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Effects on fertility

Description of key information
no data
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The materials/methods and results are described in detail and are sufficient for evaluation.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

At the dose of 750 mg/kg bw/day, weight of seminal vesicles was decreased correlating to reduced secretion in the prostate and seminal vesicle at histopathology. Additionally, a minimally increased proportion of round spermatids in the epididymides was noted in male animals after 750 mg/kg. These findings might indicate a primary systemic effect but also it is more likely that they reflect delayed sexual maturation as a sequel of poor condition of these animals. No other findings were reported for male reproduction organs. In female rats no changes (organs weights, gross or histopahtological changes) concerning the the reproduction organs were noted.

Due to the decreased weight in seminal vesicels correlating to reduced secretion in the prostate and seminal vesicle at histopathology and the minimally increased proportion of round spermatides in the epididymides a NOAEL of > 150 mg/kg bw/day is assumed for male rats.

This assumption includes that this findings indicate a primary systemic effects. However, it is more likely that they reflect delayed sexual maturation as a sequel of poor condition of these animals due to the effects in the alimentary tract caused by the local irritant and corrosive properties of the test substance.

No effects were reported for female reproduction organs at the highest applied dose. Therefore for female rats a NOAEL of > 750 mg/kg bw/day for reproduction is justified.


Short description of key information:
Bis(2-ethylhexyl) hydrogen phosphate was administered by gavage to 5 male and 5 female Wistar rats per dose group in daily doses of 0, 30, 150 or 750 mg/kg bw for 28 days to asses its toxicity. At the end of the treatment period all animals were killed and necropsied. The pathologic evaluation consisted of organ weight, gross and microscopic examination of reproductive organs, incl. testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary/oviduct.

Effects on developmental toxicity

Description of key information
For developmental toxicity an OECD Guideline 414 (Prenatal Developmental Toxicity Study) study with tris(2-ethylhexyl) phosphate as a surrogate for bis(2-ethylhexyl) hydrogen phosphate was used. Tris(2-ethylhexyl) phosphate was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg bw/day. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Rat, HanRcc: WIST(SPF Quality)
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
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 - 20 post coitum
Frequency of treatment:
Daily.
Duration of test:
At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg body weight/day (control group); Group 2: 500 mg/kg body weight/day; Group 3: 1000 mg/kg body weight/day.
Basis:

No. of animals per sex per dose:
Each group consisted of 24 mated female rats.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no data
Abnormalities:
not specified
Developmental effects observed:
not specified

Analysis of dose formulations:

The concentration of tris(2-ethylhexyl) phosphate in all samples ranged from 97.7 to 106.6% of the nominal concentrations. In addition, the homogenous distribution of tris(2-ethylhexyl) phosphate in corn oil was demonstrated. The dose formulations were demonstrated to be stable for at least 7 days when kept refrigerated, which was the maximum time of storage and use of dosing preparations in this study.

Maternal data:

Mortality and clinical signs or observations:

All females survived until the scheduled necropsy. No clinical signs or signs of discomfort were noted for any female in any group during the study.

Food consumption (g/animal/day) of dams post coitum:

Food consumption was similar in all dose groups during the treatment period.

Body weights (gram) of dams post coitum, body weight gain (gram) of dams post coitum and corrected body weight gain of dams: There were no effects on mean body weight, mean body weight gain or mean body weight corrected for uterus weight in any dose group.

Reproduction data: Reproduction processes, distribution within uterus and contents of uterus (plan view):

No treatment-related effects were noted on reproduction parameters in any dose group. Post-implantation loss and the number of embryonic resorptions per litter were statistically significantly lower at 1000 mg/kg bw/day compared to the control group. However, these values were within the range of the historical control data and treatment-related effects are typically associated with increases in post-implantation loss and embryonic resorptions. Therefore these values reflect normal variability and were not related to treatment.Macroscopical findings:

No macroscopical findings were noted for any female in any group.

Fetal data:

External examination of fetuses:

No test item-related findings were noted during external examination of the fetuses. One fetus at 1000 mg/kg bw/day had a shortened lower jaw (brachygnathia). This was within the range of historical control external findings and was considered to be incidental.

Sex ratios:

The sex ratio of the fetuses was close to 50% in all dose groups.

Body weights:

The mean body weight of the fetuses was similar in all groups and there was no effect of treatment at 500 or 1000 mg/kg/day.

