Tributyl phosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Exposure to tributyl phosphate in the diet for two generations in CD (Sprague-Dawley) rats resulted in consistent and persistent parental toxicity at 700 and 3000 ppm (and only transient and isolated adult and postnatal toxicity and histologic evidence of urinary bladder epithelial hyperplasia at 200 ppm). There was no effect of treatment on parental reproductive or offspring survival parameters at any doses tested. Reduced pup body weights per litter were observed du ring the lactation period at 3000 ppm in F1 litters, and at 3000 and occasionally at 700 ppm in F2 litters (with a single isolated observation in F2 litters at 200 ppm), associated with..and most probably caused by the matemal toxicity at these dietary dose levels.

A NOAEL for maternal toxicity was not established in this study because of the presence of transient and isolated adult toxicity and of adult urinary bladder hyperplasia at a low incidence, 0.0-7.1 %, at the low dose.

The NOAEL for reproductive toxicity (fertilityI was at least 3000 ppm (= 225 mg/kg bw) and the NOAEL for postnatal toxicity (reduced pup body weights) was at or below 200 ppm (=15 mg/kg bw).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4700 (Reproduction and Fertility Effects)

Principles of method if other than guideline:

Male and female weanling rats (F0 generation) were administered tributyl phosphate in the feed at 0, 200, 700 and 3000 ppm, 30 animals/sex/dose, for 10 weeks. Animals were then randomly mated within treatment groups for a 3 week mating period to produce the F1 generation, with exposure continued. F0 males were necropsied after the mating period, with histologic evaluation of reproductive and target tissues for high dose and control males. F1 litters were culled to 8 pups on postnatal day (pnd) 4 and weaned on pnd 21. At weaning, 10 weanlings/sex/dose were necropsied and 30/sex/dose were selected as F1 parents of the F2 generation. Selected F1 weanlings, 30 sex/sex/dose, were administerd tributyl phosphate in the diet prebreed exposure period and then mated for a 3 week mating period as described above. At weaning of F2 litters, 10 weanlings/sex/dose were necropsied.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Lab, Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: [yes/no] yes
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Housing: mostly individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-75°F
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: January 1991 To: Okober 1991

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

ad libitum via food

Details on mating procedure:

F0 (parent animals): 30 animals per sex per dose (at the age of about 8 weeks) were treated for 10 weeks followed by a 3 weeks mating period
F2: Selected F1 weanlings, 30/sex/dose were administered tributyl phosphate in the diet for a 11-week prebreed exposure period and and then mated for a 3 week mating period

The day vaignal sperm (or plug) were observed was designated gestational day 0 (GD0).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Attachment 1
Dosed feed formulations, encompassing the range of dosage concentrations employed in this
study, were homogeneous and stable for at least 28 days under refrigeration so formulations were
used within the stability limits established and were stored under refrigeration. All dietary
formulations were analyzed at 93.0-106% of target dietary concentrations prior to use. No tributyl
phosphate was detected in the vehicle control feed formulations, with an estimated detection limit of 3.6 ppm.

Duration of treatment / exposure:

F0 generation: at least 10 weeks (until they were appr. 18 weeks of age) plus mating period, gestation and lactation period
F1 generation: form birth via lactation 11 weeks (until they were approx. 14-17 weeks of age) plus mating period, gestation and lactation period.
F2 generation: during weaning

Frequency of treatment:

continuous feeding

Remarks:

Doses / Concentrations:
200, 700 and 3000 mg/kg diet (approx 15, 53 and 225 mg/kg bw/day)
Basis:

No. of animals per sex per dose:

30/sex/dose

Control animals:

yes

Positive control:

not adequate

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: initially and weekly through mating; body weights of female rats were recorded in the same manner until confirmation of matin. During gestation, females were weighed on GD 0, 7, 14 and 20. Dams producing litters were weighted on lactational days 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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Litter observations:

All pups not selected as parents or for necropsy were examined extemally, euthanized and discarded.

Postmortem examinations (parental animals):

All FO and F1 parental animals in all groups (both generations) were subjected to a complete
gross necropsy. The gross necropsy included e»amination of the extemal surfaces; all orifices;
cranial cavity; carcass; extemal and cut surfaces of the brain and spinal cord; the thoracic,
abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. All of the male
and female adults from the control and high dose groups in both generations were subjected to a
histopathologic examination. Sacrifice of the parental males occurred after the completion of the
mating period with Sponsor concurrence. Sacrifice of the matemal animals occurred after F1 or F2 litters had been weaned. Ten F1 and 10 F2 weanlings per sex per dose were also necropsied as
described above. All individual animal necropsy data are presented in Appendix II. Histopathologie evaluation was conducted on the following parental tissues from high dose and control groups: liver, kidneys (2), urinary bladder, pituitary, vagina, uterus, ovaries (2), testes (2), epididymides (2),
seminal vesicles (2) and prostate. In addition, male and female F0 and F1 urinary bladders, male
F0 and F1 kidneys and female F0 and F1 livers were evaluated microscopically for the mid and low dose groups since initial evaluation of high dose tissues indicated possible treatment-related lesions in these tissues. A complete gross necropsy and histopathologic examination was conducted for any parental animals dying on test.
The fixed (buffered neutral 10% formal in) uteri from any females of the F0 or F1 generations
failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy
status. This staining procedure does not interfere with subsequent histopathologic evaluation.

Statistics:

Both parametric and nonparametric statistical procedures were applied to selected measures
from this reproductive toxicity study. Parametric evaluations were as follows: Appropriate General
Linear Models (GLM) procedures (SAS Institute lnc., 1989a, 1989b, 1990a, 1990b, 1990c) for the
Analyses of Variance (ANOVA) were employed. Prior to GLM analysis, an arcsine-square root
transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967)
and Bartlett's test for homogeneity of variance (alpha level = 0.001) was performed on all data to be analyzed by ANOVA (Winer, 1962). GLM analyses were used to determine whether significant
dose effects had occurred for selected measures (ANOVA). When a significant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test (Dun nett, 1955; 1964) was used to compare each chemical exposed group to the vehicle control group for that measure. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons except that a two-tailed test was used for adult body weight parameters, matemal food consumption, pup body weight, and percent males per litter. Nominal scale measures were analyzed by Chi-Square Test for lndependence for differences among treatment groups. When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher's Exact Probability Test was used for pairwise comparisons between each treated group and the vehicle control group.
Nonparametric data were statistically evaluated using the Kruskal-Wallis one-way analysis of
variance by ranks (Siegel, 1956) to test for differences among dose groups. Whenever the result of a Kruskal-Wallis test was significant (p<0.05), the Mann-Whitney U test was used to make individual comparisons between vehicle and chemical dose groups for that measure (Siegel, 1956).

