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Key value for chemical safety assessment

Effects on fertility

Description of key information

For CDP (phenyl dicresyl phosphate) an OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) screening study according to OECD 422 is available. No generation study for CDP is available.

Based on the similar chemical structure and a trend in biological activity a category has been defined. The category consists of CDP (Diphenyl cresyl phosphate), TPP (Triphenyl phosphate) and TCP (Tricresyl phosphate). Structurally CDP (diphenyl cresyl phosphate) is more similar to TPP (triphenyl phosphate) than to TCP (tricresyl phosphate) because CDP (diphenyl cresyl phosphate) has only one cresyl group, whereas TPP (tricresyl phosphate) has 3 cresyl groups. A category justification document is attached in chapter 13.

Based on the category approach a read-across to studies from TPP and TCP is justified to fill the data gap of CDP concerning the sub-chronic study.

The following studies are available for category members.

Diphenyl cresyl phosphate was studied for oral toxicity in rats in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test at doses of 12, 60 and 300 mg/kg/day. Fertility and implantation indices decreased in the 300 mg/kg group. These were probably caused by dysspermatogenesis. In addition, the gestation index had a tendency to be low. However, there were no effects on other reproductive parameters such as copulation index, gestation length, number of corpora lutea, delivery index and parturition or maternal behavior.

Observation of neonates revealed no significant treatment-related effects in terms of the numbers of live offspring, the sex ratio, general condition, body weight or external features. There were no skeletal or visceral malformations. NOELs for reproductive and developmental toxicity are considered to be 60 mg/kg/day for parental males, and 300 mg/kg/day for parental females and offspring.

Fertility and developmental toxicity of triphenyl phosphate were examined in a dietary study in Sprague-Dawley rats at doses of 0, 0.25, 0.50, 0.75, 1.0% corresponding to 0, 166, 341, 516 or 690 mg/kg bw/day. Forty males and forty females per group were treated for 3 months and mated afterwards. Animals were treated further throughout mating and gestation and killed at day 20 of gestation.

Effects on fertility:  The study included treatment of males and females for three months prior to mating throughout gametogenesis and during mating and gestation. No significant differences were recorded in the number of corpora lutea, implants, implantation efficiency, viable fetuses and the number of early or late deaths between treated and control rats. No significant signs of parental toxicity were detected. As there were no effects on the litter size (indirectly measured by the number of viable fetuses and implants) and both sexes were treated in the study, these findings indicate that fertility is not adversely affected by TPP in male and female rats. The NOEL was 690 mg/kg bw/day.

The reproductive toxicity of tricresyl phosphate (<9% tri-o-cresyl phosphate) was evaluated in Long-Evans rats prior to and throughout breeding, during gestation, lactation, and the immediate post- weaning period. The results indicated that TCP adversely affects both male and female reproductive parameters.

F0 male rats showed adverse reproductive effects for all sperm parameters examined at the high dose level (200 mg/kg). Rats treated with 200 mg/kg TCP had reduced sperm concentration, motility, and velocity (65, 4, and 5% of control, respectively). A dose dependent increase in abnormal sperm morphology was observed for males in both TCP dose groups.

A 10-fold increase in the number of abnormal sperm forms was observed for high dose TCP males (200 mg/kg bw) and a 3-fold increase was observed for low dose TCP males (100 mg/kg bw) relative to controls.

TCP was not observed to have an adverse effect on mean testicular weight. Epididymal weights were reduced in 200 mg/kg dose group males.

Histopathologic evaluation revealed minimal-to-mild, but significant treatment-related lesions in the male reproductive tract. Minimal-to-mild necrosis and degeneration of seminiferous tubules, hypospermia in the epididymides, and increases in degenerate and immature spermatids in the seminiferous tubules and epididymides were observed in 200 mg/kg TCP-exposed males. Early sperm granulomas also were present in the seminiferous tubules of high dose males

Fertility rates were severely affected by TCP administration. The percent of sperm positive females littering (live or dead pups) dropped from 95% among control dams to 45 for females in the 200 mg/kg dose group. Only 1 female (5%) in the 400 mg/kg dose group delivered young; this litter contained only 3 pups (TOCP < 9%). There was no NOAEL identified in this study. The LOAEL was 100 mg/kg/day.

Based on the available data, there is a clear correlation between the exposure duration, the onset of effects concerning the applied dose and the tri-ortho-cresyl content of the applied test material.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Diphenyl cresyl phosphate was studied for oral toxicity in rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 12, 60, and 300 mg/kg/day.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on mating procedure:
no data
Duration of treatment / exposure:
Male, 45 days; Female from 14 days before mating to day 3 of lactation
Frequency of treatment:
daily
Details on study schedule:
no data
Remarks:
Doses / Concentrations:
0 (vehicle), 12, 60, 300 mg/kg/day
Basis:
other: gavage
No. of animals per sex per dose:
10 male and 10 female rats/dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Copulation index, gestation lenght, number of corpora lutea, delivery index and parturition and maternal behaviour.
Litter observations:
Number of offsprings, sex ratio, live birth index, viability index and body weights.
Postmortem examinations (parental animals):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Postmortem examinations (offspring):
External, visceral and skeletal malformations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1 JETOC/1997 (OECD 422 combined repeat dose and reproductive/developmental toxicity screening test).
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Atropie of of seminiferous tubules.
Reproductive performance:
no effects observed
Dose descriptor:
other: NOEL (fertility)
Effect level:
ca. 60 mg/kg bw/day
Sex:
male
Dose descriptor:
other: NOEL (fertility)
Effect level:
ca. 300 mg/kg bw/day
Sex:
female
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
other: Fertility and implantation indices decreased in the 300 mg/kg group. These were probably caused by dysspermatogenesis.
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No anomalies related to the test substance were detected in any of the offspring in terms of clinical signs, and external, visceral or skeletal features.
Histopathological findings:
no effects observed
Description (incidence and severity):
No anomalies related to the test substance were detected in any of the offspring in terms of clinical signs, and external, visceral or skeletal features.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Observation of neonates revealed no significant differences in the numbers of offspring or live offspring, the sex ratio, live birth index, viability index or body weights. Furthermore, no anomalies related to the test substance were detected in any of the offspring in terms of clinical signs, and external, visceral or skeletal features. The NOELs for reproductive and developmental toxicity are considered to be 60 mg/kg/day for parental males, and 300 mg/kg/day for parental females and offspring.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

