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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: continuous breeding protocol
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP, not conducted according to recognised guideline. Study is reported as literature report.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1988
Report date:
1987

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of a mixture of tricresyl phosphate isomen on reproductive performance in Swiss (CDl) mice were evaluated using a continuous breeding protocol. The design has four related elements, or Tasks. Task 1 is a 14-day range-finding study, Task 2 is the 98 day continuous breeding phase of the Fo generation, Task 3 is a crossover mating to determine the affected sex in the F0 animals, and Task 4 examines the fertility and performance of the 1st litter (F1) from the continuous breeding.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(methylphenyl) phosphate
EC Number:
215-548-8
EC Name:
Tris(methylphenyl) phosphate
Cas Number:
1330-78-5
Molecular formula:
C21H21O4P
IUPAC Name:
tris(4-methylphenyl) phosphate
Constituent 2
Reference substance name:
tricresyl phosphate
IUPAC Name:
tricresyl phosphate
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): tricresyl phosphate
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: Not specified
- Physical state: Not specified
- Analytical purity: Assessed prior to study by Midwest Research Institute
- Impurities (identity and concentrations): Karl Fischer analysis indicated 0.072 +/- 0.003% water.
- Composition of test material, percentage of components: Gas chromatography on two systems (3% Dexsil400, and an SP-2 100 capillary column) showed 20.6% pure metacresyl isomer, 3.9% para isomer, and 0.11 pure ortho isomer. Overall, pure and/or mixed ortho-, meta-, and para-cresyl isomers composed 74.9% of the total, with the remainder composed of dicresyl phenyl and di- and tricresylxylyl phosphates.
- Isomers composition: See above
- Purity test date: Not specified
- Lot/batch No.: Lot No. 1202A-2-7
- Expiration date of the lot/batch: Not specified
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: assessed prior to study initiation by Midwest Research Institute
- Storage condition of test material: Not specified
- Other:

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Mice [Swiss (1CR)BR outbred albino; also known as CDl] were obtained from Charles River Breeding Laboratories (Kingston, NY). The mice were quarantined for 2 weeks following arrival at 6 weeks of age. All the mice for the entire experiment were received in one shipment. For the 14 day quarantine period, mice were housed 10 per cage per sex in solid bottom polypropylene cages with Ab-Sorb-Dri cage litter (Laboratory Products, Garfield, NJ). The cages were kept in temperature (70 +/- 2°F) and humidity (50 +/- 15%)- controlled rooms with a 14-hr: 10-hr light: dark cycle. Two males
and two females were killed during quarantine, and sera were analyzed for 11 murine viruses (Microbiological Associates, Bethesda, MD); all tests were negative. The animals were allowed ad libitum access to NIH-07 chow and deionized/filtered water during quarantine. All mice were identified by individual ear tags and assigned to groups by a stratified randomization procedure based on body weight. The mice were housed as breeding pairs during the breeding phase of the study, and individually thereafter.