Reproductive indices:

mating index and fertility index for males and females

Offspring viability indices:

gestational index, live birth index, 4, 7, 14, and 21-day survival index, lactation index

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 males: When the data were expressed as g/kg/day, the values for all weeks in all groups were statistically equivalent.
F0 females: F0 female body weights were equivalent across all groups at the start of the study (prebreed day 0). Beginning on week 1 (prebreed day 7) and continuing through the 10-week prebreed exposure period for all weekly weighings, the mean weights at 3000 ppm were significantly lower.
Weights at 200 ppm (but not at 700 ppm) were also significantly lower at weeks 1, 2, 5 and 9.
Weekly body weights of females remaining sperm-negative during the three-week mating period,
with diminishing numberS of females over time, were statistically equivalent across all groups,
although the mean values at 3000 ppm were clear1y reduced. F0 female weekly body weight
changes exhibited only one statistically significant reduction at 3000 ppm for week 1 (days 0-7). F0
female food consumption values during the 10-week prebreed dosing period, expressed as g/day,
were significantly reduced at 3000 ppm for ~eek 4 (days 21-28) through week 1 0 (days 63-70). In
addition, the value at 700 ppm was significantly reduced for week 6 (days 35-42) and the value at 200 ppm was significantly reduced for week 9 (days 56-63). When the data were expressed on
g/kg/day, matemal food consumption values were equivalent across all groups for all intervals
evaluated. Tributyl phosphate intake (as mg/kg/day) exhibited the same incremental increases
across dosed feed groups (3.5x from 200 to 700 ppm and 4.4x from 700 to 3000 ppm) and the
same decreases with increasing age (and weight) within dosed feed groups as did the F0 males.

Maternal gestational body weights (F0 females for F1 litters): Matemal gestational body weights were statistically significantly reduced at 3000 ppm for all timepoints evaluated; gestational weight change (gd 0-20) was also significantly reduced at 3000 ppm. Matemal gestational food consumption, expressed as g/day, was significantly reduced at 3000 ppm for gd 0-7; when expressed as g/kg/day, food consumption was significantly reduced at 3000 ppm for gd 14-20 and for gd 0-20. Matemal intake of tributyl phosphate was approximately 13, 47 and 214 mg/kg/day at 200, 700 and 3000 ppm, respectively.

Maternal lactational body weights (F0 females for F1 litters): Matemal lactational body weights were statistically significantly reduced at 3000 ppm for all timepoints evaluated. Lactational weight change (postnatal days 0-21) were statistically equivalent across all groups, but with an apparent dose-related downward trend. Matemal lactational food consumption, expressed as g/day, was equivalent across all groups for all timepoints evaluated.
When the data were expressed as g/kg/day, matemal food consumption was significantly increased at 3000 ppm for pnd 4-7 and 14-21. Matemal intake of tributyl phosphate was 26, 95 and 404 mg/kg/day at 200, 700 and 3000 ppm, respectively

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F0 males: Tributyl Phosphate intake, expressed as mg/kg/day, exhibited the expected incremental
increases across dose groups, averaging approximately 13 mg/kg/day at 200 ppm, 46 mg/kg/day at 700 ppm and 194 mg/kg/day at 3000 ppm, increasing approximately 3.Sx from 200 to 700 ppm and 4.2x from 700 to 3000 ppm. The tributyl phosphate intake values also exhibited the expected
decreases within each dosed group over time. The test chemical intake ranged from 18.5, 64.7 and 263.6 mg/kg/day for the 200, 700 and 3000 ppm groups during the first week of the prebreed, to 10.6, 37.9 and 160.0 mg/kg/day for the last week of the prebreed. These decreases within groups are due to the food consumption in g/day remaining relatively constant, and the body weights increasing markedly over time. Therefore, the food consumption in g/kg/day (the basis for the test chemical intake calculations) decreased over time within each group.

F0 females: The test chemical intake within groups ranged from 17.3, 60.3 and 266.2 mg/kg/day at 200, 700 and 3000 ppm during the first week of the prebreed, to 12.2, 41.2 and 177.8 mg/kg/day at 200, 700 and 3000 ppm during the last week of the prebreed. Average intakes of test chemical were approximately 14, 49 and 209 mg/kg/day at 200, 700 and 3000 ppm, respectively

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

F0 males: Histopathologie evaluation of selected tissues from F0 males indicated clear treatment-related effects on the urinary bladder at 200 ppm (one of 30 males), 700 ppm (22 of 29 males) and at 3000 ppm (30 of 30 males) expressed as epithelial hyperplasia. Two males (out of 30, 6.7%) at 3000 ppm also exhibited renal pelvis epithelial hyperplasia, with no males in any other groups exhibiting this finding. Epididymides, liver, pituitary, prostate, seminal vesicles and testes exhibited no treatment-related lesions (see Table 8, Attachment)


F0 females: Histopathologie evaluation of selected tissues from F0 females indicated hepatic lesions at 700 and 3000 ppm, presenting as centrilobular hypertrophy in three of 30 females at 700 ppm and 28 of 30 at 3000 ppm. Urinary bladders also exhibited treatment related lesions, epithelial hyperplasia, with none at 0 ppm, two of 30 at 200 ppm, 21 of 30 at 700 ppm and 30 of 30 at 3000 ppm. F0 female kidneys, ovaries, pituitary, uterus and vagina exhibited no treatment-related lesions (see Table 18, Attachment)

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive and lactational indices for F0 animals producing F1 litters are presented in
Table 13 (see Attachment). F0 male and female mating and fertility indices were equivalent across all groups.
Gestational index, gestational length in days, number of implantation sites per dam, and number of
total, live and dead pups per litter on postnatal day 0 were all equivalent across all groups. The
numbers of litters on pnd 0 were 24, 27, 25 and 27 at 0, 200, 700 and 3000 ppm, respectively.
Prenatal mortality index and stillbirth index were statistically equivalent across all groups; there was
an apparent dose-related downward trend due to the demise of one dam at 0 ppm on gd 24 with a
full-term litter in utero. Live birth index exhibited an apparent dose-related upward trend, also due to
the dam which died on gd 24 at 0 ppm. Day 4, 7, 14, and 21 survival indices and lactation index
were equivalent across all groups.

Litter sizes and sex ratio (% males per litter) on postnatal days 0, 1, 4, 7, 14 and 21 were all
statistically equivalent across all groups.

Dose descriptor:

LOAEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: target organ: bladder (epitherial hyperplasia)

Dose descriptor:

NOAEL

Effect level:

>= 3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: The NOAEL for reproductive toxicity was at least 3000 ppm (= 225 mg/kg bw)

Critical effects observed:

no

System:

other: reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

200

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations of F1 males during the prebreed and mating periods were limited to
occasional reporting of rough coat, sores, thin fur, alopecia, and piloerection in all groups with no
treatment- or dose-related incidence or severity.