NOELs for reproductive and developmental toxicity are considered to be 60 mg/kg/day for parental males, and 300 mg/kg/day for parental females and offspring.

Conclusions:
No anomalies related to the test substance were detected in any of the offspring in terms of clinical signs, and external, visceral or skeletal features.
Executive summary:

Diphenyl cresyl phosphate was studied for oral toxicity in rats in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test at doses of 12, 60 and 300 mg/kg/day.

Fertility and implantation indices decreased in the 300 mg/kg group. These were probably caused by dysspermatogenesis. In addition, the gestation index had a tendency to be low. However, there were no effects on other reproductive parameters such as copulation index, gestation length, number of corpora lutea, delivery index and parturition or maternal behavior.

Observation of neonates revealed no significant treatment-related effects in terms of the numbers of live offspring, the sex ratio, general condition, body weight or external features. There were no skeletal or visceral malformations.

NOELs for reproductive and developmental toxicity are considered to be 60 mg/kg/day for parental males, and 300 mg/kg/day for parental females and offspring.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Also assessed by OECD.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
Method: other: see freetext
GLP compliance:
not specified
Justification for study design:
Study is used as a read-across substance in the context of a category (see category justification document). For the category members the whole database with additional fertility studies has to be considered.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
exposure period 91 days
Details on mating procedure:
TPP was administered in the diet for 91 days in a subchronic study. At the completion of this study, females were mated with males from the same group. All remained on the same diet as in the subchronic study until day 20 of gestation when dams were sacrificed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Exposure period: 91 days
Duration of test: 3 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.25, 0.5, 0.75 or 1 % in feed (= 166, 341, 516, 690 mg/kg)
Basis:
no data
No. of animals per sex per dose:
Four treated groups and an untreated control each consisting of 40 rats/sex
Control animals:
yes, concurrent no treatment
Details on study design:
TPP was administered in the diet for 91 days in a subchronic study. At the completion of this study, females were mated with males from the same group. All remained on the same diet as in the subchronic study until day 20 of gestation when dams were sacrificed.
Parental animals: Observations and examinations:
see below
Oestrous cyclicity (parental animals):
see below
Sperm parameters (parental animals):
no effects
Litter observations:
no effects observed
Postmortem examinations (parental animals):
no adverse effects noted
Postmortem examinations (offspring):
no adverse effects noted
Statistics:
no data
Reproductive indices:
see below
Offspring viability indices:
see below
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
No findings reported. Food consumption increased in pregnant dams (not dose depedant).
Dose descriptor:
NOEL
Effect level:
690 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no adverse effects were reported
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Histopathological findings:
no effects observed
No adversed effects were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
690 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Examination of the foetuses indicated no effect on body weight, crown-rump length, and sex distribution. There were no external variations or skeletal variations which could be related to TPP exposure.
Critical effects observed:
no
Reproductive effects observed:
no
OBSERVATION: no findings reported
FOOD CONSUMPTION: increased in pregnant dams (not dose-dependent)
BODY WEIGHT: decreased during pregnancy (non-significant)
NECROPSY: no significant differences in number of corpora lutea, implants, implantation efficiency, viable fetuses and number of early or late deaths.
As there was no effect on the litter size (indirectly measured by the number of viable fetuses and implants) and both sexes were treated in the study, these findings indicate that fertility is not adversely affected by TPP in male and female rats.
parental NOEL = 690 mg/kg bw
Conclusions:
Parental NOEL = 690 mg/kg bw
Executive summary:

Fertility and developmental toxicity were examined in a dietary study in Sprague-Dawley rats at doses of 0, 0.25, 0.50, 0.75, 1.0% corresponding to 0, 166, 341, 516 or 690 mg/kg bw/day. Forty males and forty females per group were treated for 3 months and mated afterwards. Animals were treated further throughout mating and gestation and killed at day 20 of gestation.

Effects on fertility- The study included treatment of males and females for three months prior to mating throughout gametogenisis and during mating and gestation. No significant differences were recorded in the number of corpora lutea, implants, implantation efficiency, viable fetuses and the number of early or late deaths between treated and control rats. No significant signs of parental toxicity were detected. As there were no effects on the litter size (indirectly measured by the number of viable fetuses and implants) and both sexes were treated in the study, these findings indicate that fertility is not adversely affected by TPP in male and female rats. The NOEL was 690 mg/kg bw/day.

