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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

None

Link to relevant study records
Reference
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.
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
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.
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
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.
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).
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: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
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).
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
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
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.
Key result
Reproductive effects observed:
not specified

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
62.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
K2
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproduction:

The following studies are included in support of the substance for reproduction effects.

Key, K1; Chapin et al (1988); Reproductive Toxicity of Tricresyl Phosphate in a Continuous Breeding Protocol in Swiss (CD-1) Mice

LOAEL: 62.5 mg/kg/day.

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.

Sup, K2; Carlton et al (1988); Examination of the Reproductive Effects of Tricresyl Phosphate Administered to Long-Evans Rats

No NOAEL is identified.

Summary:

Sperm concentration, motility, and progressive movement were decreased in the male rats that received 200mg/kg/day. A dose-dependent increase in abnormal sperm morphology was observed in the males from both treatment groups. The number of female rats delivering live pups was severely decreased by TCP exposure. Litter size and pup viability were decreased in the 400 mg/kg/day dose group. Pup body weight and developmental parameters were unaffected by TCP exposure. Significant histopathological changes were observed in the testes and epididymides of male rats and in the ovaries of female rats exposed to TCP. There was no NOAEL identified in this study.

Sup, K2; Latendresse, J. R. et al. (1994); Reproductive Toxicity of Butylated Triphenyl Phosphate and Tricresyl Phosphate Fluids in F344 Rats

No NOAEL is identified.

Summary:

The effects of tricresyl phosphate (TCP) on reproduction were studied in F344 rats using a modification of the National Toxicology Program's Continuous Breeding Protocol. Groups of breeding pairs received single daily oral doses of an equal volume of either 0 or 0.4 g TCP/kg in sesame oil for up to 135 days. A naive control group allowed to breed, but not dosed or handled daily, demonstrated that daily dosing and handling of the rats had no effect on reproduction. The fertility index and number of litters born were significantly decreased in rats exposed to 0.4 g TCP/kg. The number of pups per litter was significantly decreased in the TCP group. A crossover mating experiment using 0.4 g TCP/kg/day group mated with vehicle controls, demonstrated that TCP caused 100% infertility in male rats but did not affect reproduction in females. Both sexes of rats in the crossover experiment with TCP had significant decreases in terminal body weights and increases in adrenal gland and liver weights. TCP-dosed male rats had significantly decreased testicular and epididymal weights. TCP-dosed female rats had increased ovarian weights.

Sup, K2; Somkuti, S. G. et al. (1987); Reproductive Tract Lesions Resulting from Subchronic Administration (63 Days) of Tri-o-cresyl Phosphate in Male Rats

NOEL (Tri-o-cresyl Phosphate): 10 mg/kg/ day

Summary:

An initial dose-range pilot study where animals were gavaged with between 100 and 1600 mg tri-o-cresylphosphate (TGCP)/kg/day for 14 days resulted in decreased epididymal sperm density and disruption of the seminiferous epithelium in 100% of treated animals. A subchronic 63 day study (reflecting the 49day length of the rat seminiferous epithelium cycle plus the 14-day transit time of spermatids through the epididymis was initiated. Dose-dependent (10 to 100 mg TOCP/kg/day) decreases in cauda epididymal sperm motility and density, testicular enzyme activities, and alterations in sperm morphology were observed. Concurrent pair-fed controls (matched to the highest dose group, 100 mg TOCP/kg/day) indicated that weight loss resulting from TOCP administration had minimal contributory effects to the testicular toxicity seen. Plasma o-tocopherol acetate (vitamin E) and testosterone concentrations were unaffected. This study established 25 mg/kg as the lowest dose, which produced testicular toxicity and 10 mg/kg as a no-observable effect dose under these conditions. These data suggest that TOCP interferes with spermatogenic processes and sperm motility directly and not via an androgenic mechanism or decreased vitamin E availability.

Tri-p-cresyl phosphate (TPCP), the non-neurotoxic structural analog of TGCP, produced no toxic effects, demonstrating the necessity of the ortho-cresol moiety for induction of damage.

Sup, K2; Somkuti, S. G. et al. (1991); Light and Electron Microscopic Evidence of Tri-o-cresyl Phosphate (TOCP)-Mediated Testicular Toxicity in Fischer 344 Rats

LOAEL (Tri-o-cresyl Phosphate): Testicular toxicity at only dose tested - 150 mg/kg body weight/day

Summary:

The onset and development of testicular lesions following tri-o-cresyl phosphate (TOCP) dosing have been documented through light and electron microscopic morphological studies. Male Fischer 344 rats (190-210 g body weight) were administered 150 mg TOCP/kg/day in corn oil for 1, 3, 5, 7, 10, 14, and 21 days. Vehicle-treated rats served as the control group. Sections of formaldehyde- and glutaraldehyde fixed, methacrylate-embedded testes showed, by Day 5, numerous spermatid heads apparently detached from tails lying at oblique angles near the basement membrane of the seminiferous tubules. Columnar and spherically shaped vacuoles of the epithelium, radiating from the basement membrane to the lumen of the tubules, were also observed. Electron micrographs revealed that these were localized in Sertoli cells. Widespread dilation of Sertoli cell smooth endoplasmic reticulum was also noted. By 7 days of treatment, residual body abnormalities were noted in stage VIII tubules, along with spermatocyte-derived multinucleated giant cells. The lesion progressed with increased vacuolation of the epithelium and numbers of abnormal residual bodies and giant cells, together with spermatid karyorrhexis (Days 10, 14, and 2 I). There was also an apparent decrease in sperm density/tubule with continued exposure: 90% of the seminiferous tubules were devoid of sperm by Day 14. These morphological results indicate an initial effect of TOCP on Sertoli cells. Spermatogenesis is affected as seen by the decrease in sperm density and increase in necrotic spermatids.