Clinical observations of F1 females during the prebreed dosing period included occasional
findings of sores, inflamed ear at the site of the ear tag, chromodacryorrhea, alopecia, thin fur, and
rust-colored fur in a few animals in all groups. As reported previously, one female died at 700 ppm
on day 99. There were no treatment- or dose-related findings.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male each at 0 and 200 ppm died prior to the prebreed exposure period. At 700 ppm, one
female died prior to the prebreed exposure period and one female died on lactational day 0 having
delivered one live pup, with 11 full term fetuses in utero.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 male weekly body weights were statistically significantly reduced at 3000 ppm for the
entire 11-week (77 day) prebreed dosing period. During the three-week mating period (days 77-98),
weights were also significantly reduced at 3000 ppm for the entire period. Weekly F1 male body
weight changes were significantly reduced at 3000 ppm for week 1 (days 0-7) and were statistically equivalent across all groups for all subsequent weeks through week 14 (days 91-98). (see Table 19, Attachment 4).

F1 female body weights during the 11-week prebreed dosing period were consistently and
significantly reduced at 3000 ppm for the entire eleven weeks. At 700 ppm, mean body weights
were significantly reduced beginning on week 4 (day 28) and continuing throughout the 11-week
period. Statistical analyses of body weights du ring the mating period (for females not yet found
sperm-positive) were not reported due to the decreasing numbers of females included, as more
females became sperm-positive and were weighed and recorded based on gestational days,
although the means at 3000 ppm were lower. F1 female weekly body weight changes were
equivalent across all groups for all eleven weeks except for a significant decrease at 700 ppm for
week 3 (days 14-21) and a significant decrease at 3000 ppm for week 5 (days 28-35) (see TAble 22 in Attachment 4).

Gestation - F1 females for F2 litters: F1 matemal gestational body weights were significantly reduced at 3000 ppm for gd 0, 7, 14 and 20, and at 700 ppm for gd 0, 7 and 14; matemal gestational weight change (gd 0-20) was statistically equivalent across groups.

Lactation - F1 females for F2 litters: Matemal F1 body weights during lactation exhibited significant reductions at 3000 ppm on postnatal day 0, 4, 7, 14 and 21, significant reductions at 700 ppm on postnatal days 0, 4 and 7, and a significant reduction at 200 ppm on postnatal days 7 and 14. Matemal lactational weight change (postnatal days 0-21) exhibited no significant ditterences among groups. Matemal F1 lactational food consumption, expressed as g/day, exhibited significant reductions at 3000 ppm for all intervals evaluated throughout the lactational period, except for postnatal days 4-7. Matemal F1 lactational food consumption, expressed as g/kg/day, was significantly increased at 3000 ppm for postnatal days 4-7, 14-21 and 0-21.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 males: Food consumption, expressed as g/day, was significantly reduced at 3000 ppm for the entire 11 week period. When food consumption data were expressed as g/kg/day, food consumption was significantly increased at 3000 ppm for weeks 6 (days 35-42), week 7 (days 42-49), 8 (days 49-56), 10 (days 63-70), and 11 (days 70-77). Tributyl phosphate intake, in mg/kg/day, exhibited the expected incremental increases across dose groups, averaging approximately 14, 49 and 225
mg/kg/day at 200, 700 and 3000 ppm, respectively. Test chemical intake also exhibited the
expected decreases within each dosed group over time as the animals gained weight, with values
from all three dosed groups dropping approximately 50% over the 11-week period. The test
chemical intake ranged from 21.4, 72.4 and 327.8 mg/kg/day at 200, 700 and 3000 ppm for the first week, to 10.3, 36.4 and 166.3 mg/kg/day for the last week of the pre-breed period. (see Table 20 in Attachment).

F1 female food consumption, expressed as g/day, was significantly reduced at 3000 ppm
for all 11 prebreed dosing weeks. At 700 ppm, food consumption was significantly reduced for week 5 (days 28-35) and 6 (days 35-42) (Table 22, Attachment 4). When food consumption data were expressed as g/kg/day, values for all groups were statistically equivalent for all 11 weeks. Tributyl phosphate intake, calculated as mg/kg/day, exhibited the expected incremental increases across dosed groups, averaging approximately 16, 55 and 239 mg/kg/day at 200, 700 and 3000 ppm. The intake values also exhibited the expected declines within groups over time as the F1 females gained weight over the 11-week period. Test chemical intake ranged from 20.6, 70.2 and 314.0 mg/kg/day during the first week, to 12.6, 44.0 and 193.4 mg/kg/day during the last week of the prebreed period (see TAble 22, Attachment).

Gestation - F1 females for F2 litters: Maternal food consumption during gestation, expressed as
g/day, exhibited a significant reduction at 3000 ppm for gd 0-7, and for the gestation period, gd 0-20. When the data were expressed as g/kg/day, there was a significant reduction at 3000 ppm for
gd 7-14, 14-20 and for gd 0-20. Maternal intake of tributyl phosphate exhibited the expected
incremental increases across dose groups, averaging approximately 13, 45 and 217 mg/kg/day at
200, 700 and 3000 ppm.

Lactation - F1 females for F2 litters: Matemal intake of tributyl phosphate, expressed as mg/kg/day, exhibited the expected incremental increases across dosed feed groups, with average intakes for the lactational period of 31, 107 and 502 mg/kg/day at 200, 700 and 3000 ppm, respectively.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy findings in F1 males are presented in Table 24 (Attachment 5). There were no
treatment- or dose-related findings. One male each at O and 200 ppm died prior to scheduled
sacrifice.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

F1 males: Histopathologie evaluation of selected tissues from F1 males indicated treatment-related and dose-related lesions in the kidneys and urinary bladder. The lesion in the urinary bladder was diagnosed as epithelial hyperplasia and was observed in no animals at O ppm, two of 28 at 200
ppm, 16 of 30 at 700 ppm and 30 of 30 at 3000 ppm. The lesion in the kidneys was diagnosed as
renal pelvic epithelial hyperplasia, observed only at 3000 ppm (10 of 30 males, 33.3%). The
epididymides, liver, pituitary, prostate, seminal vesicles and testes exhibited no treatment or doserelated lesions (see Table 25, Attachment).