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
See attached justification.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
Neither clinical signs of toxicity nor body weight depression were observed among male or female Long-Evans rats exposed to 0, 100, or 200 mg/kg (males) or 0, 200, or 400 mg/kg (females) TCP in corn oil by gavage for up to 73 days. Observed sperm positive rates for TCP-exposed pairs were not different from that of controls. Fertility rates, however, were severely affected by TCP administration. The percent of sperm positive females littering (live or dead pups) dropped from 95% among control dams to 45% for females in the 200 mg/kg dose group. Only 1 female (5%) in the 400 mg/kg dose group delivered young; this litter contained only 3 pups. It was not clear if the pups had difficulty suckling or if the dam had no milk, but the pups had no milk in their stomachs, were dehydrated, and died on lactation day 5. F0 male rats showed adverse reproductive effects for all sperm parameters examined at the high dose level (200 mg/kg). Table 1 presents sperm concentration, motility, direct progressive movement (velocity), percent abnormal sperm morphology, and testicular and epididymal weights for each group. Rats treated with 200 mg/kg TCP had reduced sperm concentration, motility and velocity (65, 4, and 5% of control, respectively). A dose dependent -incease in abnormal sperm morphology was observed for males in both TCP dose groups. A 10-fold increase in the number of abnormal sperm forms was observed for high dose TCP males and a 3-fold increase was observed for low dose TCP males relative to controls. TCP was not observed to have an adverse effect on mean testicular weight. Epididymal weights were reduced (P < 0.05) in 200 mg/kg dose group males, however. Histopathologic evaluation revealed minimal-to-mild, but significant treatment-related lesions in the male reproductive tract. Minimal-to-mild necrosis and degeneration of seminiferous tubules, hypospermia in the epididymides, and increases in degenerate and immature spermatids in the seminiferous tubules and epididymides were observed in 200 mg/kg TCP exposed males. Early sperm granulomas also were present in the seminiferous tubules of high dose males. A dose-dependent diffuse vacuolar cytoplasmic alteration of ovarian interstitial cells and an impression of increased follicular and luteal activity as indicated by increased numbers of follicles and corpora lutea were observed among TCP-treated females, however.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified Generation not specified
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related
A total of 20 control pups died prior to weaning. Nineteen of the 20 pups died between days 4 and 12. The perinatal deaths included 4 males and 1female pup in 1 litter, 1 male pup in another litter, and 1 entire litter of 13 pups that died due to maternal neglect. All high dose TCP pups died. Mean litter size was decreased, but mean pup weight was unaffected by exposure to the low dose of TCP. The median day of eye opening (day 16) and the median day of observed vaginal patency (day 31-33) also were unaffected by chemical exposure.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remarks'
Reproductive effects observed:
not specified

The neurotoxicity of TCP (particularly TOCP) is well documented and has been characterized as a delayed appearance of weakness in the distal muscles (particularly of the lower extremities), gradually progressing to ataxia and weakness in upper limbs. Histologically, the lesion is characterized as a progressive dying back of the long axon of the nerves. Axons of the spinal cord and the periphery (i.e. sciatic nerve) are involved in the observed neuropathy. In contrast, the reproductive consequences of TCP exposure are only recently being explored. While Mele and Jensh described the lack of overt teratogenic effects of TOCP in Wistar rats, the animals in their study were exposed on gestation days 18 and19, too late in gestation to produce frank, grossly observable malformations. Tocco et al. (pers. commun., 1986) described an increase in minor skeletal variations in rat fetuses exposed to TOCP on gestation days 6-18. No effects on preimplantation loss, fetal death or resorption, or fetal gross or visceral morphology were observed. In the present study the reproductive toxicity of TCP(<9%TOCP) was evaluated in Long-Evans rats prior to and throughout breeding, during gestation, lactation, and the immediate post-weaning period. The results indicated that TCP adversely affects both male and female reproductive parameters. Comparable numbers of females were sperm-positive in all dose groups, indicating no adverse influence of TCP exposure on the estrus cycle, libido, or copulatory behavior. Histopathologic alterations int he ovaries of females exposed to 400 mg/kg TCP indicated a possible hormonal stimulation of the ovaries, however. Consistent with the findings of Somkuti et al who reported testicular degeneration, particularly associated with the Sertoli cells of TOCP-treated rats, as well asa decrease in sperm density and an increase in necrotic spermatids, and Carlton et al. who reported multifocal testicular degeneration in rats, the current study indicated a decrease in sperm concentration, and minimal-to mild histopathologic lesions in the testes and epididymides of TCP-exposed rats. The observed difference in the severity of response can most likely be attributed to differences in test article composition. Somkuti et al. administered pure TOCP, while the TCP used in the present study was less than 9%TOCP. The TCP mixture used in the previously described study by Carlton contained 100 x less TOCP than did the TCP used in the current study, but may have contained a higher percentage of ortho isomers other than the TOCP isomer.