Visceral examination of fetuses (microdissection technique):

A total of 4 fetuses with visceral abnormalities were noted. At 1000 mg/kg bw/day, two fetuses from a single litter were noted with multiple changes. One fetus exhibited multiple abnormalities of the craniofacial region, which correlated with the external finding of shortened lower jaw. The second fetus from this litter exhibited multiple cardiovascular changes and situs inversus. Both of these fetuses had the accompanying abnormality of pituitary small/misshapen. At 500 mg/kg bw/day, two fetuses from separate litters exhibited abnormalities; one fetus exhibited thoracic and abdominal situs inversus, and the other exhibited a heart interventricular septal defect. The low incidences of these abnormalities were within the historical control range and considered unrelated to treatment. The total incidence of visceral abnormalities and variations combined in each group was: 71 out of 151 examined fetuses (in 22 of 24 litters) of group 1 (0 mg/kg) 63 out of 144 examined fetuses (in 22 of 23 litters) of group 2 (500 mg/kg) 67 out of 143 examined fetuses (in 20 of 23 litters) of group 3 (1000 mg/kg) There was no statistically significant difference from the control group in the total incidence of abnormalities or of abnormalities and variations combined at 500 or 1000 mg/kg bw/day. For the individual variation of long thymus (cranial), the fetal incidence at 1000 mg/kg bw/day (22) was statistically significantly higher compared to the control group (10), but the litter incidence (11) was no different from the control (7). Conversely, the variation of subcutaneous haemorrhage was statistically significantly lower than the control at 1000 mg/kg bw/day on both a fetal and litter basis. Both of these variations are common in the historic control data, with the latter finding described as "localized haemorrhage".

Considering the high background incidence of these variations, and the presence of both higher and lower incidences in the 1000 mg/kg bw/day group, these variations are considered incidental to treatment.

Overall, the visceral findings noted were common findings for this species and strain of rats, and therefore, they were considered unrelated to treatment.

Skeletal examinations - bone and cartilage abnormalities and variations:

A single fetus at 1000 mg/kg bw/day was noted with a cartilage finding (severe dumbbell-shaped cervical vertebral body 1), without any abnormalities in bone structures. This single incidence is not considered treatment-related. The total incidence of bone and cartilage abnormalities and variations in each group was: 37 out of 138 examined fetuses (in 19 of 23 litters) of group 1 ( 0 mg/kg) 30 out of 134 examined fetuses (in 17 of 23 litters) of group 2 ( 500 mg/kg) 29 out of 126 examined fetuses (in 15 of 23 litters) of group 3 (1000 mg/kg) There were no statistically significant differences from the control group in the incidence of total skeletal findings or any individual skeletal findings. The bone and cartilage variations that were noted were generally observed at similar incidence in control and treated groups, and in the historic control data, and were considered not to be test item-related.

Bone examinations - ossification stage / supernumerary ribs:

Bone examination for ossification stage and/or supernumerary ribs did not reveal any test item related findings. On a fetal basis, certain isolated structures had statistically significantly lower incidences of incomplete ossification or non-ossified in 500 and/or 1000 mg/kg bw/day treatment groups. However, these were not statistically significant on a litter basis, and the direction of change and their sporadic occurrence did not indicate a treatment-related effect.

Cartilage examinations - additional variations:

Cartilage additional variations were noted at similar incidence in control and treated groups, and there was no effect of treatment. Branched xiphoid cartilage was observed at lower incidence at 1000 mg/kg bw/day than in the control group on both a fetal and litter basis, but a lower incidence of this common finding is considered normal variability and not an effect of treatment.

Conclusion:

Tris(2-ethylhexyl) phosphate was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg bw/day. No maternal toxicity was noted in any of the treatment groups. The reproduction data of the tris(2-ethylhexyl) phosphate treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any tris(2-ethylhexyl) phosphate related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this study according to OECD Guideline 414 was to evaluate any effects of tris(2-ethylhexyl) phosphate on the pregnant female rat and development of the embryo and fetus consequent to exposure of the female to tris(2-ethylhexyl) phosphate. Each group consisted of 24 mated female rats. Tris(2-ethylhexyl) phosphate was administered orally, by gavage, once daily from day 6 through to day 20 post coitum (last treatment) at dose levels of:

Group 1: 0 mg/kg bw/day (control group)

Group 2: 500 mg/kg bw/day

Group 3: 1000 mg/kg bw/day.