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive and lactational indices for F1 parents and F2 litters exhibited no treatmentrelated
changes, including no changes in mating index, fertility index (either sex), gestational index,
gestational length in days, number of implantation sites per litter, number of total, live or dead pups on postnatal day 0, prenatal mortality index of F2 offspring, stillbirth index, live birth index, 4, 7, 14 and 21 survival indices or in lactational index. The numbers of litters on postnatal day 0 were 25, 21 20 and 26 at 0, 200, 700 and 3000 ppm, respectively

Dose descriptor:

NOAEL

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

LOAEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other:

Remarks on result:

other: Bladder epithelial hyperplasia

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Critical effects observed:

no

Organ:

other: reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical observations of F1 pups during the lactation period indicated no treatment- or doserelated
findings. All findings were limited to the 0 ppm group: one (1) cyanotic female pup was found o
one (1) female was found with ectrodactyly and a shortened forelimb, and one (1) female was found with small tail on day 0. One male pup at 0 ppm exhibited a hematoma on the right hindlimb on pnd 0 (see Table 15 in Attachment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights per litter were significantly reduced at 3000 ppm on pnd 1 and 7 (all pups and females, but not males), and on 14 and 21 (all pups, and males and females separately) (see Table 14 in Attachment).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy findings of F1 pups which died during the lactation period indicated no
apparent treatment-related findings; most deaths occurred during the first four days of life, many
with patent ductus arteriosus, indicating primary atelectasis (defective expansion of the pulmonary alveoli at birth), and no milk in stomach (see Table 16 in Attachment).

Gross necropsy observations for F1 females indicated no findings that were treatment or dose
related. Findings of the one female at 700 ppm which died during delivery with fetuses in utero are
also included.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathologie evaluation of selected tissues from F1 females indicated only hepatic and
urinary bladder findings as treatment related. In the liver, the lesion described as centrilobular
hypertrophy, was observed in no females at 0 or 200 ppm (out of 30 females examined in each
group), in one of 30 at 700 ppm andin 25 of 30 at 3000 ppm. In the urinary bladder, the lesion was
described as epithelial hyperplasia. lt was observed in no females at 0 or 200 ppm, in 21 of 30 at 700 ppm and in 30 of 30 at 3000 ppm. There were no treatment-related histopathologic findings in
F1 female kidneys, ovaries, pituitaries, uteri or vaginas (see Table 35 in Attachment).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive performance

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weight

Critical effects observed:

no

Organ:

other: reproductive performance

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2 litter sizes and sex ratio (% male pups per litter) were all equivalent across groups for all
timepoints evaluated, on postnatal days 0, 1, 4, 7, 14 and 21. Pup body weights per litter (all pups
or males and females separately) were significantly reduced at 3000 ppm throughout the lactation
period beginning on postnatal day 1. Pup body weights per litter were significantly reduced at 700
ppm for pnd 1 and 21 (all pups or separately by sex); and pup weights (all pups and males only, but not females) per litter were significantly reduced at 200 ppm on pnd 14 only (see Table 31 in Attachment).

Gross pathological findings:

no effects observed

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weight

Reproductive effects observed:

no

with 700 and 3000 ppm reductions of body weights, weight
gain and food consumption during F0 and F1 prebreed dosing
periods, no signs of toxicity, no treatment related
mortality; with 200 ppm only transient effects on body
weight and food consumption. No NOAEL for adult toxicity. 
No evidence of reproductive organ histopathology at any
dose, no effect on pre- and postnatal mortality. NOAEL for
reproductive toxicity (reduced pup body weights) was at or

below 200 ppm.

Executive summary:

Exposure to tributyl phosphate in the diet for two generations in CD (Sprague-Dawley) rats resulted in consistent and persistent parental toxicity at 700 and 3000 ppm (and only transient and isolated adult and postnatal toxicity and histologic evidence of urinary bladder epithelial hyperplasia at 200 ppm). There was no effect of treatment on parental reproductive or offspring survival parameters at any doses tested. Reduced pup body weights per litter were observed du ring the lactation period at 3000 ppm in F1 litters, and at 3000 and occasionally at 700 ppm in F2 litters (with a single isolated observation in F2 litters at 200 ppm), associated with..and most probably caused by the matemal toxicity at these dietary dose levels. An adult toxicity 'no observable adverse effect level" (NOAEL) was not established in this study because of the presence of transient and isolated adult toxicity and of adult urinary bladder hyperplasia at a low incidence, 0.0-7.1 %, at the low dose.

The NOAEL for reproductive toxicity was at least 3000 ppm (= 225 mg/kg bw) and the NOAEL for postnatal toxicity (reduced pup body weights) was at or below 200 ppm (=15 mg/kg bw).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a valid 2-generation study male and female weanling rats (F0 generation) were administered tributyl phosphate in the feed at 0, 200, 700 and 3000 ppm, 30 animals/sex/dose, for 10 weeks. Animals were then randomly mated within treatment groups for a 3 week mating period to produce the F1 generation, with exposure continued. F0 males were necropsied after the mating period, with histologic evaluation of reproductive and target tissues for high dose and control males. F1 litters were culled to 8 pups on postnatal day (pnd) 4 and weaned on pnd 21. At weaning, 10 weanlings/sex/dose were necropsied and 30/sex/dose were selected as F1 parents of the F2 generation. Selected F1 weanlings, 30 sex/sex/dose, were administered tributyl phosphate in the diet prebreed exposure period and then mated for a 3 week mating period as described above. At weaning of F2 litters, 10 weanlings/sex/dose were necropsied.

The exposure resulted in consistent and persistent parental toxicity at 700 and 3000 ppm (and only transient and isolated adult and postnatal toxicity and histologic evidence of urinary bladder epithelial hyperplasia at 200 ppm). There was no effect of treatment on parental reproductive or offspring survival parameters at any doses tested. Reduced pup body weights per litter were observed du ring the lactation period at 3000 ppm in F1 litters, and at 3000 and occasionally at 700 ppm in F2 litters (with a single isolated observation in F2 litters at 200 ppm), associated with..and most probably caused by the matemal toxicity at these dietary dose levels. An adult toxicity 'no observable adverse effect level" (NOAEL) was not established in this study because of the presence of transient and isolated adult toxicity and of adult urinary bladder hyperplasia at a low incidence, 0.0-7.1 %, at the low dose.

The NOAEL for reproductive toxicity was at least 3000 ppm (= 225 mg/kg bw) and the NOAEL for postnatal toxicity (reduced pup body weights) was at or below 200 ppm (=15 mg/kg bw).

Effects on developmental toxicity

Description of key information

Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation.

