an NOAEL for dibutyl phthalate in studies by Mylchreest et al. (1998a,b, 1999); and the intermediate dose was selected to provide dose-response information.
- Rationale for animal assignment (if not random): randomization stratified by body weight, such that the body weights of all groups by sex were homogeneous by statistical analysis at study initiation
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, daily for general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily throughout the course of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, except during gestation and lactation when the females were weighed on GD 0, 7, 14 and 20 and on PD 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, although food consumption was not measured during mating due to cohabitation
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
Vaginal smears for estrous cyclicity were taken for all F0 females for the last 3 weeks of the pre-mating exposure period.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum pups/litter: 5 pups/sex/litter, excess pups were killed and examined externally and viscerally for gross malformations.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance on PD 0

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F0 and F1 animals were sacrificed at the end of the 2-week mating period.
- Maternal animals: All surviving F0 and F1 dams were sacrificed on PD 21.

GROSS NECROPSY
- All F0 and F1 parental animals were subject to a gross necropsy, which consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathologic examination was conducted on the F0 and F1 parental animal tissues taken, initially, from ten randomly selected animals per sex from the high-dose and control groups. The tissues examined were: liver, thyroid gland, ovaries, vagina, uterus with cervix, testis, prostate, epididymides, seminal vesicles, and any other tissues with gross lesions identified as being potentially treatment-related. Liver examination was extended to include 20 animals per sex in all groups (including low- and mid-dose groups). The following organs were weighed: liver, kidney, thyroid, spleen, pancreas, adrenals, pituitary, brain, ovaries, uterus, testis, prostate, epididymides and seminal vesicles.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- Of these, three F0 and three F1 weanlings per sex per litter, as well as all male pups which were positive for nipple retention, were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
There was no histopathological examination of the offspring not selected as parental animals. Organ weights were measured for the brain, thymus, spleen, ovaries, uterus, testes and epididymides.
Statistics:
Various statistical techniques used including Levene's test, standard one-way analysis of variance (ANOVA), Dunnett's test, chi-square test, Cochran-Armitage test, Fisher's exact probability, and analysis of covariance (ANCOVA). For all tests, 0.05 (one- or two-tailed) was taken as the criterion for statistical significance.
Reproductive indices:
Male and female mating indices, male and female fertility indices, male pregnancy index, female gestational index, live birth index.
Offspring viability indices:
4-, 7-, 14- and 21-day survival indices, and lactation index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Two F0 males and one F0 female died (two at the lowest dose, one at the top dose), but the deaths were not considered to be treatment-related. The only clinical sign in this generation was an increased incidence of alopecia in the females (1, 6, 5 and 7 in control, low-, mid- and high-dose groups respectively), although the incidence in male controls was also 6 (compared with 2, 4 and 8 in the low-, mid- and high-dose males) suggesting that this is not a treatment-related effect. In the F1 generation, both males and females from various dose groups showed low incidences of clinical signs, including alopecia, chromodacryorrhoea, pale appearance, red ears and small size, while two high-dose F1 males had hypospadias and four had nipples.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight was statistically significantly reduced in the high-dose F0 females in comparison with controls throughout the 70-day pre-mating period, while a slight reduction in the high-dose F0 males was not statistically significant except on study day 7. In the F1 generation, male and female body weights were significantly reduced at the top dose throughout the pre-mating period (and during the 2-week mating period in the case of the males). F0 and F1 maternal body weights during pregnancy and lactation were significantly reduced at the top dose, on days 7, 14 and 20 of pregnancy and days 0, 4, 7 and 14 (but not day 21) of lactation.
There were no clear effects on food consumption in the F0 generation, but at the high dose F1 males showed a statistically significant decrease over the 70-day pre-mating period (when expressed in mg/kg bw/day). No such effect was seen in high-dose F1 females; in fact they showed a statistically significant increase in food consumption over this period and throughout pregnancy.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
In the F0 and F1 males and females, BBP intake increased in line with dietary concentrations across the different dose groups, and showed the expected reductions over time within dose groups as body weights increased. Based on food consumption and body weight data, daily intake during the pre-mating period (days 0-70) was as follows for the low-, mid- and high-dose groups respectively: F1 males, around 50, 230 and 720 mg/kg bw/day; F1 females, around 56, 290 and 840 mg/kg bw/day; F2 males, around 56, 280 and 890 mg/kg bw/day; F2 females, around 65, 320 and 1050 mg/kg bw/day. During mating (days 77-84), the equivalent figures were: F1 males, around 40, 180 and 590 mg/kg bw/day; F2 males, around 40, 200 and 630 mg/kg bw/day. BBP intake during pregnancy (GD 0-20) was similar in F1 and F2 females, at around 50, 250 and 800 mg/kg bw/day, but was much higher during lactation (PND 0-21) at around 120, 615 and 1900 mg/kg bw/day.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
In the F0 females there were no treatment-related effects on estrous cycle length or on the percentage of females cycling or with an abnormal cycle. In the F1 generation, one low- and one mid-dose female was not cycling, and abnormal cycles were observed in 5, 2, 9 and 2 females in the control, low-, mid- and high-dose groups, respectively. Cycle length was equivalent across all dose groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 males showed no treatment-related effects on a range of sperm measures (% motile or progressively motile sperm, epididymal sperm concentration, % abnormal epididymal sperm, testicular homogenization-resistant spermatid head counts, and efficiency of daily sperm production). Daily sperm production per se was unaffected at the top dose, but significantly increased in the low- and mid-dose groups. High-dose F1 males showed significant decreases in % motile and progressively motile sperm and in epididymal sperm concentrations, but there were no treatment-related effects on testicular spermatid head concentration, daily sperm production, efficiency of daily sperm production and % abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the F0 generation there were no treatment-related effects on mating, fertility or pregnancy indices, or on total implantation sites per litter, % postimplantation loss per litter, or number of total, dead and live pups per litter at birth. In the F1 generation, male and female mating and fertility indices were significantly reduced at the top dose, as were the numbers of implantation sites per dam, live pups per litter and total pups per litter.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the F0 generation, relative liver and kidney weights were statistically significantly increased in high-dose males and females (as were relative kidney weights in mid-dose females), and relative ovarian and uterine weights were significantly reduced in high-dose females. In the F1 generation, relative uterine weight was increased in high-dose females, while high-dose males showed decreases in the absolute weights of the brain, testes, epididymides, prostate and seminal vesicles. Relative liver and kidney weights were unaffected at the top dose, but were increased at the mid-dose.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At scheduled sacrifice, there were no abnormal gross necropsy findings in the F0 males or females or the F1 females (other than alopecia in the mid- and high-dose females). F1 males (predominantly in the high-dose group) showed small or missing reproductive organs (testis, epididymis and/or seminal vesicles), hypospadias, and the presence of one or more nipples.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the F0 generation, treatment-related histopathological findings were seen only in the liver, with cytological alteration of the hepatocytes in high-dose males and females. This liver effect was also seen in the high-dose F1 females, but not in the F1 males. The only effects observed in the F1 males (high-dose only) were in the reproductive organs, with aspermia in the epididymis, chronic epididymal inflammation, atrophy of the seminiferous tubules and dilation of the rete testis duct.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
ca. 250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Effect level:
ca. 50 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Reduced anogenital distance at 250 mg/kg bw/day and above in F1 and F2 males, missing epididymides and retained nipples at 750 mg/kg bw/day in F2 males
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
There was no evidence of a treatment-related effect on F1 litter size, as the average number of live pups per litter on postnatal day (PND) 0 was 13.8, 12.2, 13.4 and 13.1 in the control, low-, mid- and high-dose groups respectively. The mean number throughout lactation was unaffected by treatment. F1 live litter sizes were also equivalent across all dose groups, except on PND 0 and 4 when they were statistically significantly reduced at the top dose. The F1 and F2 pup sex ratio (% male pups/litter) was unaffected at any dose for any interval (PND 0, 4, 7, 14 and 21). In F1 and F2 males, mean anogenital distance on PND 0 was significantly reduced in the mid- and high-dose groups (absolute or adjusted for individual body weights), while no effect was seen in F1 and F2 females.