Sup, K2; Somkuti, S. G. et al. (1987); Testicular Toxicity Following Oral Administration of Tri-O-Cresyl Phosphate (TOCP) In Roosters

LOAEL (Tri-o-cresyl Phosphate):  Testicular toxicity at only dose tested - 100 mg/kg body weight/day

Summary:

Tri-o-cresyl phosphate (TOCP) is a neurotoxic organophosphorus compound. This organophosphorus compound-induced delayed neurotoxicity (OPIDN) has been previously studied in the chicken. Reports of neurotoxic agents causing adverse effects on the male reproductive system initiated the present study which was designed to examine the effects of TOCP on the rooster. Previous studies have demonstrated 100 mg TOCP/kg/day to be an OPIDN-inducing dose with minimal mortality in roosters. This dose level was administered to adult leghorn roosters (p.o., n= 10) for 18 consecutive days. By days 7-10 of the study, TOCP-treated birds exhibited limb paralysis characteristic of OPIDN. Analysis at termination revealed significant inhibition of neurotoxic esterase activity (NTE) in both brain and testis. There was also a slight decrease in brain acetylcholinesterase (Ache) activity. Sperm motility was shown to be greatly decreased. In addition, sections of formalin-fixed, methacrylate-embedded testes from TOCP-treated birds showed vacuolation of, and disorganization in the seminiferous epithelium. The marginal body weight decreases (17%) in treated animals were not considered to contribute to the testicular toxicity induced by TOCP.

Sup, K2; Somkuti, S. G. et al. (1987); Time Course of the Tri-o-cresyl Phosphate-induced Testicular Lesion in F-344 Rats: Enzymatic, Hormonal, and Sperm Parameter Studies

LOAEL (Tri-o-cresyl Phosphate):  Testicular toxicity at only dose tested - 150 mg/kg body weight/day

Summary:

This study was designed to follow the onset of the testicular lesion through possible changes in sperm numbers and production, serum hormones, and various enzyme activities. Rats were administered TOCP daily (150 mg/kg) for periods of 3, 7, 10, 14, or 21 days. Vehicle-treated animals served as controls. Sperm motility and sperm number per milligram cauda epididymis were both lower in treated animals by Day 10. Testicular weight to body weight ratio was significantly decreased only in the longest treatment duration animals (21 days). Testicular neurotoxic esterase and nonspecific esterase activities were also inhibited, while Beta-glucuronidase activity was not affected. Luteinizing and follicle stimulating hormone levels were normal, as were both serum and interstitial fluid testosterone concentrations. Sertoli cell fluid secretion, as measured by testis weight increase after efferent duct ligation, showed no significant changes. Other organs (spleen, liver, kidney, pancreas, small intestine, adrenal and pituitary glands) had no overt signs of pathology as observed by light microscopy in animals treated for 21 days. A separate group of animals was treated for 21 days and subsequently examined after 98 days of observation (two cycles of the rat seminiferous epithelium). No recovery of spermatogenesis was seen, indicating that the toxicity was irreversible at the dose used.

Conclusion:

Tricresyl phosphate has been demonstrated to induce 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 (Chapinet al. 1988).

 

In the study by Carltonet al. (1987), sperm concentration, motility, and progressive movement were decreased in the males given 200 mg/kg tricresyl phosphate, and a dose-dependent increase in abnormal sperm morphology was observed. The number of females delivering live young was severely reduced by exposure to 200 mg/kg tricresyl phosphate or more. Histological changes were observed in the testes and epididymides of the treated males and in the ovaries of treated females at 200 mg/kg tricresyl phosphate or more.

 

Tri-o-cresyl phosphate has been shown to interfere with spermatogenic processes and sperm motility. The threshold dose for observable testicular toxicity is 10-25 mg/kg per day for 63 days, with irreversible testicular toxicity occurring at 150 mg/kg per day for 21 days (Somkutiet al. 1987).

 

Atrophy of the seminiferous tubules in male rats and ovarian interstitial cell hypertrophy was noted in 90-day subchronic toxicity studies.

 

The LOAEL for reproductive toxicity of tricresyl phosphate appears to be 0.05 per cent in the feed or 62.5 mg/kg/day. The LOAEL for tri-o-cresyl phosphate has been measured as 10-25 mg/kg/day.

 

Short description of key information:

Reproduction effects are discussed below.

Justification for selection of Effect on fertility via oral route:

Effects were noted at all dose levels in both studies utilised for the assessment; in the main, these were related to male reproductive effects and included such effects as sperm concentration and morphology and sperm motility being decreased in the dose groups tested. As effects were noted at all dose levels, it was not possible to determine a NOEC for this this endpoint on the basis of the studies included.The LOAEL for reproductive toxicity of tricresyl phosphate appears to be 0.05 per cent in the feed or 62.5 mg/kg/day.

