Bis(2-ethylhexyl) hydrogen phosphate

Endpoint summary

Description of key information

Bis(2-ethylhexyl) hydrogen phosphate was administered orally by gavage to 5 male and 5 female Wistar (HsdRCCHan:Wist) rats per dose group using corn oil as vehicle, in daily doses of 0, 30, 150, 750 mg/kg bw for a period of 4 weeks. This GLP study was performed according to OECD guideline 407.
The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation. 
Additional, a 90 day and a 2 years study in rats (and mice) for tris(2-ethylhexyl) phosphate are on hand. In analogy to other phosphoric acid esters a hydrolysis of the triester to the di- and the monoester can be assumed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Bis(2-ethylhexyl) hydrogen phosphate was administered orally by gavage to 5 male and 5 female Wistar (HsdRCCHan:Wist) rats per dose group using corn oil as vehicle, in daily doses of 0, 30, 150, 750 mg/kg bw for a period of 4 weeks. This GLP study was performed according to OECD guideline 407.
The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 30, 150, or 750 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female rats/dose

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local toxicity

Critical effects observed:

not specified

Survival was not affected by the treatment with bis(2-ethylhexyl) hydrogen phosphate up to a dose of 750 mg/kg bw/day in males and up to a dose of 150 mg/kg bw/day in females. One female animal of the high dose group had to be killed in moribund condition. It showed a massive gastritis in the forestomach, which was related to the treatment with the test article.

Several clinical findings observed at the high dose group of 750 mg/kg bw/day are regarded to reflect or to be secondary to the poor general condition of the animals of the high dose group, e.g. decreased motility, piloerection, bloody muzzle, uncoordinated gait, flaccid muscle tone, decreased body temperature, diminished motor and locomotor activity.

Furthermore, body weight development of males at the dose of 750 mg/kg bw/day was retarded and food intake per animal was lower. The increase in relative weight of testes at the dose of 750 mg/kg bw/day is regarded to be related to the body weight development of males of as well as the decrease in weight of thymus. The water intake was increased in males starting at 30 mg/kg bw/day and in females at 750 mg/kg bw/day. This effect is attributed to the effects on the alimentary tract.

The findings in the alimentary tract at necropsy were discolored areas, alteration and white covering of the mucosal surface of the forestomach as well as thickening of the gastric wall, which were evident in male animals starting at the dose of 30 mg/kg bw/day, and in female animals from the dose of 150 mg/kg bw/day onwards. At histopathology hyper- and parakeratosis and edema of the forestomach, gastritis in the forestomach and glandular stomach (all treated groups) associated with erosions (at 150 and 750 mg/kg) and ulcers of the forestomach (at 750 mg/kg), partially leading to peritonitis were noted. These findings indicate a local irritating and corrosive effect of the test substance.

The investigation gave evidence of treatment-related effects on liver function. In males, protein concentration was significantly decreased at 750 mg/kg bw/day, relative weights of liver were increased in males starting at 30 mg/kg b.w./day and at 750 mg/kg bw/day in females, hepatocellular hypertrophy was present starting at the dose of 150 mg/kg bw/day. These findings are regarded as a common sequel of metabolic activation and enzyme induction in response to a xenobiotic.

Hypertrophy of the follicular epithelia in the thyroid gland starting at the dose of 150 mg/kg bw/day is regarded to be secondary to the impact of the metabolic activation of the hepatocytes onto the thyroxine metabolism.

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.

At necropsy absolute and relative weights of adrenals were increased in males at the dose of 750 mg/kg bw/day. At histopathologic evaluation diffuse hyperplasia of the zona fasciculata in the adrenal gland was noted starting at the dose of 150 mg/kg bw/day in males and at the dose of 750 mg/kg bw/day in females.

Executive summary:

Bis(2-ethylhexyl) hydrogen phosphate was administered orally by gavage to 5 male and 5 female Wistar (HsdRCCHan:Wist) rats per dose group using corn oil as vehicle, in daily doses of 0, 30, 150, 750 mg/kg bw for a period of 4 weeks. This GLP study was performed according to OECD guideline 407.