CLINICAL SIGNS (OFFSPRING)
Clinical signs were observed occasionally in the F1 and F2 generations in all dose groups, but were not considered treatment-related. In the F2 generation, there were a number of pups found dead or missing and presumed dead on PND 0 and 4, but with no relation to treatment (28, 22, 17 and 13 in control, low-, mid- and high-dose groups, respectively).

BODY WEIGHT (OFFSPRING)
Mean F1 and F2 pup body weights per litter (both sexes) were significantly reduced at the top dose on PND 0 and 4 (F1 generation) and on PND 7, 14 and 21 (both generations).

SEXUAL MATURATION (OFFSPRING)
An assessment of sexual maturation during the F1 pre-mating exposure period in 30 females and 30 males per group revealed statistically significant delays in the acquisition of vaginal opening and preputial separation at the top dose (absolute or adjusted for body weight).

ORGAN WEIGHTS (OFFSPRING)
F2 weanlings (PND 21) in the top dose group showed statistically significant reductions in relative spleen weight and, in males, absolute testis weight.

GROSS PATHOLOGY (OFFSPRING)
Out of the 54 high-dose F2 male weanlings (PND 21), 20 had missing epididymides, five had missing or small seminal vesicles and 13 had between one and six retained nipples.

HISTOPATHOLOGY (OFFSPRING)
[See F1 parental generation effects described above.] No histopathological examination of F2 generation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a 2-generation reproductive toxicity study in which BBP was given in the diet of male and female rats at doses of around 0, 50, 250 or 750 mg/kg bw/day, the top dose (which induced systemic toxicity in the parents) had an adverse effect on reproduction in the F1 generation and caused growth impairment and delayed sexual maturation in the F1 and F2 offspring. Anogenital distance was reduced in F1 and F2 male pups from 250 mg/kg bw/day.
Executive summary:

In a reliable two-generation reproductive toxicity study, male and female rats received Santicizer 160 in the diet at 0, 50, 250 or 750 mg/kg bw/day for 10 weeks prior to mating and during the 2-week mating period. The males were then killed, while the females continued with the same treatment throughout gestation, lactation and weaning, until sacrifice on postnatal day (PND) 21. The F1 offspring (reduced to 10 pups/litter on PND 4) were reared by their dams to PND 21, at which point at least one pup/sex/litter was randomly selected to produce the second (F2) generation and continued under the same exposure regime as the parents.

Systemic toxicity was seen in all three generations at the top dose, which also produced reproductive toxicity. In the F0 generation this was limited to the females (with reductions in the relative weights of the ovary and uterus), while in the F1 generation both males and females showed a reduction in mating and fertility indices. In the F1 and F2 offspring, growth was impaired and sexual maturation delayed at 750 mg/kg bw/day, while anogenital distance (an indication of foetal androgen action) was reduced in F1 and F2 male pups from 250 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity was 250 mg/kg bw/day, while that for developmental toxicity was 50 mg/kg bw/day (based on the reduced anogenital distance in F1 and F2 male pups).