Effects on developmental toxicity

Description of key information

Developmental effects are discussed below.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 19 2004 to March 21 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
minor deviations in timing of 3 observations; animal room humidity exceeded the limits specified in the guideline by 3% for 1 day; in the opinion of the Study Director, these minor deviations did not affect the quality or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
On February 3 (41 males) and February 10 (165 females), 2004, Sprague-Dawley (Crl: CD (SD)IGS BR) rats were received from Charles River Laboratories, Raleigh, North Carolina. All female rats were approximately nine weeks old at arrival, and ten weeks old at breeding. The male rats, utilized for mating purposes only, were approximately ten weeks old at arrival and 12 weeks old at breeding. Only females with positive evidence of mating (vaginal plug or sperm) were selected for study. These animals were weighed on Day 0 of gestation which was defined as the day when evidence of mating was observed. The females weighed between 187 to 267 g on Day 0 of gestation. During the nine to 16-day acclimation period, all rats were observed daily for any clinical signs of disease.
Mated female rats were given a clinical examination on Day 0 of gestation and only females considered suitable, based on the results of these examinations, were included in the selection process. Animals were sorted into treatment groups using a simple randomization procedure on the day they mated. After the required numbers of mated females were sorted among the groups, the males were euthanized via carbon dioxide inhalation and discarded. Extra female rats obtained but not used on study, were either euthanized via carbon dioxide inhalation and discarded or transferred to the stock colony.
Each female rat was assigned an animal number to be used in Provantis™ and implanted with a microchip bearing a unique identification number. Each cage was identified by the study number, animal number, group number, and sex. The individual animal number plus the study number comprised a unique identification for each rat. Animal identification was verified during the conduct of the study, as documented in the study data.
From acclimation until euthanasia, the rats were individually housed in suspended, stainless steel, wire-mesh type cages, except during pairing when the females were housed in similar cages overnight with males (1 :1). Fluorescent lighting was provided for approximately 12 hours per day via automatic timer. Throughout the study, all rats were kept in an environmentally controlled room. Temperature and relative humidity in the animal room were monitored and recorded daily, and maintained between 64 to 77°F and 32 to 73%, respectively.
Diet (meal lab Diet Certified rodent diet #5002, PMI Nutrition International, Inc.) and tap water were available ad libitum during the course of the study. Documentation of lot numbers of the basal diet used is retained in the study file. Analytical certifications of each diet lot were performed by the manufacturer and are maintained in the Archives. Water was supplied using an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of this study.

Mated female rats were given a clinical examination on Day 0 of gestation and only females considered suitable, based on the results of these examinations, were included in the selection process. Animals were sorted into treatment groups using a simple randomization procodure on the day they mated. After the required numbers of mated females were sorted among the groups, the males were euthanized via carbon dioxide inhalation and discarded. Extra female rats obtained but not used on study, were either euthanized via carbon dioxide inhalation and discarded or transferred to the stock colony.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Justification for Route of Administration: the oral route is one of the potential routes of human exposure to this test article.

Justification of Dose Levels: the dose levels were selected by the Sponsor, or in consultation with the Sponsor, on the basis of available date from previous studies.

Vehicle and Test Article Preparation: To prepare the vehicle, the required amount of corn oil was dispensed into amber glass containers prior to handling the test article. The vehicle was dispensed weekly and stored refrigerated. To prepare the test article formulations, the required amount of test article, TCP, was weighed directly into a beaker. Vehicle was added to the beaker and the contents were stirred using a magnetic stir bar and stir plate until well mixed. The mixture was transferred into a graduated cylinder. The beaker was rinsed with additional vehicle and the rinse was transferred into the cylinder. More vehicle/was added to the cylinder to yield the required amount of prepared test article. The cylinder was shaken thoroughly and the mixture was dispensed into a beaker. The contents of the beaker were stirred using a magnetic stir bar and stir plate, and dispensed into amber glass containers using a syringe. The test article formulations were prepared weekly and stored refrigerated. Each formulation was prepared separately. The test article was used as received from the Sponsor and no adjustment was made for purity.

Administration: Test article and vehicle control administration began on Gestation Day 0 and continued through to include Day 19. The test article was administered to the treated groups via oral gavage once per day at approximately the same time each day at respective dose levels of 20, 100, 400, and 750 mg/kg/day at a dose volume of 5 mL/kg/dose. The control animals received the vehicle, com oil, at the same volume, duration, and dosing regimen as the treated animals. Individual doses were based on the most recent body weight. The vehicle and test article were administered via oral gavage using an appropriately sized plastic disposable syringe attached to a Fuchigami dosing needle. The test article formulations were stirred continuously during test article administration using a magnetic stir bar and stir plate.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dosing Formulations
Homogeneity: Homogeneity of the test article in the vehicle at similar concentrations and batch sizes used in this study was established in a previously conducted study (MPI Research Study Number 1038-002).
Stabilty: Refrigerated stability of the test article formulations for at least 14 days at concentration levels of 10 to 200 mg/mL was established in a previously conducted study (MPI Research Study Number 1038-001). Samples (5 mL/sample) of the low concentration formulation (4.0 mg/mL) from Week 1 were collected from the container with a syringe and placed in an amber glass bottle. The samples were stored refrigerated for up to 14 days, and delivered to MPI Analytical after 7 and 14 days for analysis of the stability of the test article in the vehicle.
Concentration: Samples (5 mL/sample) of the test article formulations at each concentration were collected from the preparations for Weeks 1 and 4 of study. The samples were collected from the container, while stirring, with a syringe, placed into amber glass bottles, and stored refrigerated until analyzcd for test article concentrations. Additional samples (2 mL/sample) of the test article formulations at each concentration were collected from Week 3 preparations and retained frozen for possible further analysis.
Analyses: All analytical work was conducted by MPI Analytical, a division of MPI Research, using the methodology from an analytical method validated by KAR Laboratories, Kalamazoo, Michigan. MPI Analytical conducted a partial (transfer) validation in accordance with the appropriate SOPs, using validated procedures from KAR Laboratories. All samples were analyzed using the procedures validated by MPI Analytical.
Reserve Sample and Test Article Disposition: a reserve sample from the lot ofTCP used in this study was taken and archived. The remaining test article will be returned to a Sponsor-designated location after completion of the study.