A standard dose volume of 4 mL/kg bw with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females in the main groups were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations.

Maternal data:

General tolerability:

All females survived until the scheduled necropsy. No clinical signs or signs of discomfort were noted for any female in any group during the study.

Food consumption:

Food consumption was similar in all dose groups during the treatment period.

Body weights:

Absolute body weights, mean body weight gain and corrected body weight gain were not affected by treatment with the test item. Reproduction data:

No effects were noted due to treatment with the test item in the reproduction data.

Macroscopical data:

No macroscopical findings were noted for any female in any group.

Fetal data:

External examination of fetuses:

At scheduled Caesarean section, no test item-related findings were observed.

Sex ratios:

The sex ratio of the fetuses was close to 50% in all dose groups.

Body weights:

The mean body weight of the fetuses was similar in all groups.

Visceral examination of fetuses (microdissection technique):

No test item-related findings were noted.

Skeletal examinations - bone and cartilage abnormalities and variations:

There were no treatment-related effects on the incidence of skeletal abnormalities and variations.

Bone examinations - ossification stage / supernumerary ribs:

Skeletal examination (ossification stage), which was tabulated separately to evaluate changes in degree of ossification and thus the relative stage of development of fetuses, did not reveal any test item-related findings. There was no effect of treatment on the incidence of supernumerary ribs.

Cartilage examinations - additional variations:

Cartilage additional variations were noted at similar incidence in control and treated groups, and there was no effect of treatment.

Conclusion:

Tris(2-ethylhexyl) phosphate was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg bw/day. No maternal toxicity was noted in any of the treatment groups. The reproduction data of the tris(2-ethylhexyl) phosphate treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any tris(2-ethylhexyl) phosphate related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The materials/methods and results are described in detail and are sufficient for evaluation.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No maternal toxicity was noted in any of the treatment groups. The reproduction data of the test item-treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any test item-related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg bw/day.

Tris(2-ethylhexyl) phosphate (CAS No. 78-42-2) was used as a surrogate for bis(2-ethylhexyl) hydrogen phosphate (CAS No. 298-07-7) as in analogy to other phosphoric acid esters a hydrolysis to the corresponding di- and the monoester can be assumed.

As metabolites, bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexanol are expected (BUA Report No. 172 (Advisory Committee on Existing Chemicals of Environmental Relevance (BUA)) and ECETOC JACC Report No. 20 (Tris-/Bis-/Mono-(2-ethylhexyl)phosphate, 1992).

The expected metabolite 2-ethylhexanol is in maternal tolerable doses in rats and mice after oral, dermal and inhalative application not embryotoxic or teratogene. (BUA Report No. 172 (Advisory Committee on Existing Chemicals of Environmental Relevance (BUA)).

Therefore a read across with tris(2-ethylhexyl) phosphate (CAS No. 78-42-2) as a surrogate for bis(2-ethylhexyl) hydrogen phosphate (CAS No. 298-07-7) is justified.


Justification for selection of Effect on developmental toxicity: via oral route:
For developmental toxicity an OECD Guideline 414 (Prenatal Developmental Toxicity Study) study with tris(2-ethylhexyl) phosphate as a surrogate for bis(2-ethylhexyl) hydrogen phosphate used.

Toxicity to reproduction: other studies

Additional information

no data

Justification for classification or non-classification

In the repeated dose toxicity study at 750 mg/kg bw/day a decreased weight in seminal vesicels correlating to reduced secretion in the prostate and seminal vesicle at histopathology and the minimally increased proportion of round spermatides in the epididymides was found. These findings might indicate a primary systemic effect but also it is more likely that they reflect delayed sexual maturation as a sequel of poor condition of these animals.Therefore for fertility a NOAEL of 150 mg/kg bw/day for male rats and for female rats a NOAEL of 750 mg/kg bw/day is assumed.

In the developmental study no maternal toxicity was noted in any of the treatment groups. The reproduction data of the tris(2-ethylhexyl) phosphate treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any tris(2-ethylhexyl) phosphate related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg bw/day.

A classification is therefore not justified based on the reulsts of the repeated dose and the developmental study.

Additional information