Conclusions:
The reproductive toxicity of TCP (<9% TOCP) was evaluated in Long-Evans rats prior to and throughout breeding, during gestation, lactation, and the immediate post- weaning period. The results indicated that TCP adversely affects both male and female reproductive parameters. Comparable numbers of females were sperm-positive in all dose groups, indicating no adverse influence of TCP exposure on the estrus cycle, libido, or copulatory behavior. Histopathologic alterations in the ovaries of females exposed to 400 mgkg TCP indicated a possible homo& stimulation of the ovaries, however. A decrease in sperm concentration, and minimal-to mild histopathologic lesions in the testes and epididymides of TCP-exposed
rats were observed.
Executive 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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study OECD 422
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Based on the similar chemical structure and a trend in biological activity a category has been defined. The category consists of CDP (Diphenyl cresyl phosphate), TPP (Triphenyl phosphate) and TCP (Tricresyl phosphate). Structurally CDP (diphenyl cresyl phosphate) is more similar to TPP (triphenyl phosphate) than to TCP (tricresyl phosphate) because CDP (diphenyl cresyl phosphate) has only one cresyl group, whereas TPP (tricresyl phosphate) has 3 cresyl groups. A category justification document is attached.

Based on the category approach a read-across to studies from TPP and TCP is justified to fill the data gap of CDP concerning the sub-chronic study.

The following studies are available for category members (rat and rabbit studies).

The influence of diphenyl cresyl phosphate upon the progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain in accordance with the guidelines of the OECD 414. For this purpose diphenyl cresyl phosphate was administered by gavage at dosages of 100, 300 or 900 mg/kg/day to groups of 22 pregnant rats from Day 6 to 15 of gestation inclusive. Control animals received the vehicle, maize oil, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Satellite females, five per group, were similarly treated and killed on Day 16 of gestation, for determination of haematology and blood chemistry parameters.

Post-dose salivation was observed on a regular basis at 300 and 900 mg/kg/day in a dosage-related manner. Other signs observed at 900 mg/kg/day included brown staining, hair loss, urogenital staining, piloerection and ungroomed coat. The general condition of females receiving 100 and 300 mg/kg/day was similar to that of the Controls. No deaths occurred.

Bodyweight stasis or loss was observed over Days 7 to 10 of gestation at 900 mg/kg/day, although the deficit was recouped by Day 20 of gestation. Weight gains at 100 and 300 mg/kg/day were unaffected by treatment.

Food consumption was reduced at 900 mg/kg/day for the first few days of the treatment period. Food consumption at 100 and 300 mg/kg/day was similar to that of the Controls.

Water consumption showed a marked increase throughout the treatment period, and up to termination, for females receiving 300 and 900 mg/kg/day. Water consumption at 100 mg/kg/day was unaffected by treatment.

With the exception of four females exhibiting hair loss at 900 mg/kg/day, necropsy of females on Day 20 of gestation, revealed no macroscopic findings that were considered to be an effect of treatment.

Litter survival, growth and development in utero was unaffected by treatment with Diphenyl cresyl phosphate at all dosages.

On day 16 of gestation, packed cell volume, haemoglobin concentration and red blood cell count were lower for all treated groups, although females receiving 100 mg/kg bw were only marginally affected. Increased total leucocyte counts, neutrophil and platelet counts were recorded at 300 and 900 mg/kg/day. Polychromasia and/ or hypochromasia were observed for all females at 900 mg/kg/day.

Blood chemistry of females receiving 300 and 900 mg/kg/day revealed low albumin concentrations, slightly high alpha-globulin and high beta-globulin concentrations, and a lower albumin to globulin ratio. At 900 mg/kg/day, there were high alanine and aspartate amino-transferase activities, and marginally low plasma glucose concentrations.

Females receiving 100 mg/kg/day were considered to be unaffected by treatment.

It was concluded from this investigation that oral administration of diphenyl cresyl phosphate to pregnant rats during the period of organogenesis at a dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ.

At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree.

At the lowest dosage of 100 mg/kg/day, Diphenyl cresyl phosphate was well tolerated with no signs of overt toxicity, and this was considered to be the maternal NOAEL.

Litter parameters were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.

Triphenyl phosphate was tested in a prenatal developmental toxicity study in pregnant New Zealand rabbits following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 32, 80 and 200 mg/kg bw/day in 1% aqueous carboxymethyl cellulose. The doses were chosen on the basis of dose range finding studies that showed strong toxicity (including mortality) at 750 and 250 mg/kg bw/day.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations.

No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination). No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations).

Based on the results of this prenatal developmental toxicity study, both the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for triphenyl phosphate were established as being at least 200 mg/kg bw/day, since no adverse effect was observed. No higher doses could be tested in pregnant rabbits based on dose range finding studies.