Tributyl phosphate was administrated by gastric intubation to mated New Zealand White rabbits (18 mated females/group) at dose levels of 50, 150 and 400 mg/kg/day during the day 6-18 gestation period. On day 30 of gestation, surviving females were sacrificed, given a postmortem evaluation at which time gravid uterus and liver were weighed and corpora lutea/uterine implantation data recorded. Fetuses recovered at this time were weighed and evaluated for external and visceral malformations/variations

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Tributyl phosphate was administrated by gastric intubation to mated New Zealand White rabbits (18 mated females/group) at dose levels of 50, 150 and 400 mg/kg/day during the day 6-18 gestation period. Included in this study was a vehicle-treated control group. During gestation, body weights and food consumption were recorded.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products Inc. Denver Pennlylvania
- Weight at study initiation: 4019 g mean
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 75 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 58-74°F
- Humidity (%): 30-67%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1990

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity analyses for the batch sizes used in this study were
performed at the low- and high-concentration levels (50 and 400 mg/ml,
respectively). The mean percents of nominal± standard deviation (S.O.) for
this set of six samples at the low- and high-concentration 1eve1s were 103 ±
1.03% and 102 ± 1.22%, respective1y. On the basis of these data, dosing
solutions at these concentration levels as prepared were considered homogeneous.
Analyses of dosing solutions used for the study demonstrated a mean
percent of nominal ± S.O. of 106 ± 3.21% for Group II, 103 ± Q.84% for Group III
and 105 ± 2.74% for Group IV.

Details on mating procedure:

each female selected for mating was paired with 2 males.

Duration of treatment / exposure:

day 6 to 18 of gestation

Frequency of treatment:

once daily

Duration of test:

sacrifice on day 30 of gestation

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

18 pregnant females per dose

Control animals:

yes

Details on study design:

Sex: female
Duration of test: up to day 30 of gestation

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 12, 15, 19, 24, and 30 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined:
Complete gross postmortem examinations
were perfonned on all mated rabbits
including those dying spontaneously
during the study. Only abnormal tissues
were saved (10% neutral buffered
fonnalin). Females showing signs of
abortion (passing of placental/fetal
tissue prior to Day 26 of gestation) or
premature delivery (presence of
fetuses/pups at Day 26 of gestation or
later) were killed via intravenous
injection of sodium pentobarbital in the
marginal ear vein on the day such
evidente was observed. Reproductive
systems were examined. Fetuses/
delivered pups recovered during the Day
26-30 gestation interval were evaluated
for external malfonnations, eviscerated
and processed for staining of the
skeletal structures with Alizarin Red S.
These stained specimens were evaluated
for ske1etal malfonnations only. Intact
fetuses recovered from females aborting
their pregnancies were eva1uated for
external malfonnations and discarded.
Liver weights were also recorded for
females surviving to Day 30 gestation
sacrifice.

Ovaries and uterine content:

The intact uterus (ovaries attached) was
removed from the abdominal cavity,
weighed and the number and location of
the following were recorded for each
uterine horn:
live fetuses
dead fetuses (no evidence of tissue degeneration)
late resorptions {recognizable dead fetus undergoing degeneration
regardless of size) early resorptions (evidence of
implantation but no recognizable
fetus) implantation sites
The ovaries were dissected free from the
uterus and evaluated for the presence
and number of corpora lutea.
When no uterine implants were grossly
apparent, the uterus was stained with
ammonium sulfide (method of Salewski). Foci visualized
following this staining procedure were
counted and females with such foci were
included as pregnant in the calculation
of pregnancy rate. The numbers of foci
were not included in the calculation of
uterine implantation data. When no foci
were visualized post-staining, the
female was considered not pregnant.

Fetal examinations:

All fetuses were given a gross examination for external malformations/variations to include observation for palatal defects. Subsequently, each fetus was weighed and tagged
individually for identification.
All fetuses were examined for visceral and skeletal malformations.

Statistics:

Data were analyzed between control (Group I) and treatment groups (Groups
II-IV}. Statistically significant differences between the control and treatment group data are presented in summary and mean tables of the appendices.

Clinical signs:

no effects observed

Description (incidence and severity):

Findings noted during the physical examinations of the females in
the treatment groups occurred at low incidence or with comparable frequency as
in the control group and no adverse effects of treatment at doses up to and
including 400 mg/kg/day were evident.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality occurred in the control (Group I) or treatment Groups
II and III.
In Group IV, two femates died (mortality rate= 10.5%, Appendix G).
The death of one of these females (No. 4503) was not considered tobe related to
treatment. This female which died on Day 8 of gestation, was difficult to
handle at the time of dosing and was set aside tobe dosed later. However, she
was found dead prior to the second attempt to administer the dose. The death of
this female was attributed to injuries/complications incurred while handling to
administer the dose. The other death in Group IV was female No. 4505. This
female, which was found dead on Day 28 of gestation, had lost 441 grams during
the Oay 6-19 gestation interval and 188 grams during the Day 19-24 posttreatment
gestation interval. This female had also experienced a reduction in
food consumption from Day 10 of gestation to Day 24 (the last recording
interval). At gross postmortem examination, tesions of the tungs (edema) and
bronchi (presence of dark red fluid) were noted and 10 fetuses were recovered in
utero; however, the cause of death could not be determined from these findings.
Thus, only at the 400 mg/kg/day dose level was a low incidence of
maternat mortality encountered. (see Table G in Attachment)

Description (incidence and severity):

Mean body weights for each recording interval during gestation
(Days 0, 6, 9, 12, 15, 19, 24 and 30) were comparable between the control and
each treatment group (Table C in Attachment).
No adverse effect of treatment at doses up to and including
150 mg/kg/day was evident from maternal weight data or weight change data during
gestation. At the 400 mg/kg/day dose level, maternal toxicity was suggested
from the weight losses experienced during the several weighing intervals of the
treatment period, albeit, only for the Oay 6-9 gestation interval was this
difference from control data statistically significant.

Description (incidence and severity):

no adverse effect of treatment at doses up to and including
150 mg/kg/day was evident from gestation food consumption data. At the 400
mg/kg/day dose level, a slight reduction in food consumption seen over the Oay
7-14 gestation interval was considered suggestive of a treatment-related
response.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no adverse effect of treatment at doses up to and including
400 mg/kg/day was evident from liver weight data. (see Table J-2 in Attachment)

Gross pathological findings:

no effects observed

Description (incidence and severity):

Tan/red foci in the lungs were observed in some animals from all
groups with a slightly greater incidence of red foci seen in the treatment
animals. The toxicological significance of this finding on the basis of gross
examination only is unclear.
Other postmortem findings observed grossly occurred sporadically
and were not considered tobe related to the test article.