A High-Performance Liquid Chromatography - Ultraviolet (HPLC-UV) assay has been validated for the test article, tricresyl phosphate (TCP), in corn oil for concentrations ranging from 2.5 to 200 mg/mL. The validation procedure consisted of four separate validation runs occurring over separate days. The first run consisted of six injections of a working RF standard, a check standard, a diluent blank, a matrix blank, and system performance checks. The first run included triplicate dilutions taken from each of three Quality Control (Qc) dose preparations at approximately 2.5 and 20 mg/mL. The second run included triplicate dilutions taken from each of three QC dose preparations at approximately 2.5, 20, and 200 mg/mL. The third run included re-prepared triplicate dilutions of the 200 mg/mL QC dose concentration. Autosampler stability samples and samples for determining the stability under the conditions of administration were analyzed during the second, third, and fourth analytical runs. The selectivity, linearity, range, accuracy, and precision of the method were evaluated. Linearity around working response factor (RF) was determined at +/- 30%. All coefficients of
determination (R2) values were greater than 0.99. The calculated concentrations of the response factor standards over the four runs ranged from - 1.2 to 1.0% RE. Analysis of the matrix was performed over three validation runs and analysis of the diluents blank was performed over all validation runs. None of the matrix blank or diluent assays contained any interferences within the retention region ofTCP, indicating that the method was selective for quantitation. The recoveries of TCP in the QC preparations ranged from 97.7 to 101.6% at 2.5 mg/mL, 96.4 to 103.2% at 20 mg/mL, and 94.1 to 102.3 % at 200 mg/mL, over the three validation runs. The intra-day accuracy (%RE) of the QC samples ranged -0.1 to 1.6% at 2.5 mg/mL and -0.1 to 3.2% at 20 mg/mL for the first validation run and ranged -2.3 to -1.0% at 2.5 mg/mL, -3.6 to -3.3% at 20 mg/mL, and -5.9 to -3.1 % at 200 mg/mL for the second validation run, and -2.4 to 2.3% at 200 mg/mL for the third validation run. The intra-day precision (%RSD) of the QC samples were 0.9 and 0.7% RSD at 2.5 mg/mL, 1.7 and 0.1 % RSD at 20 mg/mL, and 2.6 and 1.5% at 200 mg/mL, for the validation runs. Pre-processed QC samples were stable at ambient temperature over the 6 days tested. The post-processed samples and selected standards were stable at ambient temperature over the 7 days tested.
Details on mating procedure:
Female rats were housed together with an untreated male rat (1 :1i) used specifically for breeding. Males were of the same strain and from the same source as the females. Mating was established by daily inspection for a copulatory plug in the vagina or microscopic observation of sperm in the vaginal rinse. Evaluations for evidence of mating was performed early each morning before 9:00 AM. The day evidence of mating was confirmed wil be designated Day 0 of gestation. Once all mated females have been identified each day they weresorted into appropriate groups.
Duration of treatment / exposure:
Test article and vehicle control administration began on Gestation Day 0 and continued through to include Day 19.
Frequency of treatment:
The test article was administered to the treated groups via oral gavage once per day at approximately the same time each day.
Duration of test:
28 days
Remarks:
Doses / Concentrations:
20 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Number of animals
Group Dose level mg/kg/day Initial Laparohysterectomy/necroscopy Microscopic pathology
1 0 25 25 As required
2 20 25 25 As required
3 100 25 25 As required
4 400 25 25 As required
5 750 25 25 As required
Maternal examinations:
In-life Examinations
Mortality and Cageside Observations: All rats were observed twice each day for morbidity, mortality, signs of injury, and availability of food and water.
Detailed Clinical Examinations: Detailed clinical examinations were conducted daily from Gestation Days 0 through 20. Each rat was removed from the cage and given a detailed clinical examination, approximately one hour postdose. Any pretest observations and observations conducted on the male rats used for breeding purposes are not reported, but are maintained in the study file.

Body Weights and Body Weight Changes: Individual body weights were recorded on Gestation Days 0, 3,6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following gestation day intervals: 0-3, 3-6, 6-9, 9- 1 2, 12-15, 15-18, 18-20, and 0-20. Adjusted body weight (Day 20 gestation body weight minus the gravid uterine weight) and adjusted body weight change (Days 0-20 of gestation) were also calculated. Any pretest body weights and body weights recorded for the male rats used for breeding purposes are not reported, but are maintained in the study file.
Food Consumption: Food consumption was recorded on the corresponding body weight days and calculated for the following intervals: Gestation Days 0-3, 3-6,6-9,9-12, 12-15, 15-18, 18-20, and 0-20.

Postmortem Study Evaluations
Maternal Necropsy: A complete necropsy was performed on all dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. Gross lesions were saved in 10% neutral buffered formalin, and the carcasses were discarded.