Administration / exposure

Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
other: Not applicable.
Vehicle:
other: NIH-07 powdered feed
Details on exposure:
Weighed aliquots of TCP were mixed with weighed aliquots of NIH-07 powdered feed, and homogenized in a Patterson-Kelly Twin Shell blender. Feed concentrations for the range-finding study (Task 1) were 0.0, 0.437, 0.875, 1.75, 3.5, and 7% TCP (w/w) in the diet; and 0.0, 0.05, 0.1, and 0.2% TCP (w/w) for the remainder of the study. Neat and feed-formulated TCP was stable for >14 days at room temperature; dosed-feed formulations were made every 2 weeks, and stored at 4 deg C until added to the feed cups at weekly intervals.
Details on mating procedure:
The animals were housed as breeding pairs for 98 days, following 7 days of premating consumption of dosed feed. Endpoints during Task 2 were clinical signs, parental body weight, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hr of birth, and parental body weights after the last litter. This last litter was reared by the dam until weaning after which dosed feed was provided to the F1 mice at the same concentrations as their parents had consumed during Task 2.
Because of the effect on fertility during Task 2, a crossover mating trial (Task 3) was subsequently performed to determine the affected sex in the F0 mice. This was done after the last Task 2 litter was weaned. Treatment with TCP continued between the end of Task 2 and the crossover mating. There were three groups: control males X control females, control males X 0.2% TCP-treated females, and 0.2% TCP-treated males X control females.
The animals were assigned to their mating partner by the stratified randomization procedure and cohabited for 1 week, during which time they consumed control feed without TCP. The pairs were then separated and the females were allowed to deliver their litters.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of dosed feed were assayed for TCP content by gas chromatography four times during the continuous breeding phase of the study, and were found to be 94 - 107% of target concentration.
Duration of treatment / exposure:
Task 2: The animals were housed as breeding pairs for 98 days, following 7 days of premating consumption of dosed feed.
Task 3: Treatment with TCP continued between the end of Task 2 and the crossover mating. There were three groups: control males X control females, control males X 0.2% TCP-treated females, and 0.2% TCP-treated males X control females
Frequency of treatment:
As described above; within diet for set periods, dependant on the phase (task) of the study.
Details on study schedule:
Because of the effect on fertility during Task 2, a crossover mating trial (Task 3) was subsequently performed to determine the affected sex in the Fo mice. This was done after the last Task 2 litter was weaned. Treatment with TCP continued between the end of Task 2 and the crossover mating. There were three groups: control males X control females, control males X 0.2% TCP-treated females, and 0.2% TCP-treated males X control females.

The animals were assigned to their mating partner by the stratified randomization procedure and cohabited for 1 week, during which time they consumed control feed without TCP. The pairs were then separated and the females were allowed to deliver their litters. During the postcohabitation phase, animals were given either control or TCPdosed feed as during Task 2.