Description (incidence and severity):

Pregnancy rates were 88.9% for the control and treatment Groups II
and III and 100.0% for Group IV.
No adverse effect of treatment at doses up to and including 400
mg/kg/day was evident from pregnancy rates. (see Table G-1 in Attachment)

Number of abortions:

no effects observed

Description (incidence and severity):

The incidences of females that aborted or delivered prematurely for
the control group and treatment Groups II, III and IV were 6.3% (1/16), 12.5%
(2/16), 0% (16 pregnancies) and 5.9% {1/17) respectively. These incidences
were considered comparable between the control and each treatment group.
Thus, no adverse effect of treatment at doses up to and including
400 mg/kg/day was evident from abortion or premature delivery data. (see Table G-1 in Attachment)

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

no adverse effect of treatment at doses up to and including
150 mg/kg/day was evident from uterine implantation data. At the 400 mg/kg/day
dose level, the mean number of resorptions and the mean resorption/implant ratio
were increased sufficiently to be suggestive of a treatment-related response. (see Table G-1 in Attachment)

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

no adverse effect of treatment at doses up to and including
400 mg/kg/day was evident from fetal weight data. (see Table G-1 in Attachment)

Changes in sex ratio:

no effects observed

Description (incidence and severity):

up to and including the 400 mg/kg/day dose level, no adverse
effect of treatment was evident from fetal sex distribution data. (see Table G-1 in Attachment)

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were seen in the 124 control fetuses
(15 litters), 117 Group II fetuses {14 litters} or 134 Group III fetuses (16
litters) recovered and evaluated at Day 30 gestation maternal sacrifice. In
Group IV, the incidence of fetuses with external malformations was 2.6% (3/117
fetuses). The incidence of litters containing fetuses with external
malformations for Group IV was 12.5% (2/16 litters). These incidences of
external malformations for treatment Group IV on both a per fetus and per litter
basis, did not differ statistically from the concurrent control data.
no clear adverse effect of treatment at doses up to and
including 400 mg/kg/day was evident from fetal external malformation data. (see Table L-1 in Attachment)

Description (incidence and severity):

The incidences of fetuses with skeletal malfonnations for the
control and treatment Groups II, III and IV were 3.2% (4/124 fetuses), 6.0%
(7/117 fetuses), 0.8% (1/125 fetuses) and 4.3% (5/117 fetuses), respectively.
The incidences of litters containing fetuses with skeletal malfonnations for
these same groups were 26.7% (4/15 litters), 50.0% (7/14 litters), 6.7% (1/15
litters) and 25.0% (4/16 litters), respectively. These incidences on aper
fetus and per litter basis did not differ statistically between the control and
treatment groups and were considered comparable between these same groups.
no adverse effect of treatment at doses up to and
including 400 mg/kg/day was evident from fetal skeletal malformation data. (see Table N-1 in Attachment)

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were seen in the 124 control fetuses
(15 litters), 116 Group II fetuses (14 litters) or 134 Group III fetuses (16
litters) evaluated. In Group IV, the incidence of fetuses with visceral
11alfonoations was 1.7% {2/117) and the incidence of litters containing affected
fetuses was 6.3% (1/16 litters}. These incidences on both aper fetus and per
litter basis, did not differ statistically from control data.
The only visceral malformation seen was distention of the
lateral ventricle of the brain. This malfonnation was seen in the two fetuses
from a single Group IV litter noted externally with domed cranium. In the
absence of other visceral malfonnations among the Group IV fetuses, the
relatively low incidence of this one finding was not considered indicative of a
treatment-related response. (see Table M-1 in Attachment)

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

at the 400 mg/kg/day dose level maternal toxic effect
(mean weight loss) and not statistically significant
increase of resorptions, no fetotoxic or teratogenic
effects, at the 50 and 150 mg/kg/day dose level no maternal
toxicity, no embryotoxic, fetotoxic or teratogenic
effects.

Executive summary:

Under the conditions of this study, tributyl phosphate mixed in corn oil and administered by gastric intubation to pregnant New Zealand White rabbits over the Day 6-18 gestation interval was not maternally toxic, embryotoxic, fetotoxic or teratogenic at dose levels of 50 and 150 mg/kg/day.
At the 400 mg/kg/day dose level, one female died late in gestation (Day 28) but the cause of death could not be determined on the basis of gross postmortem observations. The death of a second female in this group was attributed to injuries incurred while handling to administer the dose. Maternal toxicity at the 400 mg/kg/day dose level was indicated from mean weight loss experienced during the Day 6-9 gestation interval; this difference in comparison to the mean weight gain experienced by the concurrent control group during this interval was statistically significant. Other changes seen at the 400 mg/kg/day dose level which did not differ statistically from control data but which were considered suggestive of a maternal toxic response were as follows: mean weight losses during several intervals of the treatment period (Days 9-12 and 12-15) in comparison to mean weight gains for the control; mean weight loss over the entire Day 6-19 gestation interval in comparison to a mean weight gain for the control group; and reduced levels of food consumption over the Day 7-14 gestation interval. At Day 30 of gestation, the mean number of resorptions per pregnant female and the mean resorption/implant ratio for the 400 mg/kg/day dose level were increased in cornparison to concurrent control and recent historical control data for this laboratory, and while these differences were not statistically significant, an embryotoxic response rnay be suggested. The 400 mg/kg/day dose level was not considered tobe fetotoxic or teratogenic.

Thus, the no-observable-adverse-effect level (NOAEL) for developmental toxicity of tributy phosphate in the rabbit for this study was 150 mg/kg/day.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Lab, Kingston, NY
- Age at study initiation: females 64 days
- Weight at study initiation: 241 g
- Fasting period before study:
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67-74 °F
- Humidity (%): 31-68%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1990

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dosing volume: 5 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

homogeneity of the dosing solutions was determined in the low- and high-dose concentration levels.
Stability of the test item in corn oil at concentration levels of 16 and 750 mg/mL for a 10-day storage interval was verified.
Concentration in dosing solutions was determined.

Homogeneity analyses for the batch sizes used in this study were
performed at the low- and high-concentration levels (Groups II and IV). The
mean analytical values, expressed as a percent of nominal concentrations ±
standard deviation (S.O.) for this set of six samples at the low- and highconcentration
Jevels, were 106 ± 2.25% and 104 ± 1.97%, respectively.
Analyses of dosing solutions used for the study indicated mean values
(percent of nominal± S.O.) of 104 ± 2.43% for Group II, 103 ± 1.94% for Group
III and 100 ± 3.62% for Group IV.

Details on mating procedure:

Females were placed with male rats nightly in a 1:1 ratio. Vaginal smears were taken early in the morning following intervals of nightly co-habitation and females were considered to have mated if sperm was noted at microscopic evaluation or a vaginal plug was observed.

