Tributyl phosphate

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

publication

Title:

Two generation study of dietary tributyl phosphate in CD rats

Author:

Gerhart JM, Tyl RW, Myers CB, Marr MC, Brine DR, Seely JC

Year:

1993

Bibliographic source:

Toxicologist 13, 76 (1993)

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl phosphate

EC Number:

204-800-2

EC Name:

Tributyl phosphate

Cas Number:

126-73-8

Molecular formula:

C12H27O4P

IUPAC Name:

tributyl phosphate

Details on test material:

purity 99.7 %

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 function / performance (P0)

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.

Effect levels (P0)

1

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)

2

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)

Target system / organ toxicity (P0)

1

Critical effects observed:

no

System:

other: reproductive performance

2

Critical effects observed:

yes

Lowest effective dose / conc.:

200

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Results: P1 (second parental generation)

General toxicity (P1)

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 function / performance (P1)

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

Effect levels (P1)

1

Dose descriptor:

NOAEL

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

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

Target system / organ toxicity (P1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

2

Critical effects observed:

no

Organ:

other: reproductive performance

Results: F1 generation

General toxicity (F1)

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).

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive performance

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weight

Target system / organ toxicity (F1)

Critical effects observed:

no

Organ:

other: reproductive performance

Results: F2 generation

General toxicity (F2)

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

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weight

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).