For Tricresyl phosphate a developmental study according to EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study) is available. 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 spillage 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
In an OECD 414 study diphenyl cresyl phosphate was administered by gavage at dosages of 100, 300 or 900 mg/kg/day to groups of 22 pregnant rats from Day 6 to 15 of gestation inclusive. All females were killed on Day 20 of gestation. The aim of the study was to assess the effects of oral administration of diphenyl cresyl phosphate during the organogenesis phase of gestation upon the progress and outcome of pregnancy in the rat.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD
Route of administration:
oral: gavage
Vehicle:
maize oil
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Females were paired on a one-to-one basis with stock males of the same strain. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.
Duration of treatment / exposure:
From day 6 to Day 15 of gestation inclusive.
Frequency of treatment:
daily
Duration of test:
All females were killed on Day 20 of gestation for examination of their uterine content.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
900 mg/kg bw/day
No. of animals per sex per dose:
Groups of 22 female rats/dose
Control animals:
yes
Maternal examinations:
Serial: Clinical signs, maternal body weight, food consumption, water consumption.
Terminal: Haematology, blood chemistry.
Ovaries and uterine content:
Number of corpora lutea, number of implantation sites, number of resorption sites, number and distribution of foetuses in each uterine horn.
Fetal examinations:
External and internal examination at necropsy. Skeletal examination at necropsy.
Statistics:
The significance of sugestive inter-group differences was tested using appropriate statistical tests.
Indices:
Pre-implantation loss, post-implantation loss, foetal observations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Post-dose salivation was observed on a regular basis at 300 and 900 mg/kg/day in a doseage-related manner. Other signs observed at 900 mg/kg/day included brown staining, hailoss, urogenital staining, piloerection and ungroomed coat. The general condition of females receiving 100 and 300 mg/kg/day was similar to that of the Controls.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight stasis or loss was observed over Days 7 to 10 of gestation at 900 mg/kg/day, although the deficit was recouped by Day 20 of gestation. Weight gains at 100 and 300 mg/kg/day were unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced at 900 mg/kg/day for the first few days of the treatment period. Food consumption at 100 and 300 mg/kg/day was similar to that of the controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption showed a marked increase throughout the treatment period, and up to termination, for females receiving 300 and 900 mg/kg/day. Water consumption at 100 mg/kg/day was unaffected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
On day 16 of gestation, packed cell volume, haemoglobin concentration and red blood cell count were lower for all treated groups, although females receiving 100 mg/kg bw were only marginally affected. Increased total leucocyte counts, neutrophil and platelet counts were recorded at 300 and 900 mg/kg/day. Polychromasia and/ or hypochromasia were observed for all females at 900 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry of females receiving 300 and 900 mg/kg/day revealed low albumin concentrations, slightly high alpha-globulin and high beta-globulin concentrations, and a lower albumin to globulin ratio. At 900 mg/kg/day, there were high alanine and aspartate amino-transferase activities, and marginally low plasma glucose concentrations.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
With the exception of four females exhibiting hairloss at 900 mg/kg/day, necropsy of females on Day 20 of gestation, revealed no macroscopic findings that were considered to be an effect of treatment.
Neuropathological findings:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
It was concluded from this investigation that oral administration of diphenyl cresyl phosphate to pregnant rats during the period of organogenesis at a dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ.
At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree.
At the lowest dosage of 100 mg/kg/day, diphenyl cresyl phosphate was well tolerated with no signs of overt toxicity, and this was considered to be the maternal NOAEL.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
A dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ.
At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter survival, growth and development in utero was unaffected by treatment with diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter survival, growth and development in utero was unaffected by treatment with diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Litter survival, growth and development in utero was unaffected by treatment with diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter survival, growth and development in utero was unaffected by treatment with diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Litter survival, growth and development in utero was unaffected by treatment with diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 900 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Satellite females, five per group, were similarly treated and killed on Day 16 of gestation, for determination of haematology and blood chemistry parameters.

Post-dose salivation was observed on a regular basis at 300 and 900 mg/kg/day in a doseage-related manner. Other signs observed at 900 mg/kg/day included brown staining, hailoss, urogenital staining, piloerection and ungroomed coat. The general condition of females receiving 100 and 300 mg/kg/day was similar to that of the Controls. No deaths occurred.

Bodyweight stasis or loss was observed over Days 7 to 10 of gestation at 900 mg/kg/day, although the deficit was recouped by Day 20 of gestation. Weight gains at 100 and 300 mg/kg/day were unaffected by treatment.

Food consumption was reduced at 900 mg/kg/day for the first few days of the treatment period. Food consumption at 100 and 300 mg/kg/day was similar to that of the Controls.

Water consumption showed a marked increase throughout the treatment period, and up to termination, for females receiving 300 and 900 mg/kg/day. Water consumption at 100 mg/kg/day was unaffected by treatment.

With the exception of four females exhibiting hairloss at 900 mg/kg/day, necropsy of females on Day 20 of gestation, revealed no macroscopic findings that were considered to be an effect of treatment.

Litter survival, growth and development in utero was unaffected by treatment with Diphenyl cresyl phosphate at all dosages.

On day 16 of gestation, packed cell volume, haemoglobin concentration and red blood cell count were lower for all treated groups, although females receiving 100 mg/kg bw were only marginally affected. Increased total leucocyte counts, neutrophil and platelet counts were recorded at 300 and 900 mg/kg/day. Polychromasia and/ or hypochromasia were observed for all females at 900 mg/kg/day.

Blood chemistry of females receiving 300 and 900 mg/kg/day revealed low albumin concentrations, slightly high alpha-globulin and high beta-globulin concentrations, and a lower albumin to globulin ratio. At 900 mg/kg/day, there were high alanine and aspartate amino-transferase activities, and marginally low plasma glucose concentrations.

Females receiving 100 mg/kg/day were considered to be unaffected by treatment.

Conclusions:
It was concluded from this investigation that oral administration of Diphenyl Cresyl phosphate to pregnant rats during the period of organogenesis at a dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ. At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree. At the lowest dosage of 100 mg/kg/day, Diphenyl cresyl phosphate was well tolerated with no signs of overt toxicity, and this was considered to be the maternal NOAEL.