Task 4, offspring assessment, was also conducted for TCP. In this phase, the last liner from Task 2 was reared, weaned, and kept to sexual maturity while housed by sex two or three per cage, consuming the same concentration of TCP in the feed as consumed by their parents. Because of parental infertility, there were no young in the high dose group, so the last litter from both low- and middose groups (0.05 and 0.1% TCP) were reared to sexual maturity (74 +/- 10 days). At this time, a male and a female from different liners within the same treatment group were cohabited for 7 days; the mice were then housed singly until delivery. The endpoints for this mating trial were the same as those in Task 3. Three weeks after the end of cohabitation, F1, mice were necropsied, the endpoints were the same as for the necropsy of the F0 mice
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
62.5 mg/kg/day
Basis:
nominal in diet
Over the course of this continuous breeding phase, the breeding pairs consumed an average of 5 g food per day per mouse, producing an estimated dose of TCP equal to approximately 62.5, 124, and 250 mg/kg/day for the 0.05, 0.1, and 0.2% TCP groups.
Remarks:
Doses / Concentrations:
124 mg/kg/day
Basis:
nominal in diet
Over the course of this continuous breeding phase, the breeding pairs consumed an average of 5 g food per day per mouse, producing an estimated dose of TCP equal to approximately 62.5, 124, and 250 mg/kg/day for the 0.05, 0.1, and 0.2% TCP groups.
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
nominal in diet
Over the course of this continuous breeding phase, the breeding pairs consumed an average of 5 g food per day per mouse, producing an estimated dose of TCP equal to approximately 62.5, 124, and 250 mg/kg/day for the 0.05, 0.1, and 0.2% TCP groups.
No. of animals per sex per dose:
Control group: 20
Test groups: 10 per dose level.
Control animals:
yes, plain diet
Details on study design:
Task 1 used five doses and control (n = 8/sex/group) as a range-finding study, to set doses for the breeding phase (Task 2). Endpoints for Task 1 were clinical signs of toxicity, percentage mortality, body weight, and food and water consumption.
Task 2 was the continuous breeding phase, consisting of a control group (40 breeding pairs) and three dose groups (20 pairs per group). Task 2 dose levels were set so that the highest dose would be expected not to depress weight gain by more than 10% and permit >90% survival. The middle dose was selected to assess reproductive toxicity with little or no systemic toxicity, while the low dose was designed to be a no-effect level. Dose levels for Task 2 were 0.0, 0.05, 0.1, and 0.2% TCP by weight in the diet. The animals were housed as breeding pairs for 98 days, following 7 days of premating consumption of dosed feed. Endpoints during Task 2 were clinical signs, parental body weight, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hr of birth, and parental body weights after the last litter. This last litter was reared by the dam until weaning after which dosed feed was provided to the F1 mice at the same concentrations as their parents had consumed during Task 2.
Because of the effect on fertility during Task 2, a crossover mating trial (Task 3) was subsequently performed to determine the affected sex in the F0 mice. This was done after the last Task 2 litter was weaned. Treatment with TCP continued between the end of Task 2 and the crossover mating. There were three groups: control males X control females, control males X 0.2% TCP-treated females, and 0.2% TCP-treated males X control females.
The animals were assigned to their mating partner by the stratified randomization procedure and cohabited for 1 week, during which time they consumed control feed without TCP. The pairs were then separated and the females were allowed to deliver their litters. During the postcohabitation phase, animals were given either control or TCP dosed feed as during Task 2. Endpoints for Task 3 were the same as for Task 2, with the addition of daily checking for the presence of a copulatory plug. After the litters were delivered at the end of Task 3 and litter data were collected, F0 males and females were necropsied; the endpoints evaluated were organ weight body weight, sperm motility, morphology, and epididymal sperm number, and estrous cyclicity as monitored by vaginal lavage for the preceding 5 days. Sperm morphology was scored by the criteria of Wyrobek and Bruce (1 975), and for these data, all rnorphologic abnormalities were lumped together as "abnormal." Selected organs were evaluated microscopically after fixation in 10% neutral buffered formalin (Bouin's fixative for testes) and embedding in paraffin; sections were stained with hematoxylin and eosin according to standard procedures. Tissue damage in the histology sections was graded using a scoring system as follows: 1- present; 2-slight; 3-moderate; 4-moderately severe; 5-severe, high.
Task 4, offspring asessment, was also conducted for TCP. In this phase, the last liner from Task 2 was reared, weaned, and kept to sexual maturity while housed by sex, two or three per cage, consuming the same concentration of TCP in the feed as consumed by their parents. Because of parental infertility, there were no young in the high dose group, so the last litter from both low- and middose groups (0.05 and 0.1% TCP) were reared to sexual maturity (74 +/- 10 days). At this time, a male and a female from different litters within the same treatment group were cohabited for 7 days; the mice were then housed singly until delivery. The endpoints for this mating trial were the same as those in Task 3. Three weeks after the end of cohabitation, F1 mice were necropsied, the endpoints were the same as for the necropsy of the F0 mice.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
Endpoints for Task 1 were clinical signs of toxicity, percentage mortality, body weight, and food and water consumption.
Endpoints during Task 2 were clinical signs, parental body weight, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hr of birth, and parental body weights after the last litter.
Endpoints for Task 3 were as per Task 2, but with the with the addition of daily checking for the presence of a copulatory plug.
Enpoints for Task 4 Twere the same as those in Task 3.
Oestrous cyclicity (parental animals):
At the end of Task 3 estrous cyclicity was monitored by vaginal lavage for 5 days.
Sperm parameters (parental animals):
At the end of Task 3 sperm motility, morphology, and epididymal sperm number was evaluated in necropsied animals. Sperm morphology was scored by the criteria of Wyrobek and Bruce (1 975), and for these data, all morphologic abnormalities were lumped together as "abnormal."
Litter observations:
Fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hr of birth,
Postmortem examinations (parental animals):
After the litters were delivered at the end of Task 3 and litter data were collected, F0 males and females were necropsied; the endpoints evaluated wereorgan weight, body weight, sperm motility, morphology, and epididymal sperm number, and estrous cyclicity as monitored by vaginal lavage for the preceding 5 days. Selected organs were evaluated microscopically after fixation in 10% neutral buffered formalin (Bouin's fixative for testes) and embedding in paraffin; sections were stained with hematoxylin and eosin according to standard procedures. Tissue damage in the histology sections was graded using a scoring system as follows: 1- present; 2-slight; 3-moderate; 4-moderately severe; 5-severe, high.
Postmortem examinations (offspring):
F1 mice were necropsied, the endpoints were the same as for the necropsy of the F0 mice, i.e. organ weight, body weight, sperm motility, morphology, and epididymal sperm number, and estrous cyclicity as monitored by vaginal lavage for the preceding 5 days. Selected organs were evaluated microscopically after fixation in 10% neutral buffered formalin (Bouin's fixative for testes) and embedding in paraffin; sections were stained with hematoxylin and eosin according to standard procedures. Tissue damage in the histology sections was graded using a scoring system as follows: 1- present; 2-slight; 3-moderate; 4-moderately severe; 5-severe, high.
Statistics:
Statistical analyses were performed as follows. The level of significance for all tests was set at p < 0.05. The Cochran-Annitage test (Armitage, 1971) was used to test for a dose-related trend in fertility (Task 2). Pairwise comparisons involving mating and fertility indices were performed using Fisher's exact test (Tasks 2 and 4). The number of litters and the number of live pups per litter were computed on a per fertile pair basis and then treatment group means were determined. The proportion of live pups was defined as the number of pups born alive, divided by the total number of pups produced by each pair. The sex ratio was expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair. Dose group means for these parameters were tested for overall differences by using the Kruskal-Wallis test (Conover, 1980) and for ordered differences using Jonekheere's test (Jonckheere, 1954). Pairwise comparisons of treatment group means were performed by applying the Wilcoxon-Mann-Whitney U test (Conover, 1980). Since the number of pups in a litter may affect the average weight of the litter, an analysis of covariance (Neter and Wasserman, 1974) was used to test for treatment differences in average pup weight, adjusting for average litter size (live and dead pups). Pairwise comparisons were done using a two-sided t test. A Kruskal-Wallis test was also performed. For the organ weights, least-squares treatment group means were generated from an analysis of covariance (with body weight as the covariate) and were tested for overall equality using the F test, and for pairwise equality using a t test. All comparisons were twosided. The Kruskal-Wallis and Wilcoxon-Mann-Whitney U tests were also employed.
Reproductive indices:
Pairwvise comparisons involving mating and fertility indices were performed using Fisher's exact test (Tasks 2 and 4).
Offspring viability indices:
Pairwise comparisons involving mating and fertility indices were performed using Fisher's exact test (Tasks 2 and 4).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

