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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(2-ethylhexyl) phosphate
EC Number:
201-116-6
EC Name:
Tris(2-ethylhexyl) phosphate
Cas Number:
78-42-2
Molecular formula:
C24H51O4P
IUPAC Name:
tris(2-ethylhexyl) phosphate
Details on test material:
Identification: CA3842 / TEHP
Product code: CA3842
Description: Clear, colourless liquid

Batch number: CH2/27047
Purity: 99.4%
Stability of test item in vehicle: At least 7 days in the refrigerator (2 - 8 °C)
Expiry date (retest date): End of July 2009
Storage conditions: Room temperature (20 ± 5 °C)
Safety precautions: Routine hygienic procedures (gloves, goggles, face
mask).
The test item was also used as the analytical standard for analysis of the dose formulations.

Test animals

Species:
rat
Strain:
other: Rat, HanRcc: WIST(SPF Quality)

Administration / exposure

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.
Doses / concentrations
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.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no data

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.