Litter survival, growth and development in utero was unaffected by treatment with Diphenyl cresyl phosphate at all dosages. Litter parameters growth and development were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.
Executive summary:

The influence of Diphenyl cresyl phosphate upon the progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain in accordance with the guidelines of the OECD 414. For this purpose , Diphenyl cresyl phosphate was administered by gavage at dosages of 100, 300 or 900 mg/kg/day to groups of 22 pregnant rats from Day 6 to 15 of gestation inclusive. Control animals received the vehicle, maize oil, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Satellite females, five per group, were similarly treated and killed on Day 16 of gestation, for determination of haematology and blood chemistry parameters.

Post-dose salivation was observed on a regular basis at 300 and 900 mg/kg/day in a doseage-related manner. Other signs observed at 900 mg/kg/day included brown staining, hailoss, urogenital staining, piloerection and ungroomed coat. The general condition of females receiving 100 and 300 mg/kg/day was similar to that of the Controls. No deaths occurred.

Bodyweight stasis or loss was observed over Days 7 to 10 of gestation at 900 mg/kg/day, although the deficit was recouped by Day 20 of gestation. Weight gains at 100 and 300 mg/kg/day were unaffected by treatment.

Food consumption was reduced at 900 mg/kg/day for the first few days of the treatment period. Food consumption at 100 and 300 mg/kg/day was similar to that of the Controls.

Water consumption showed a marked increase throughout the treatment period, and up to termination, for females receiving 300 and 900 mg/kg/day. Water consumption at 100 mg/kg/day was unaffected by treatment.

With the exception of four females exhibiting hairloss at 900 mg/kg/day, necropsy of females on Day 20 of gestation, revealed no macroscopic findings that were considered to be an effect of treatment.

Litter survival, growth and development in utero was unaffected by treatment with Diphenyl cresyl phosphate at all dosages.

On day 16 of gestation, packed cell volume, haemoglobin concentration and red blood cell count were lower for all treated groups, although females receiving 100 mg/kg bw were only marginally affected. Increased total leucocyte counts, neutrophil and platelet counts were recorded at 300 and 900 mg/kg/day. Polychromasia and/ or hypochromasia were observed for all females at 900 mg/kg/day.

Blood chemistry of females receiving 300 and 900 mg/kg/day revealed low albumin concentrations, slightly high alpha-globulin and high beta-globulin concentrations, and a lower albumin to globulin ratio. At 900 mg/kg/day, there were high alanine and aspartate amino-transferase activities, and marginally low plasma glucose concentrations.

Females receiving 100 mg/kg/day were considered to be unaffected by treatment.

It was concluded from this investigation that oral administration of Diphenyl Cresyl phosphate to pregnant rats during the period of organogenesis at a dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ.

At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree.

At the lowest dosage of 100 mg/kg/day, Diphenyl cresyl phosphate was well tolerated with no signs of overt toxicity, and this was considered to be the maternal NOAEL.

Litter parameters were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
See attached justification.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
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): one femaled in the high dose group delivered early
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination) in the tested doses of up to and including 200 mg/kg bw/day. Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at 200 mg/kg bw/day resulted in a higher incidence of lungs with absent accessory lung lobe(s). While in the control and mid dose groups only one fetus (A015-03 and A048-02, respectively) each was noted with this malformation, the incidence increased to 3(3) fetuses (litter) in the high dose
group (A067-03, A069-07, A074-04). This resulted in a litter proportion of 1.6% which was at the upper limit of the available historical control range for this finding (MAX: 1.7%). In addition, 2 dead fetuses from litter 78 that were delivered preterm on Day 29 post-coitum (A078-03 and A078-05) had absent
accessory lung lobes. The total litter proportion of fetuses at 200 mg/kg bw/day with absent accessory lung lobe(s) thus increased from 1.6% (given in Table 1.15; APPENDIX 1) to 2.4%. This litter proportion was clearly above the range of available historical control data. Lungs with absent accessory lung lobe(s) is a more common finding in New Zealand White rabbits. The historical control data from this laboratory consisted of 17 developmental studies with this strain in which in total 2787 (315) control fetuses (litters) were examined. In 10 of these studies fetuses with absent accessory lung lobe(s) were found, i.e. in total 20 (17) control fetuses (litter). The highest incidence was 3(3) fetuses (litter) seen in 2 studies. As in the high dose group of the present study the incidence of lungs with absent accessory lobe(s) was only slightly higher with in total 5(4) fetuses (litter) including the 2 fetuses from the preterm delivered litter, it was considered as an incidence finding and thus not to be toxicologically relevant.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no developmental findings up to and including 200 mg/kg bw/day that were considered to be toxicologically relevant.
No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations). Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study.
Abnormalities:
not specified
Developmental effects observed:
no

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Conclusions:
Triphenyl phosphate was tested in a prenatal developmental toxicity study in pregnant New Zealand rabbits following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 32, 80 and 200 mg/kg bw/day in 1% aqueous carboxymethyl cellulose. The doses were chosen on the basis of dose range finding studies that showed strong toxicity (including mortality) at 750 and 250 mg/kg bw/day.
Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.
All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations.
No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination). No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations).