Task 2: Over the course of this continuous breeding phase, the breeding pairs consumed an average of 5g food per day per mouse, producing an estimated dose of TCP equal to approximately 62.5, 124, and 250mg/kg/day for the 0.05, 0.1, and 0.2% TCP groups, respectively. During Task 2, female mice in the high dose group exhibited hind limb weakness; no clinical signs of toxicity were noted in any male mice. The fertility index (the number of pairs producing one or more litters of live or dead pups, divided by the number of pairs cohabited, X 100) was not changed by exposure to TCP. For the 0.0, 0.05, 0.1, and 0.2% groups, respectively, these values were 97, 95, 100, and 89%. Female postpartum body weight was decreased at the high dose (Table l), but not at lower doses. The numbers in parentheses in Table 1 also indicate that there were fewer pairs in the 0.1%TCP group that produced 4 and 5 litters, while a decrease in number of fertile pairs occurred by litter 2 in the high dose group. Table 2 shows the mean number of live and dead pups at each litter. This table shows more clearly that TCP increased the number of dead pups and decreased the number of live pups in the 0.2% group at the first litter and also in the last two litters in the 0.1% group; the number of pairs having litters declined with TCP exposure and time.
Table 3 summarises the treproduction data for all of Task 2. The proportion of pups born alive shows a decreasing trend and is significantly affected at the high dose. Again, the fertility effects at the high dose occurred in the presence of maternal weight depression and hindlimb weakness. Because of the effects seen on reproduction in this task, a crossover mating was carried out to determine the affected sex.