The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Daily oral treatment of rats with bis(2-ethylhexyl) hydrogen phosphate at doses of 0, 30, 150, 750 mg/kg bw for a period of 4 weeks resulted in effects in the alimentary tract (hyper- and parakeratosis, edema, gastritis, erosions and ulcers), partially leading to peritonitis, partly starting at the low dose of 30 mg/kg bw/day and indicating a local irritating and corrosive effect of bis(2-ethylhexyl) hydrogen phosphate.

Most of further findings are regarded to reflect or to be secondary to the poor general condition of the animals due to this effect, e.g.: the death of one animal (at 750 mg/kg), several clinical findings including findings at FOB and motor activity measurement (at 750 mg/kg), retarded body weight development of males (at 750 mg/kg), increased water intake (in males starting at 30 mg/kg and in females at 750 mg/kg ), decreased weight of seminal vesicles (at 750 mg/kg) and reduced secretion in the prostate and seminal vesicle and a minimally increased proportion of round spermatids in the epididymides (at 750 mg/kg).

Furthermore, findings giving evidence for activation of the liver metabolism and enzyme induction were observed starting at 150 mg/kg, e.g. increase in liver weights, hepatocellular hypertrophy. Secondary to the impact of this metabolic activation, hypertrophy of the follicular epithelia in the thyroid gland was noted.

Finally, weights of adrenals were increased (at 750 mg/kg in males) and diffuse hyperplasia of the zona fasciculata in the adrenal gland was observed (in males starting at 150 mg/kg and in females at 750 mg/kg). This effect is seen as a sequel of the stress of the animals of this dose group.

Endpoint conclusion

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.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Daily oral treatment of rats with bis(2-ethylhexyl) hydrogen phosphate at doses of 0, 30, 150, 750 mg/kg bw for a period of 4 weeks resulted in effects in the alimentary tract (hyper- and parakeratosis, edema, gastritis, erosions and ulcers), partially leading to peritonitis, partly starting at the low dose of 30 mg/kg bw/day and indicating a local irritating and corrosive effect of bis(2-ethylhexyl) hydrogen phosphate.

Most of further findings are regarded to reflect or to be secondary to the poor general condition of the animals due to this effect, e.g.: the death of one animal (at 750 mg/kg), several clinical findings including findings at FOB and motor activity measurement (at 750 mg/kg), retarded body weight development of males (at 750 mg/kg), increased water intake (in males starting at 30 mg/kg and in females at 750 mg/kg ), decreased weight of seminal vesicles (at 750 mg/kg) and reduced secretion in the prostate and seminal vesicle and a minimally increased proportion of round spermatids in the epididymides (at 750 mg/kg).

In the 90 day and the 2 years study with tris(2-ethylhexyl) phosphate comparable results were found:

In a 13 week oral repeated dose study male and female four-week-old F344/N rats were administered by gavage with tris(2-ethylhexyl) phosphate (5 days per week). The study was conducted to evaluate the cumulative toxicity of tris(2-ethylhexyl) phosphate and to determine the doses to be used in the 2 year studies. Groups of rats of each sex were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw tris(2-ethylhexyl) phosphate, 5 days per week for 13 weeks by gavage. The animals were checked twice daily for signs of moribundity and mortality; moribund animals were killed. Animal weights were recorded weekly. At the end of the 13 week studies, survivors were killed. Necropsies were performed on all animals, except those excessively autolyzed or cannibalized. No hematology or clinical biochemistry was performed.

The following tissues were examined microscopically in vehicle controls, highest dose group, and all animals that died during the study: gross lesions and tissue masses, mandibular lymph node, salivary gland, sternebrae, including marrow, thyroid, parathyroids, small intestine, colon, liver, prostate/testes or ovaries/uterus, lungs and mainstem bronchi, mammary gland, skin, heart, esophagus, stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary, spinal cord if neurological signs were present, eyes if grossly abnormal.

No compound related deaths, slight-moderate depression of weight gain at 2000 or 4000 mg/kg bw in female rats and at 4000 mg/kg bw in male rats could be seen. No histopathologic effects could be observed.