Duration of treatment / exposure:

day 6 to 15 of gestation

Frequency of treatment:

once daily

Duration of test:

sacrifice on day 20 of gestation

Dose / conc.:

750 mg/kg bw/day

Dose / conc.:

375 mg/kg bw/day

Dose / conc.:

188 mg/kg bw/day

No. of animals per sex per dose:

24 pregnant females per dose and control

Control animals:

yes

Details on study design:

Sex: female
Duration of test: up to day 20 of gestation

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 8, 12, 16, and 20 of gestation. Day 20 bw are presented as actual and corrected (minis gravid uterus weight)

FOOD CONSUMPTION : Yes
- recorded for days 0-6, 6-11, 11-16, and 16-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Complete gross postmortem examinations were performed on all mated females
including those dying spontaneously or killed in a moribund condition.
External surface, all orifices, the cranial cavity, carcass, the external
surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and
paranasal sinuses, the thoracic, abdominal and pelvic cavities and their
viscera and the cervical tissues and organs were examined for all animals.
The carcass of each female was discarded at completion of the gross postmortem
evaluation.

Liver weights were also recorded for all females sacrificed on day 20 of gestation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
When no uterine implants were grossly apparent, the uterus was stained with
alllllOnium sulfide (method of Salewski). Foci visualized following this staining
procedure were counted and females with such foci were included as pregnant in
the calculation of pregnancy rate. The numbers of foci were not included in the
calculation of uterine implantation data. When no foci were visualized
post-staining, the female was considered not pregnant.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: yes

Statistics:

Data were analyzed between control and treatment groups. Statistically significant differences between the control and treatment goup data are presented in summary and mean tables.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at all dose levels evaluated, maternal toxicity was evident
from physical observation data. These effects (A-G Stains, staining of the skin/fur in the abdominal area and wetness of the skin/fur in the ventral
abdominal region) were most prevalent among animals at the 375 and 750 mg/kg/day
dose levels.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality occurred in the control group (Group I) or treatment Groups II and III (188 and 375 mg/kg/day, respectively). In Group IV (750 mg/kg/day), the maternal mortality rate was 29.2% (7/24 females) and this difference from the control incidence was statistically significant. In Group IV, six females (Animal Nos. 4504, 4516, 4517, 4518, 4520 and 4523)
died during the Oay 6-15 treatment interval of gestation after having received
five to nine days of treatment. Group IV female No. 4512 died on Day 17 of
gestation having completed the entire 10-day dosing regimen. All the Group IV
animals that died experienced weight loss during the Day 6-9 gestation interval.
During the physical evaluations, these same animals were noted with observations
suggestive of toxicity, i.e., ventral surface wet with yellow/brown staining
and/or staining of the skin/fur in the ano-genital area (A-G Stains), and these
findings were noted soon after treatment initiated. Ouring the gross postmortem
evaluations, only one of these animals (No. 4520) had evidence of an intubation
injury, i.e., perforation of the trachea. Therefore, of the seven deaths encountered at the 750 mg/kg/day dose level, six were considered to be directly related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

over the entire treatment period encompassed by the Day 6-16
gestation interval, mean weight gain for each of the treatment groups was lower
than control, and these differences were statistically significant and
demonstrated a dose-response relationship (see Table D-1 in Attachment 1)
Mean corrected Day 20 gestation weights (Appendix E-1) were lower
than control in each of the treated groups and these differences (Group
II, -4.9%; Group III, -5.6%; and Group IV, -11.9%) were statistically
significant. Mean weight gains over the Oay 6-20 gestation interval using the
corrected Day 20 gestation weights were lower than control for each of the
treatment groups and these differences (Group II, -25.2%; Group III, -43.1%; and
Group IV, -86.8%) were also statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption data during the pre-treatment (Days 0-6),
treatment (Days 6-11 and 11-16) and post-treatment (Days 16-20) intervals of
gestation were comparable between the control and Group II.
In Groups III and IV, mean food consumption data for the pretreatment
interval of gestation and for the Day 11-16 interval of the treatment
period were comparable to control data. During the Day 6-11 gestation interval
of the treatment period, mean food consumption data for Groups III and IV were
lower than control and these differences (-12.1% and -37.4%, respectively) were
statistically significant. During the post-treatment interval, rnean food
consumption data for Groups III and IV were higher than control and these
differences (+7.9% and +25.8%, respectively), which were statistically
significant, were considere'd indicative of compensatory feeding following
reductions in food consumption early in the treatment period.
Thus, no adverse effect of treatment was evident from food
consumption data at the 188 mg/kg/day dose level. At the higher dose levels
evaluated (375 and 750 mg/kg/day), a reduction in food consumption was noted
early in the treatment period (Days 6-11 of gestation) and this was considered
indicative of an adverse effect of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean absolute liver weights were considered comparable between the
control and each treatment group. (see Table J2 in Attachment)

Gross pathological findings:

no effects observed

Description (incidence and severity):

No adverse effect of treatment at doses up to and including 750
mg/kg/day was evident from the gross postmortem evaluations.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 88 females (23 control, 24 Group II, 24 Group III and 17
Group IV) survived to scheduled sacrifice. Eighty-five of these females (22
control, 24 Group II, 23 Group III and 16 Group IV) were considered pregnant on
the basis of grossly visible uterine implantation sites (resorptions, fetuses).
One control female (No. 1518) delivered on what was considered Day 19 of
gestation. A total of 13 pups were delivered and these pups weighed between 5.9
and 7.1 grams. Due to the size of these pups, this pregnancy was not considered
representative of a 19 day gestation, but could be more appropriately identified
as a Day 21 or 22 gestation. Apparently, the actual Day 0 gestation for this
female was incorrectly identified. The uteri of three females that did not have
grossly visible implants (Group I female No. 1501, Group III female No. 3515 and
Group IV female No. 4513) were stained (Appendix A) to ascertain the presence of
foci suggestive of early fetal death in utero and complete resorption. No
uterine foci were visualized post-staining of the Group I (control) or Group III
females and they were considered not pregnant. Foci were visualized poststaining
of the uterus for the one Group IV female evaluated. This female (No.
4513) was considered pregnant only for the purpose of calculating pregnancy
rate.
The mean numbers of corpora lutea and uterine implants per pregnant
female and the mean pre-implantation loss indices were comparable between the
control and each treatment group.
The mean number of viable fetuses per pregnant female was also
comparable between the control and each treatment group. One dead fetus was
recovered in utero from one Group I and one Group IV female.
The mean number of resorption sites per pregnant female, the mean
ratio of resorptions/implants and the incidence of females with one or more
resorptions in utero were comparable between the control and each treatment
group.

Dose descriptor:

LOAEL

Effect level:

188 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: food consumption

Abnormalities:

no effects observed

Description (incidence and severity):

only general toxicity observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weights, distinguished by sex and as a composite for
both sexes, were considered comparable between the control and treatment Groups
II and III. In Group IV, mean fetal weights were lower than control and these
differentes as a composite for both sexes (-9.8%) and when distinguished by sex
(males -8.8% and female -10.3%), were statistically significant.
Thus, no adverse effect of treatment at doses up to and including
375 rng/kg/day was evident from fetal weight data. At the 750 mg/kg/day dose
level, mean fetal weights were reduced, indicative of a fetotoxic response to
either treatment or the severe maternal toxicity evident at this dose level.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean number of male and female fetuses per pregnant female was
comparable between the control and each treatment group. Likewise, the ratio of
total viable male/female fetuses per group was similar between these same
groups.