Task 3: For this task, high-dose males were co-habited with control females, control males with high-dose females and control males were paired with high-dose females for 7 days. The results of this crossover mating (Table 4) indicate that fertility in both males and females was affected by TCP exposure. Although the number of detected matings was not decreased by TCP treatment, the fertility index was decreased (Table 4). And while the fertility was affected equally in both sexes,the reproductive performance (pups per litter, proportion born alive) was more severely affected in the treated females, although treated males did produce fewer live pups per litter. At the end of this task, both sexes of F0 control and high dose animals were necropsied. Table 5 shows the male and female body and organ weight changes seen after TCP exposure. Female paired kidney/adrenal weights were decreased by0.2%TCP. Although male liver and kidney/adrenal weights were not affected by0.2 % TCP, right testis and right epididymis weights were decreased, aswere combined left testis/epididymis weights (not shown). Prostate and seminal vesicle weights were not affected by TCP. At the time of termination, the number of moving vs non-moving sperm was determined, and the percentage of motile sperm was calculated.
Table 6 shows that epididymal sperm motility and concentration were decreased by TCP, while the percentage of abnormal sperm was increased. Histopathologic examination revealed no treatment-related changes in the prostate, seminal vesicles, liver, or kidney of the male F0 mice, or in the ovaries, uterus, vagina, liver, or kidneys of the females. Treatment-related atrophy of the seminiferous tubules was seen in the testes of the TCP-treated males, ranging from scattered foci of decreased germ cell number, to widespread bilateral loss of germ cells in the high dose mice. There was also TCP-related hypertrophy of the adrenal zona fasciculata cells, and brown degeneration of the cells in the adrenal juxtamedullary zone which was foundinboth sexes. Male mice showed an increase in both the incidence and the severity of these adrenal changes, while females showed changes only in the severity (Table 7).

Effect levels (P0)

Key result
Dose descriptor:
LOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The LOAEL for reproductive toxicity of tricresyl phosphate appears to be 0.05 per cent in the feed or 62.5 mg/kg/day
Remarks on result:
other: See comments below

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See below
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See below
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
See below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See below
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
See below
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Task 4: Because of the effects on both sexes of the F0 generation, the last litter from Task 2 was reared and given the same TCP containing diet as its parents between weaning and Day 74 (+/-10) of age. These mice were then mated to nonsiblings from the same dose group at approximately 74 days of age. There were insufficient mice in the high dose group, so Task 4 consisted of mice from the control, 0.05, and 0.1% TCP groups. No adverse clinical signs were noted for any TCP-treated F1 mice. There was a significant trend toward decreased mating and fertility indexes and the number of live pups per litter also decreased (Table8). The proportion of live pups per litter was significantly decreased in the 0.1% TCP group compared to controls (Table8), but there were no treatment-related changes in the live pup weight (not shown). Three weeks after the end of this cohabitation period, the F1 adult mice were terminated and necropsied. While female organ weights (liver and kidney/adrenals) were not decreased (not shown), mean female body weights were significantly decreased for both the 0.05 and 0.1% TCPgroups vs controls (29.67 +/-0.51g for controls, 27.96 +/-0.51, and 27.16 +/-0.63g for the 0.05 and 0.1% TCP groups, respectively, mean +/- SEM, n=20). For the F1 males, body weight was not decreased; combined left testis/epididymis weights were significantly decreased from 181+/-5 mg for controls to 168 +/- 3mg for the 0.1% group. Other organ weights were not decreased (not shown). Sperm motility was decreased in both 0.05 and 0.1% TCP groups vs control (Table 9), although epididymal sperm concentration was not decreased, and the percentage of abnormal sperm was significantly increased only at the low dose (Table 9). Histopathologic examination of tissues found exposure-related changes in the adrenals of both sexes (Table 7, and above); there was no pathology in the reproductive organs of either sex as detected by these methods.

Effect levels (F1)

Key result
Dose descriptor:
other: Dose level.
Generation:
F1
Effect level:
62.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: See remarks
Remarks:
The fertility index (number of pairs producing litters divided by the number of pairs cohabitated, X 100) was not affected by exposure to TCP. However, the number of litters per pair decreased in a dose related manner, and the proportion of pups born live in the high dose group was significantly lower than the control. In the crossover mating phase, impaired fertility was found in both male and female mice treated with 0.2% TCP, with greater effect in the females. The high dose group also demonstrated significantly lower body weights and changes in adrenal morphology. An examination of sperm from the F1 males at necropsy found normal sperm concentration and morphology in all dose groups. Sperm motility was significantly decreased in the 0.05% and 0.1% males (0.2% males not examined for sperm motility). TCP impaired fertility in both sexes of mice and adversely affected sperm motility even at the lowest dose. Tricresyl phosphate induces functional and structural effects in the male reproductive system and functional reproductive impairment in females. Effects on fertility were noted at doses of 0.1 per cent or more in the feed. Observed male gonad pathologies included seminiferous tubule atrophy and decreased testis and epididymal weights at 0.05 per cent and above, with sperm motility reduced in both the 0.05 per cent and 0.1 per cent tricresyl phosphate groups compared to controls.