The NOEL was 1000 mg/kg bw in female rats and 2000 mg/kg bw in male animals (NTP, 1984).

In a 2 year study in male and female rats the animals were treated orally by gavage 5 days per week for 103 weeks. The doses of tris(2-ethylhexyl) phosphate administered were 1000 or 2000 mg/kg in female rats and 2000 or 4000 mg/kg in male rats. Survival rates and mean body weight gains of dosed female rats were comparable to those of their respective vehicle controls. Microscopic evaluation was performed on the same organs as in the sub-chronic study discussed above. Survival rates of dosed male rats were comparable to that of the vehicle controls; but body weight gains were depressed at higher doses. The NOEL was 2000 mg/kg bw in female rats and <2000 mg/kg bw in male rats (NTP, 1984).

In a 13 week oral repeated dose study male and female four-week-old B6C3F1 mice were administered by gavage with tris(2-ethylhexyl) phosphate (5 days per week). The study was conducted to evaluate the cumulative toxicity of tris(2-ethylhexyl) phosphate and to determine the doses to be used in the 2 year studies. Groups of 10 mice of each sex were administered 0, 500, 1000, 2000, 4000 or 8000 mg/kg bw tris(2-ethylhexyl) phosphate, 5 days per week for 13 weeks by gavage. The animals were checked twice daily for signs of moribundity and mortality; moribund animals were killed. Animal weights were recorded weekly. At the end of the 13 week studies, survivors were killed. Necropsies were performed on all animals, except those excessively autolyzed or cannibalized.

The following tissues were examined microscopically in vehicle controls, highest dose group, and all animals that died during the study: gross lesions and tissue masses, mandibular lymph node, salivary gland, sternebrae, including marrow, thyroid, parathyroids, small intestine, colon, liver, prostate/testes or ovaries/uterus, lungs and mainstem bronchi, mammary gland, skin, heart, esophagus, stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary, spinal cord if neurological signs were present, eyes if grossly abnormal, gallbladder. No hematology or clinical biochemistry was performed.

No compound related deaths could be seen. Slight-moderate depression of weight gain at 4000 mg/kg bw (4.5%) in female mice and at 8000 mg/kg bw (7.1%) in male mice could be observed. Inflammatory lesions in the gastric mucosa were observed in all dose groups, with increased severity in the higher dose groups. Ulceration was observed in the forestomach of 1/10 males at 2000 mg/kg bw/day, 1/10 females at 4000 mg/kg bw/day and 1/10 males and 3/10 females at 8000 mg/kg bw/day.

The LOEL was 500 mg/kg bw based on the local inflammation in the gastric mucosa (NTP, 1984).

In a 2 year study in male and female B6C3F1 mice the animals were treated orally by gavage 5 days per week for 103 weeks. The doses of tris(2-ethylhexyl) phosphate administered were 500 or 1000 mg/kg. In male mice the survival was statistically significantly diminuated in the lower dose group (500 mg/kg). In the high dose group also deaths were observed, but not statistically significant compared to the control group. The NOEL therefore was <500 mg/kg bw for male mice and 1000 mg/kg bw for female mice (NTP, 1984).

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-ethylhexan-1-ol (CAS No. 104-76-7) 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-ethylhexan-1-ol (CAS No. 104-76-7) 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.

A prolongation of the duration of the study (from 90 day to 2 years) revealed no additional scientific knowledges or findings.

Therefore an additional testing of bis(2-ethylhexyl) hydrogen phosphate in a 90 day study (additional to the 28 day study) is not justified either scientifically and on grounds of animals welfare as no additional scientific knowledges or findings are expected.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The repeated dose systemic toxicity study in wistar rats (4 weeks daily administration by gavage) with the test item bis(2-ethylhexyl) hydrogen phosphate was used as the study with the best reliablility.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: other

Justification for classification or non-classification

Due to the results of the 28 day systemic toxicity study with bis(2-ethylhexyl) hydrogen phosphate and the results of the 90 day and 2 years study with tris(2-ethylhexyl) phosphate as a surrogate for bis(2-ethylhexyl) hydrogen phosphate a classification is not justified.