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were seen in the 349 control (Group
I) fetuses (22 litters), 370 Group II fetuses (24 litters) or 362 Group III
fetuses (23 litters) evaluated. In Group IV, the incidence of fetuses with
external malformations was 0.4% (1/258 fetuses) and the incidence of litters
containing fetuses with external malformations was 6.3% (1/16 1itters); these
incidences did not differ statistically from the control data.
The only malformation seen during the fetal external
examinations was a flexure defect of the hindlimb, i.e., hindlimb extension
defect. This malformation was seen in one fetus recovered from the in .u..t..e.cQ
litter of female No. 4509. The low incidence in occurrence of this one
malformation was not considered indicative of a treatment-related response.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidences of fetuses with skeletal malformations for the
control (Group I) and treatment Groups 11, III and IV were 0% (168 fetuses
evaluated), 0.6% (1/177 fetuses), 0% (173 fetuses evaluated) and 1.6% (2/124
fetuses), respectively. The incidences of litters containing fetuses with
skeletal malfonnations for these same groups were 0% (22 litters), 4.2% (1/24
litters), 0% (23 litters) and 12.5% (2/16 litters), respectively. These
incidences on aper fetus and per litter basis did not differ statistically
between the control and treatment groups and were considered comparable between
these same groups. (see Table M1 in Attachment)

Description (incidence and severity):

The incidences of fetuses with visceral malformations for the
control (Group I) and treatment Groups II, III and IV were 0.6% (1/181 fetuses),
0.5% (1/193 fetuses), 1.1% (2/189 fetuses) and 0.7% (1/134 fetuses),
respectively. The incidences of litters containing fetuses with visceral
malformations for these same groups were 4.5% (1/22 litters), 4.2% (1/24
litters), 8.7% (2/23 litters) and 6.3% (1/16 litters), respectively. These
incidences on a per fetus and per litter basis were comparable between the
control and each treatment group. (see Table L-1 in Attachment)

Description (incidence and severity):

A potential fetotoxic response to treatment at all dose levels evaluated
was suggested from an increase in both the incidence of fetuses with one or more
ossification variations in comparison to concurrent control data andin the
increased incidence, both on aper fetus and per litter basis, of particular
ossification variations. Ingroups treated at the 188 and 375 mg/kg/day dose
levels, only mild developmental delays were seen in regard to both concurrent
control data and recent historical control data for this laboratory. A more
significant developmental delay was observed at the 750 mg/kg/day dose level but
was not unexpected since fetal weights were reduced and maternal toxicity was
observed.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: embryotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation. Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation.

No embryotoxic effects were seen at any of the dose levels evaluated and only at the 750 mg/kg/day dose level was a reduction in mean fetal weight seen.

In groups treated at the 188 and 375 mg/kg/day dose levels, only mild developmental delays were seen in regard to both concurrent control data and historical control data.

No embryotoxic effects were seen at any of the dose levels evaluated and only at the 750 mg/kg/day dose level was a reduction in mean fetal weight seen.

External, visceral and skeletal examination of the fetuses recovered from females in the treatment groups at day 20 of gestation revealed no teratogenic response at any of the dose levels evaluated.

The NOAEL was 750 mg/kg bw for teratogenicity and embryotoxicity in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Species:

rabbit

Quality of whole database:

The lowest NOAEL for developmental toxicity was obtained in the rabbit with 150 mg/kg bw/day.

Additional information

Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation. Tributyl phosphate was administered by gastric intubation to rats during the day 6-15 gestation interval. Dose levels were 188, 375 and 750 mg/kg/day. Each study group was comprised of 24 mated female CD rats. Animals were sacrificed on day 20 of gestation and given a gross postmortem evaluation. No embryotoxic effects were seen at any of the dose levels evaluated and only at the 750 mg/kg/day dose level was a reduction in mean fetal weight seen. In groups treated at the 188 and 375 mg/kg/day dose levels, only mild developmental delays were seen in regard to both concurrent control data and historical control data. No embryotoxic effects were seen at any of the dose levels evaluated and only at the 750 mg/kg/day dose level was a reduction in mean fetal weight seen. External, visceral and skeletal examination of the fetuses recovered from females in the treatment groups at day 20 of gestation revealed no teratogenic response at any of the dose levels evaluated. The NOAEL was 750 mg/kg bw for teratogenicity and embryotoxicity in this study on rats.

 

In a second developmental toxicity study (Noda et al., 1994) in rats, the no-observed-adverse-effect levels (NOAEL) for teratogenicity was considered to be more than 500 mg/kg bw/day.

 

Tributyl phosphate was and administered by gastric intubation to pregnant New Zealand White rabbits over the Day 6-18 gestation interval. Tributyl phosphate was not maternally toxic, embryotoxic, fetotoxic or teratogenic at dose levels of 50 and 150 mg/kg/day.
At the 400 mg/kg/day dose level, one female died late in gestation (Day 28) but the cause of death could not be determined on the basis of gross postmortem observations. The death of a second female in this group was attributed to injuries incurred while handling to administer the dose. Maternal toxicity at the 400 mg/kg/day dose level was indicated from mean weight loss experienced during the Day 6-9 gestation interval; this difference in comparison to the mean weight gain experienced by the concurrent control group during this interval was statistically significant. Other changes seen at the 400 mg/kg/day dose level which did not differ statistically from control data but which were considered suggestive of a maternal toxic response were as follows: mean weight losses during several intervals of the treatment period (Days 9-12 and 12-15) in comparison to mean weight gains for the control; mean weight loss over the entire Day 6-19 gestation interval in comparison to a mean weight gain for the control group; and reduced levels of food consumption over the Day 7-14 gestation interval. At Day 30 of gestation, the mean number of resorptions per pregnant female and the mean resorption/implant ratio for the 400 mg/kg/day dose level were increased in cornparison to concurrent control and recent historical control data for this laboratory, and while these differences were not statistically significant, an embryotoxic response may be suggested. The 400 mg/kg/day dose level was not considered to be foetotoxic or teratogenic.

Thus, the no-observable-adverse-effect level (NOAEL) for developmental toxicity of tributyl phosphate in the rabbit was 150 mg/kg/day.

 

 

Justification for classification or non-classification

Based on a 2-generation reproduction toxicity study and developmental toxicity studies in rats and rabbits a classification is not justified.