Ovaries and uterine content:
Ovarian and Uterine Examinations: On Day 20 of gestation, each female was euthanized by carbon dioxide inhalation and immediately subjected to a laparohysterectomy. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous abnormalities. The abdominal cavity was then opened and the uterus was exposed. The uterus was excised and the gravid uterine weight recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, position of the cervix, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded. The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were grossly examined. Before the umbilical cord was cut
on each fetus, it was momentarly clamped with forceps to prevent bleeding and promote clotting. After examination, the uterus was discarded. Each implant was characterized as either a viable or nonviable fetus, or either an early or late resorption. Viable fetuses responded to touch while nonviable fetuses did not and showed no signs of autolysis. Early resorptions were characterized as implantation sites consisting of tissues but no recognizable fetal characteristics, while late resorptions displayed recognizable fetal characteristics, but were undergoing the process of autolysis. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. If no foci were seen, the female was considered not pregnant and all data were excluded from statistical analysis.
Fetal examinations:
Teratologic Examinations: Fetuses were individually weighed, sexed externally, tagged for identification, and examined for external malformations and variations. Approximately one-half of the fetuses in each litter were placed in Bouin's solution for subsequent soft tissue examination using the Wilson razor-blade sectioning technique. The remaining fetuses were fixed in alcohol, processed for Alizarin Red Sand Alcian blue staining, and cleared with glycerin for subsequent skeletal examination of bone and cartilage. Fetal findings were classified as malformations or developmental variations under the supervision of a developmental toxicologist.
Statistics:
The statistical analyses employed for various endpoints are listed below. (Control group = 1, treatment groups = 2,3,4,5)
Parental in-life data
Endpoints: gestation body weights, gestation body weight increases, gestation food consumption, adjusted body weights, adjusted body weight changes - statistical test: group pair-wise comparisons.
Fertility Indices
Endpoint: Pregnancy index - statistical test: Fisher's exact test
Uterine and Ovarian exam
Endpoints: Gravid uterine weights, Corpora lutea/dam, total implantations/dam, Litter size (per dam), viable fetuses/dam, total number resorptions/dam, number early resorptions/dam, number late resorptions/dam - statistical test: group pair-wise comparisons.
Endpoints: fetal sex ratio (% males/litter), % preimplantation loss (mean/dam), % postimplantation loss (mean/dam) - statistical test: arcsin-square root transformation.
Endpoints: malformations by finding and exam type (external, visceral and skeletal)-litter incidence, variations by finding and exam type (external, visceral and skeletal)-litter incidence, total malformations by finding and exam type (external, visceral and skeletal combined)-litter incidence - statistical test: Fisher's exact test (Fetal and litter incidences were reported but only the litter incidences were statistically analysed).
Endpoint: non-viable fetuses per dam - statistical test: descriptive statistics.
Endpoint: mean fetal body weights - statistical test: covariate analysis.

Indices:
Mean Fetal Body Weights

The mean fetal body weights and litter sizes were collected from each dam. As outlined in the protocol, the analysis for fetal bodyweights included tests to determine if it was appropriate to conduct an analysis of covariance using litter size as a covariate. If the assumptions for the covariate failed, the model was run with just treatment as an effect and a follow-up group pair-wise analysis was run. If the assumptions did not fail, the model was run with treatment and litter size and pairwise comparisons were made using Dunnett's test.

Upon examination of the data from the above analysis, a follow-up trend analysis was requested.

A dose-response analysis (linear trend test) was conducted using linear contrasts under the appropriate statistical modeL. The dose-response analysis was run for males, females and pooled sexes.

Males
The assumptions on the analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. Treatments 2, 4 and 5 were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattem in the data across treatments (p = -(0.0001).