Based on the results of this prenatal developmental toxicity study, both the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for triphenyl phosphate were established as being at least 200 mg/kg bw/day, since no adverse effect was observed. No higher doses could be tested in pregnant rabbits based on dose range finding studies.
Executive summary:

Triphenyl phosphate was tested in a prenatal developmental toxicity study in pregnant New Zealand rabbits following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 32, 80 and 200 mg/kg bw/day in 1% aqueous carboxymethyl cellulose. The doses were chosen on the basis of dose range finding studies that showed strong toxicity (including mortality) at 750 and 250 mg/kg bw/day.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations.

No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination). No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations).

Based on the results of this prenatal developmental toxicity study, both the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for triphenyl phosphate were established as being at least 200 mg/kg bw/day, since no adverse effect was observed. No higher doses could be tested in pregnant rabbits based on dose range finding studies.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Nulliparous, non-pregnant and untreated females were used at inititation of the study.
- Age at delivery: 17-19 weeks; mating with adult and proven fertile males at WIL Research
- Fasting period before study: none
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was grinded to a fine powder in a grinding mill. Subsequently, the powder was suspended in the vehicle. Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous carboxymethyl cellulose was chosen based on trial formulations performed at WIL.
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (26 November 2014), according to a validated method (Project 506608). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (lowest concentration only).
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
a) A copulation plug was observed, and / or
b) The daily vaginal smear was sperm positive.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating. These male rats are in the possession of RCC and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
Day 6 - 28 post coitum inclusive
Frequency of treatment:
once daily
Duration of test:
All animals surviving to the end of the observation period (Day 29 post-coitum) and the female with premature delivery were euthanised by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs and the fetuses.
No. of animals per sex per dose:
Each group consisted of 22 mated female rabbits.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose levels
Dose levels were selected based on the results of a dose range finding study (Project 505943; see APPENDIX 6). In this latter study dose levels of 83, 250 and 750 mg/kgmg/kg bw/day were tested. Due to severe toxicity, all females at 750 mg/kgmg/kg bw/day and one female at 250 mg/kgmg/kg bw/day had to be euthanized before scheduled necropsy. Another female at 250 mg/kgmg/kg bw/day was noted with clinical signs (pale appearance on Days 22 and 23 post-coitum and reduced production of (pale) faeces from Day 17 post-coitum). She had no food consumption from Days 16-23 post-coitum and body weight loss (up to -6%). No signs of toxicity were noted for the remaining females at 250 mg/kgmg/kg bw/day and all females at 83 mg/kgmg/kg bw/day. Based on these data, dose levels of 32, 80 and 200 mg/kgmg/kg bw/day were selected for the present prenatal developmental toxicity study.
Maternal examinations:
Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.
All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups.
Ovaries and uterine content:
The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref. 1). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses.

The specimens of all groups will be archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) divided by the number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) divided by the number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = number of viable fetuses affected/litter divided by the number of viable fetuses/litter x 10
Historical control data:
available
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
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): one femaled in the high dose group delivered early
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination) in the tested doses of up to and including 200 mg/kg bw/day. Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at 200 mg/kg bw/day resulted in a higher incidence of lungs with absent accessory lung lobe(s). While in the control and mid dose groups only one fetus (A015-03 and A048-02, respectively) each was noted with this malformation, the incidence increased to 3(3) fetuses (litter) in the high dose
group (A067-03, A069-07, A074-04). This resulted in a litter proportion of 1.6% which was at the upper limit of the available historical control range for this finding (MAX: 1.7%). In addition, 2 dead fetuses from litter 78 that were delivered preterm on Day 29 post-coitum (A078-03 and A078-05) had absent
accessory lung lobes. The total litter proportion of fetuses at 200 mg/kg bw/day with absent accessory lung lobe(s) thus increased from 1.6% (given in Table 1.15; APPENDIX 1) to 2.4%. This litter proportion was clearly above the range of available historical control data. Lungs with absent accessory lung lobe(s) is a more common finding in New Zealand White rabbits. The historical control data from this laboratory consisted of 17 developmental studies with this strain in which in total 2787 (315) control fetuses (litters) were examined. In 10 of these studies fetuses with absent accessory lung lobe(s) were found, i.e. in total 20 (17) control fetuses (litter). The highest incidence was 3(3) fetuses (litter) seen in 2 studies. As in the high dose group of the present study the incidence of lungs with absent accessory lobe(s) was only slightly higher with in total 5(4) fetuses (litter) including the 2 fetuses from the preterm delivered litter, it was considered as an incidence finding and thus not to be toxicologically relevant.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no developmental findings up to and including 200 mg/kg bw/day that were considered to be toxicologically relevant.
No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations). Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study.
Abnormalities:
not specified
Developmental effects observed:
no

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Executive summary:

Triphenyl phosphate was tested in a prenatal developmental toxicity study in pregnant New Zealand rabbits following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 32, 80 and 200 mg/kg bw/day in 1% aqueous carboxymethyl cellulose. The doses were chosen on the basis of dose range finding studies that showed strong toxicity (including mortality) at 750 and 250 mg/kg bw/day.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations.

No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination). No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations).

Based on the results of this prenatal developmental toxicity study, both the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for triphenyl phosphate were established as being at least 200 mg/kg bw/day, since no adverse effect was observed. No higher doses could be tested in pregnant rabbits based on dose range finding studies.

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:
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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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
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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In life examinations
No effect of treatmcnt 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.

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
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.

Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.
Abnormalities:
not specified
Developmental effects observed:
not specified

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.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
See attached justification.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In life examinations
No effect of treatmcnt 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.

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
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.

Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.
Abnormalities:
not specified
Developmental effects observed:
not specified

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

The influence of Diphenyl cresyl phosphate upon the progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain in accordance with the guidelines of the OECD 414. For this purpose, Diphenyl cresyl phosphate was administered by gavage at dosages of 100, 300 or 900 mg/kg/day to groups of 22 pregnant rats from Day 6 to 15 of gestation inclusive. Control animals received the vehicle, maize oil, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Satellite females, five per group, were similarly treated and killed on Day 16 of gestation, for determination of haematology and blood chemistry parameters.

Post-dose salivation was observed on a regular basis at 300 and 900 mg/kg/day in a dosage-related manner. Other signs observed at 900 mg/kg/day included brown staining, hair loss, urogenital staining, piloerection and ungroomed coat. The general condition of females receiving 100 and 300 mg/kg/day was similar to that of the Controls. No deaths occurred.

Bodyweight stasis or loss was observed over Days 7 to 10 of gestation at 900 mg/kg/day, although the deficit was recouped by Day 20 of gestation. Weight gains at 100 and 300 mg/kg/day were unaffected by treatment.

Food consumption was reduced at 900 mg/kg/day for the first few days of the treatment period. Food consumption at 100 and 300 mg/kg/day was similar to that of the Controls. Water consumption showed a marked increase throughout the treatment period, and up to termination, for females receiving 300 and 900 mg/kg/day. Water consumption at 100 mg/kg/day was unaffected by treatment. With the exception of four females exhibiting hair loss at 900 mg/kg/day, necropsy of females on Day 20 of gestation, revealed no macroscopic findings that were considered to be an effect of treatment.

Litter survival, growth and development in utero was unaffected by treatment with Diphenyl cresyl phosphate at all dosages. On day 16 of gestation, packed cell volume, haemoglobin concentration and red blood cell count were lower for all treated groups, although females receiving 100 mg/kg bw were only marginally affected. Increased total leukocyte counts, neutrophil and platelet counts were recorded at 300 and 900 mg/kg/day. Polychromasia and/ or hypochromasia were observed for all females at 900 mg/kg/day.

Blood chemistry of females receiving 300 and 900 mg/kg/day revealed low albumin concentrations, slightly high alpha-globulin and high beta-globulin concentrations, and a lower albumin to globulin ratio. At 900 mg/kg/day, there were high alanine and aspartate amino-transferase activities, and marginally low plasma glucose concentrations. Females receiving 100 mg/kg/day were considered to be unaffected by treatment.

It was concluded from this investigation that oral administration of diphenyl cresyl phosphate to pregnant rats during the period of organogenesis at a dosage of 900 mg/kg/day resulted in several findings indicative of toxicity. These included reduced weight gain, increased water consumption and an effect on erythrocytic parameters. The liver was identified as a possible target organ. At a dosage of 300 mg/kg/day, water consumption was increased, and erythrocytic parameters were affected as for the highest dosage, but to a lesser degree.

At the lowest dosage of 100 mg/kg/day, Diphenyl cresyl phosphate was well tolerated with no signs of overt toxicity, and this was considered to be the maternal NOAEL. Litter parameters were unaffected by treatment at all dosages, and the NOEL for foetuses was therefore considered to be 900 mg/kg/day, which was the highest dose tested (BG Chemie, 1996).

Justification for classification or non-classification

There is a trend in the repeated dose toxicity of the category members (see category justification document).

With triphenyl phosphate (TPP) no adverse effects on fertility and development were found.

With diphenyl cresyl phosphate fertility and implantation indices decreased in the 300 mg/kg group in a repeated dose/reproductive/developmental screening test with a diphenyl cresyl phosphate test material with a purity of 41.9. These were probably caused by dysspermatogenesis, which is a typical effect of tri-orthocresyl phosphate which was maybe included in the test material. No adverse effects on development were observed.

With tricresyl phosphate fertility rates were severely affected even at the lowest dose group (200 mg/kg bw).

The reproductive toxicity of tricresyl phosphate is mainly triggered by the tri-ortho-cresy phosphate as component or impurity of the tri-cresyl phosphate mixture.

The investigations of Somkuti et al., demonstrate the adverse effects of tri-ortho cresyl phosphate on spermatogenesis. Tri-para-cresyl-phosphate (TPCP), the structural analog of TOCP, produced no such effects, demonstrating the role of the ortho-cresol moiety for induction of damage.

No effect on treatment was evident in the developmental study with tricresyl phosphate in dose up to 750 mg/kg bw (tri-ortho-cresyl phosphate < 0.04).

Triphenyl phosphate (TPP) has the lowest toxicity, whereas tricresyl phosphate (TCP) has the highest toxicity. The toxicity of diphenyl cresyl phosphate (CDP) is between TPP and TCP.

Based on these data a category formation and read-across of diphenyl cresyl phosphate with TPP and TCP is scientifically justified.

As nowadays technical diphenyl cresyl phosphates (and tricresyl phosphates) have only negligible amounts of tri-ortho-cresyl phosphate the classification as Rep Cat 2 (H361) for TCP should not be applied to diphenylcresyl phosphate.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification for CDP (diphenyl cresyl phosphate) is not justified.