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Any other information on results incl. tables

Tabulated data is attached below under "Background Information" for use.

Task1: For the dose range finding Task 1, the levels of TCP in the diet were 0.0, 0.437, 0.875, 1.75,3.5,and 7.0% by weight; aliquots of dosed feed contained 95 -100% of the target concentration. No clinical signs of toxicity were observed in the lowest two dose groups, and their food consumption was equal to controls, although body weight gain was slightly depressed. All animals in the 1.75, 3.5, and 7.0% groups exhibited piloerection, tremors, diarrhea, and lethargy. By the end of the 14 day dosing period, all the animals in the 1.75, 3.5, and 7.0% groups had died. On the basis of these data, dose levels for Task 2 were set at 0.0, 0.05, 0.1, and 0.2% TCP in the diet by weight.

The results above indicate that at these doses, a tricresyl phosphate mixture was generally toxic to male and female mice,as evidenced by body weight depression and adrenal histopathology. In addition, there were functional and structural effects in the male reproductive system, and functional reproductive impairment in the females. The Task 3 fertility data (Table 4) suggest that TCP is more toxic to the females than to the males; this is supported by the more severe body weight effects for the females and the increase in dead pups as soon as the first litter. Generally, these changes occurred in the presence of adverse clinical signs (hind limb weakness in females) and body weight depression. Although these females appeared more affected by TCP, it is difficult to weigh the relative contribution of generalized toxicity to the reproductive toxicity. The lack of pathology in the female reproductive tracts supports the concept of a functional change without structural damage, a change whose link to the generalized toxicity cannot be determined from these data. Clear effects were also demonstrated on the male reproductive system. These occurred in the absence of clinical signs for both F0 and F1males; F0 high dose males showed changes in sperm motility, morphology, epididyrnal sperm concentration, and reproductive organ weights. The F1males also showed changes in sperm motility at lower doses, in the absence of changes in sperm concentration or morphology. Similar effects have been reported for an NTP-sponsored 13 -week subchronic study of an isomeric mixture of TCP gavaged to rats and mice at and above (up to 800mg/kg/day for 13 weeks) the high dose estimated consumption in our study; females were more prone to the neurotoxicity, and adrenal lesions were present in both sexes (Deskinet al. 1985; Carlton et al.,1986a). Higher doses of TCP also produced ovarian vacuolation and/or hyperplasia (Carltonet al.,1986a), and extensive peripheral neural damage. Additionally, an EPA-sponsored fertility study found that mixed TCP isomers gavaged to male and female rats (for 56 and 14 days, respectively, before mating) adversely sited sperm parameters (motility, morphology, concentration) and fertility (Carltonet al.,1986b). In the F1 males, sperm motility was lowered in both the 0.05 and 0.1% TCP dose groups, in the absence of changes in clinical signs or body weight changes. Similar effects on sperm motility have been reported for other organophosphates, trimethylphosphate (Harbisonet al.,1976), and dimethyl methylphosphonate (DMMP; Dunnicket al.,1984a) in rats. Trimethylphosphate, TOCP, DMMP, and mixed TCP isomers have been reported to produce structural damage to the testis of rats (Hanna and Kerr, 1981 for TMP; Somkutiet al.,1986 for TOCP; Dunnick etal..1984a for DMMP, and Carlton etal.,1986a,b for TCP). Studies on the development of the testicular lesion support the concept that TOCP exposure affects the Sertoli cell and inhibits spermatogenesis (Somkutiet al.,1986). Dunnicket al.(1984b) reported that B6C3F1 mice are less responsive than are Fischer rats to the reproductive toxicity of DMMP. The fact that the F1mice in the study reported above showed decreased sperm motility, with no change in epididymal sperm number or histopathology, indicates that sperm motility is more sensitive to TCP than is the process of spermatogenesis in the second generation of CD-1 mice. We should note that because the fertility of F1 females was not evaluated, the contributions of a female effect to the decreased F1fertility cannot be determined.