Females
The assumptions on analysis of covariance were met and thus, an analysis of covariance was performed with the litter size as the covariate. Pair-wise comparisons showed that all treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p = -(0.0001).
Historical control data:
Yes; documented in Appendix L of the report.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, the only maternal toxicity seen was an increased frequency of salivation following dosing. At the higher dose levels evaluated (400 and 750 mg/kg/day), the frequency of animals with sparsc amounts of hair in the abdominal and lumbar regions, ventral surface, and hind limbs, and unkempt appearance was also increased. Lower body weights and body weight gain during gestation were seen at 400 and 750 mg/kg/day and reduced food consumption was seen at 750 mg/kg/day. Excessive feed spilage was also seen with increased frequency among females in the 400 and 750 mg/kg/day groups. No effect of treatment was evident from maternal macroscopic findings, uterine implantation data, fetal sex ratios, or fetal malformation data.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
See below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See below
Urinalysis findings:
no effects observed
Description (incidence and severity):
See below
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
no effects observed
Description (incidence and severity):
See below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
See below
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See below
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See below
Early or late resorptions:
no effects observed
Description (incidence and severity):
See below
Dead fetuses:
no effects observed
Description (incidence and severity):
See below
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See below
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See below
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See below
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In life examinations
No effect of treatment with TCP was evident from mortality data. One female (animal number 339) in the 20 mg/kg/day group was found dead on Gestation Day (GD) 4. Clinical findings seen in this animal included rapid breathing (GO 2-3), red material around nose (GD 1-3), skin cold to touch (GD 1-3), skin discolored red on the forepaws (GD 1-2). At necropsy, adhesions involving the heart, lungs and thoracic cavity, and a lung mass were noted. These were considered suggestive of a dosing injury. All remaining animals in the control and treated groups survived to scheduled euthanasia.
No effect of treatment at the 20 mg/kg/day dose level was evident from clinical evaluations. Findings seen in these animals occurred at low incidence or with similar frequency as controls and were considered unrelated to treatment. Salivation was seen with increased frequency in groups treated with TCP at dose levels of 100,400, and 750 mg/kg/day. Salivation was seen at least once during the daily clinical examinations in 11, 24, and 25 animals in the 100, 400, and 750 mg/kg/day groups, respectively. It was not seen among the control or 20 mg/kg/day animals. Other clinical findings seen with increased frequency in the 400 and 750 mg/kg/day groups and considered related to treatment were sparse amounts of hair in the abdominal and lumbar regions, ventral surface and hind limbs, and unkempt appearance.
No effect of treatment with TCP at dose levels of 20 and 100 mg/kg/day was evident from gestation body weights or body weight gain. At the 400 and 750 mg/kg/day dose levels, body weights on GD 20 were statistically lower than controls and body weight gain was statistically lower than controls over GD 15-18, 18-20, and 0-20. A dose-responsiveness was evident in these data and the 750 mg/kg/day group actually experienced a mean weight loss of 0.3 grams over GD 18-20 in comparison to a 35 gram weight gain in controls. The 400 mg/kg/day animals experienced a weight gain of 22 grams over this period. Weight gain over the entire GD 0-20 period was 126.5 grams in the 400 mg/kg/day group, about 14% lower than controls (146.6 grams), and 94.9 grams in the 750 mg/kg/day group, a 35% reduction from controls.
No effect of treatment with TCP at dose levels of20 and 100 mg/kg/day was evident from gestation food consumption data. No clear effect on gestation food consumption was evident at the 400 mg/kg/day dose level, but at 750 mg/kg/day, food consumption was statistically lower than controls over GD 0-3 and 18-20. The lower food consumption over this latter interval (15.7 g/animal/day vs.24.1 g/animal/day in controls) was consistent with the weight loss for this group over this interval. Noteworthy from the food consumption data for these treatment groups (400 and 750 mg/kglday) was the increased spillage seen among these animals. Eleven animals (44%) in the 400 mg/kg/day group and 10 animals (40%) in the 750 mg/kg/day group were noted with excessive spillage of feed at one or more recording intervals over the 20-day gestation period. Similar spillage was not seen among the 25 control females or among the females in the 20 and 100 mg/kg/day groups. The increased frequency of spillage at these dose levels appeared to be treatment-related but its toxicological significance, if any, is unclear.
Postmortem study evaluations
No effect of treatment was evident from maternal macroscopic examinations. The few macroscopic findings seen among the treated animals occurred at low incidence and were considered unrelated to treatment.
Pregnancy rates ranged from 92 to 100% among all the groups and provided 24, 23, 24, 25, and 25 GD 20 litters for evaluation in the control, 20, 100, 400, and 750 mg/kg/day groups, respectively. No effect of treatment with TCP at a dose level up to and inclusive of750 mg/kg/day was evident from uterine implantation data. The mean number of corpora lutea, uterine implantation sites, fetuses, and resorption sites in the treated groups was comparable to controls. Likewise, the mean pre- and post-implantation loss indices for the treated groups were comparable to controls.
Gravid uterine weights, adjusted GD 20 body weights, and adjusted body weight gain over GD 0-20 were comparable to controls in the 20 and 100 mg/kg/day groups. In the 400 and 750 mg/kg/day groups, gravid uterine weights were statistically lower than controls. Adjusted GD 20 body weights and adjusted body weight gain (GD 0-20) were also lower than control in these same groups; however, only at the 750 mg/kg/day dose level were these differences statistically significant. The lower gravid uterine weights in the 400 and 750 mg/kg/day groups were consistent with lower fetal body weights.

Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights in the treated groups were lower than controls. At the 20 and 100 mg/kg/day dose levels these differences were slight (-4.4 to -6.3%) in comparison to controls and of greater magnitude, -9% and -18% in the 400 and 750 mg/kg/day groups, respectively. In most cases these differences in fetal weights, distinguished by sex and for both sexes combined, were statistically significant in comparison to controls. A dose-response analysis (linear trend test) of fetal body weights was also conducted for males, females, and pooled (combined) sexes. For all of these comparisons (males, females, pooled sexes), the trend analyses revealed a significant linear pattern in the data across treatments (p=<0.0001). Mean fetal body weights in the treated groups were outside the low range of recent historical control data for this laboratory, while mean fetal body weights for controls were within this historical range. The lower fetal body weights seen in the treated groups were considered indicative of a treatment-related response.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See below
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See below
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See below
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See below
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
See below
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No effect of treatment with TCP was evident from the fetal skeletal malformation data. The few skeletal malformations seen among fetuses in the 100, 400, and 750 mg/kg/day groups occurred at low incidence and were considered spontaneous and unrelated to treatment. Skeletal malformations of the head (squamosal misshapen and small mandible) were seen in the one fetus in the 100 mg/kg/day group noted externally with mouth, jaw, and eye malformations. In the 400 mg/kg/day group, one fetus was noted with skeletal malformations involving the head (jugal absent and squamosals small). In the 750 mg/kg/day group, absent rib and defects of the lumbar and thoracic vertebrae were seen in one fetus. No skeletal malformations were seen among fetuses in the control and 20 mg/kg/day group. A delay in ossification, as indicated from an increase in unossified/incompletely ossified bones (i.e., ossification variations), was apparent at the 750 mg/kg/day dose level. The incidence of litters containing at least one fetus with an ossification variation in this group was 100%, which differed statistically from the 75% incidence in controls. There was also an increase in litter incidence of incompletely ossified skull bones (occipitals, frontals, parietals, and supraoccipitals) and unossified sternebrae in this group, consistent with a delay in ossification, but only for the latter was the difference from controls statistically significant. At the lower dose levels, some variability was seen in the litter incidence of several ossification variations and in the incidence of litters containing fetuses with variations, but these differences from controls were not statistically significant, and no clear effect of treatment was evident.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See below
Visceral malformations:
no effects observed
Description (incidence and severity):
See below
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal body weights in the treated groups were lower than controls. At the 20 and 100 mg/kg/day dose levels these differences were slight (-4.4 to -6.3%) in comparison to controls and of greater magnitude, -9% and -18% in the 400 and 750 mg/kg/day groups, respectively. In most cases these differences in fetal weights, distinguished by sex and for both sexes combined, were statistically significant in comparison to controls. A dose-response analysis (linear trend test) of fetal body weights was also conducted for males, females, and pooled (combined) sexes. For all of these comparisons (males, females, pooled sexes), the trend analyses revealed a significant linear pattern in the data across treatments (p=<0.0001). Mean fetal body weights in the treated groups were outside the low range of recent historical control data for this laboratory, while mean fetal body weights for controls were within this historical range. The lower fetal body weights seen in the treated groups were considered indicative of a treatment-related response.
No effect of treatment with TCP at a dose level up to and inclusive of 750 mg/kg/day was evident from fetal sex ratios (% male fetuses/litter). Mean sex ratios in the treated groups ranged from 44.2 to 53.7% and were considered comparable to the 47.6% in controls.
No effect of treatment with TCP was evident from fetal external examinations. Gastroschisis was seen in one fetus in the 20 mg/kg/day group (litter incidence 4.3%) and with edema in three fetuses from a single litter in the 400 mg/kg/day group (litter incidence 4.0%). Gastroschisis has not been noted in recent historical control data for the laboratory, but in the absence of this or similar malformations among the 750 mg/kg/day fetuses, its occurrence in this study was considered spontaneous and unrelated to treatment. Malformations of the mouth, jaw, and eyes were seen in one fetus in the 100 mg/kg/day group. In the absence of similar malformations among fetuses at the higher dose levels, this too was considered a spontaneous occurrence and unrelated to treatment. No malformations were secn in the control and 750 mg/kg/day fetuses at exteral examination.
No effect of treatment with TCP was evident from the fetal visceral examinations. No malformations were seen at visceral examination of the control and treated fetuses. Increased renal pelvic cavitation, a visccral variation, was seen with low incidence of occurrence in the control, 100, 400, and 750 mg/kg/day groups. These incidences in the treated groups were comparable to controls and no effect of treatment was evident. This finding was not seen among fetuses in the 20 mg/kg/day group.
No effect of treatment with TCP was evident from the fetal skeletal malformation data. The few skeletal malformations seen among fetuses in the 100, 400, and 750 mg/kg/day groups occurred at low incidence and were considered spontaneous and unrelated to treatment. Skeletal malformations of the head (squamosal misshapen and small mandible) were seen in the one fetus in the 100 mg/kg/day group noted externally with mouth, jaw, and eye malformations. In the 400 mg/kg/day group, one fetus was noted with skeletal malformations involving the head (jugal absent and squamosals small). In the 750 mg/kg/day group, absent rib and defects of the lumbar and thoracic vertebrae were seen in one fetus. No skeletal malformations were seen among fetuses in the control and 20 mg/kg/day group. A delay in ossification, as indicated from an increase in unossified/incomplete1y ossified bones (i.e., ossification variations), was apparent at the 750 mg/kg/day dose level. The incidence of litters containing at least one fetus with an ossification variation in this group was 100%, which differed statistically from the 75% incidence in controls. There was also an increase in litter incidence of incompletely ossified skull bones (occipitals, frontals, parietals, and supraoccipitals) and unossified sternebrae in this group, consistent with a delay in ossification, but only for the latter was the difference from controls statistically significant. At the lower dose levels, some variability was seen in the litter incidence of several ossification variations and in the incidence of litters containing fetuses with variations, but these differences from controls were not statistically significant, and no clear effect oftreatment was evident.
No adverse effect of treatment with TCP at a dose level up to and inclusive of 750 mg/kg/day was evident from fetal malformation data. The overall incidence of litters containing at least one fetus with a malformation from either the external, visceral, or skeletal examinations was low in each of the treated groups (ranging from 4.0 to 4.3%) and did not differ statistically from the 0% incidence in controls.

Key result
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: see explanation below
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
yes

The mean fetal body weights and litter sizes were collected from each dam. As outlined in the protocol, the analysis for fetal bodyweights included tests to determine if it was appropriate to conduct an analysis of covariance using litter size as a covariate. If the assumptions for the covariate failed, the model was run with just treatment as an effect and a follow-up group pair-wise analysis was run. If the assumptions did not fail, the model was run with treatment and litter size and pairwise comparisons were made using Dunnett's test. Upon examination of the data from the above analysis, a follow-up trend analysis was requested. A dose-response analysis (linear trend test) was conducted using linear contrasts under the appropriate statistical model. The dose-response analysis was run for males, females and pooled sexes.

 

Males: The assumptions on the analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. Treatments 2, 4 and 5 were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattem in the data across treatments (p = <0.0001).

 

Females: The assumptions on analysis of covariance were met and thus, an analysis of covariance was performed with the litter size as the covariate. Pair-wise comparisons showed that all treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p = <0.0001).

 

Males and Females Combined: The assumptions on analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. All treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p<0.0001).

Conclusions:
In this oral rat developmental toxicity study with tricresyl phosphate (TCP), the No-Observable-Effect Level (NOEL) for maternal toxicity was 20 mg/kg/day. At 100 mg/kg/day, the only maternal toxicity seen was an increased frequency of salivation following dosing. At the higher dose levels evaluated (400 and 750 mg/kg/day), the frequency of animals with sparse amounts of hair in the abdominal and lumbar regions, ventral surface, and hind limbs, and unkempt appearance was also increased. Lower body weights and body weight gain during gestation were seen at 400 and 750 mg/kg/day and reduced food consumption was seen at 750 mg/kg/day. Excessive feed spilage was also seen with increased frequency among females in the 400 and 750 mg/kg/day groups. No effect of treatment was evident from maternal macroscopic findings, uterine implantation data, fetal sex ratios, or fetal malformation data. Lower fetal body weights were seen at all dose levels evaluated. An increase in variations during the skeletal examinations indicative of a delay in fetal ossifìcation was seen at 750 mg/kg/day. Thus, the 20 mg/kg/day dose level was determined to be the Lowest-Observable-Adverse-Effect Level (LOAEL) for developmental toxicity as a NOEL could not be determined due to decreased fetal body weights in all treated groups.
Executive summary:

In this oral rat developmental toxicity study with tricresyl phosphate (TCP), the No-Observable-Effect Level (NOEL) for maternal toxicity was 20 mg/kg/day. The 20 mg/kg/day dose level was also determined to be the Lowest-Observable-Adverse-Effect Level (LOAEL) for developmental toxicity as a NOEL could not be determined due to decreased fetal body weights in all treated groups.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the oral rat developmental toxicity study with tricresyl phosphate (TCP), the No-Observable-Effect Level (NOEL) for maternal toxicity was 20 mg/kg/day. At 100 mg/kg/day, the only maternal toxicity seen was an increased frequency of salivation following dosing. At the higher dose levels evaluated (400 and 750 mg/kg/day), the frequency of animals with sparse amounts of hair in the abdominal and lumbar regions, ventral surface, and hind limbs, and unkempt appearance was also increased. Lower body weights and body weight gain during gestation were seen at 400 and 750 mg/kg/day and reduced food consumption was seen at 750 mg/kg/day. Excessive feed spilage was also seen with increased frequency among females in the 400 and 750 mg/kg/day groups. No effect of treatment was evident from maternal macroscopic findings, uterine implantation data, fetal sex ratios, or fetal malformation data. Lower fetal body weights were seen at all dose levels evaluated. An increase in variations during the skeletal examinations indicative of a delay in fetal ossifìcation was seen at 750 mg/kg/day. Thus, the 20 mg/kg/day dose level was determined to be the Lowest-Observable-Adverse-Effect Level (LOAEL) for developmental toxicity as a NOEL could not be determined due to decreased fetal body weights in all treated groups.

 

In the study by Chapinet al. (1988) the last litter born in the 98-day breeding phase was reared to 74 days and then mated within the control and two of the treatment groups (0.05 per cent and 0.1 per cent tricresyl phosphate). A decreased proportion of liveborn and reduced number of liveborn pups per litter was observed.

 

In the study by Carltonet al. (1987) described above, litter size and pup viability were decreased in the high-dose (400 mg/kg) group but pup bodyweight and developmental landmarks were unaffected by tricresyl phosphate exposure.

 

In the study by Latendresseet al. (1994) described above, repeated exposure to 0.4 g/kg tricresyl phosphate resulted in a significant decrease in numbers of live births when both parents were continuously treated. In the crossover phase, this parameter was not affected when treated females were considered but untreated females mated with treated male rats produced no litters.

Justification for selection of Effect on developmental toxicity: via oral route:

K1 study

Justification for classification or non-classification

Regulation (EC) No 1272/2008 - the CLP Regulation, states as follows:

 

3.7.2.5. Animal and experimental data

 

3.7.2.5.3. Adverse effects or changes, seen in short- or long-term repeated dose toxicity studies, which are judged likely to impair reproductive function and which occur in the absence of significant generalised toxicity, may be used as a basis for classification, e.g. histopathological changes in the gonads.

 

Furthermore, classification as Category 2 states as follows:

 

Suspected human reproductive toxicant Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.

 

Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

 

The effects of TCP on reproductive performance were evaluated in mice using a continuous breeding protocol.At the lowest dose investigated (equal to 62.5 mg/kg bw)sperm concentration and morphology were normal at termination, although motility was decreased compared to controls. Only at higher dosestricresyl phosphate has been demonstrated to induce functional and structural effects in the male reproductive system and functional reproductive impairment in females. This is in the form of atrophy of the seminiferous tubules in male rats and ovarian interstitial cell hypertrophy in females. Apart from effects on numbers of liveborn pups (probably related to maternal toxicity), only minor treatment-related developmental effects have been observed in the studies reported. 

 

The observations above were reported at systemically toxic TCP doses evidenced by body weight depression and adrenal histopathology. Effects on adrenals were reported in repeated dose toxicity studies at significantly (ca. 10-fold) lower doses ( LOAEL 7 mg/kg was taken for DNEL calculation) indicating that TCP is not a selective reproductive toxicant and effects on fertility are observed in the presence of systemic toxicity.

 

In addition, this data indicates that reproductive organs, and in particular male testicular effects are noted. However the data indicates that there is no clear evidence of an adverse effect on sexual function and fertility or on development, particularly as the majority of the testicular studies are conducted on the ortho isomer, which is not present in current TCP products, as discussed elsewhere in this dossier. As such, it is not appropriate to specifically state that the substance fulfils the criteria of Category 1B and instead, the substance should be placed into category 2 for reproduction effects. 

 

The above results triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

Repr. Cat 2; H361: Suspected of damaging fertility or the unborn child <testicular effects>

Additional information

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