The tri-ortho-substituted cresyl phosphate is the most neurotoxic isomer of TCP. An ortho-substituted ring can be metabolized in vivo to the active metabolite saligenin cyclic o-tolyl phosphate (Etoet al.,1962), which is approximately five times as neurotoxic as the parent compound (Bleiburg and Johnson, 1965). The metabolic pathway for TOCP proposed by Etoet al.(1962) suggests that if one of the three cresyl rings is ortho-substituted, the o-cresyl ring can be hydroxylated, allowing ring closure, and formation of the active intermediate. This is supported by the thorough literature review by Abou-Donia (1981). There was less than 0.1% of pure triorthecresyl phosphate in the mixture tested in the above study. However, approximately 50% of the isomers was mixed tricresyls; it is thus possible that a significant amount of the TCP consumed was metabolized to the active saligenin phosphate intermediate. This is consistent with the studies of Somkutiet al.(1987), which reported that administration of pure tri-para-cresyl phosphate to rats produced no significant toxic effects on the male reproductive system, although similar doses and durations of TOCP produced severe effects. Although there have been numerous theories about the neurotoxic mechanisms of TCP (e.g., Mannoet al.,1979; Pattonet al.,1986; Suwitaet al.,1986; Eto, 1974, for review), few studies have addressed the relationship between metabolism and reproductive toxicity. However, Somkuti (1986) analyzed numerous rat tissues after dosing with [14C]TOCP, and found that testis contained more saligenin cyclic-o phosphate per gram wet weight than did liver, kidney, brain, or plasma. Although the species differences in responses to tricresyl phosphates (Abou-Donia, 1981) make it difficult to extrapolate from rats to the CD-1 mice used above, the preliminary metabolic data from Somkuti (1986) suggest that there may be a toxicokinetic component to the male reproductive effects. These studies have demonstrated that tricresylphosphate can produce reproductive toxicity without functional neurologic impairment in males, and can impair reproductive performance in both sexes of CD-1 mice.

Applicant's summary and conclusion

Conclusions:
The fertility index (number of pairs producing litters divided by the number of pairs cohabitated, X 100) was not affected by exposure to TCP.
However, the number of litters per pair decreased in a dose related manner, and the proportion of pups born live in the high dose group was
significantly lower than the control. In the crossover mating phase, impaired fertility was found in both male and female mice treated with 0.2%
TCP, with greater effect in the females. The high dose group also demonstrated significantly lower body weights and changes in adrenal morphology. An examination of sperm from the Fl males at necropsy found normal sperm concentration and morphology in all dose groups.
Sperm motility was significantly decreased in the 0.05% and 0.1% males (0.2% males not examined for sperm motility). TCP impaired fertility in
both sexes of mice and adversely affected sperm motility even at the lowest dose.

Tricresyl phosphate induces functional and structural effects in the male reproductive system and functional reproductive impairment in females. Effects on fertility were noted at doses of 0.1 per cent or more in the feed. Observed male gonad pathologies included seminiferous tubule atrophy and decreased testis and epididymal weights at 0.05 per cent and above, with sperm motility reduced in both the 0.05 per cent and 0.1 per cent tricresyl phosphate groups compared to controls.

The LOAEL for reproductive toxicity of tricresyl phosphate appears to be 0.05 per cent in the feed or 62.5 mg/kg/day.
Executive summary:

This study has demonstrated that tricresylphosphate can produce reproductive toxicity without functional neurologic impairment in males, and can impair reproductive performance in both sexes of CD-1 mice. Tricresyl phosphate induces functional and structural effects in the male reproductive system and functional reproductive impairment in females. Effects on fertility were noted at doses of 0.1 per cent or more in the feed. Observed male gonad pathologies included seminiferous tubule atrophy and decreased testis and epididymal weights at 0.05 per cent and above, with sperm motility reduced in both the 0.05 per cent and 0.1 per cent tricresyl phosphate groups compared to controls. The LOAEL for reproductive toxicity of tricresyl phosphate appears to be 0.05 per cent in the feed or 62.5 mg/kg/day.