Phosphoric acid, butyl ester, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl hydrogen phosphate

EC Number:

203-509-8

EC Name:

Dibutyl hydrogen phosphate

Cas Number:

107-66-4

Molecular formula:

C8H19O4P

IUPAC Name:

dibutyl hydrogen phosphate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: SD (Crj:CD(SD))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co., Ltd. (795 Shimo-furusawa, Atsugi-shi, Kanagawa-ken)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 9 weeks, females: 8 weeks
- Weight at study initiation: males 345 (331-362) g, females 203 (192-215) g
- Housing: During acclimation and the dosing period, the rats were individually housed in stainless steel mesh cages (276 mm wide × 426 mm deep × 200 mm high) on 5 stainless steel racks in a barrier system animal house. The females confirmed as having copulated were housed in polycarbonate cages (276 mm wide × 426 mm deep × 200 mm high) containing bedding (White Flake, Charles River Japan Co., Ltd. (batch number 4-1-24, 4-8-6).
- Diet: Clea-Japan, Inc. CE-2 solid food, batch number E-2072-UB, E-2092-S4, ad libitum
- Water: tap water, used on ultraviolet irradiation after filtration through a 1 μm cartridge filter, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

administration of the dosing solution into the stomach of the rat using a gastric tube and syringe

Vehicle:

other: Japanese Pharmacopoeia sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item starts to gradually degrade in the dosing solution from 7 hours after preparation, therefore the dosing solution was prepared daily, immediately before use.

VEHICLE
- Concentration in vehicle: (30 mg/kg group: 0.6 w/v%; 100 mg/kg group: 2 w/v%; 300 mg/kg group: 6 w/v%; 1000 mg/kg group: 20 w/v%)
- Amount of vehicle: 5 mL
- Lot/batch no.: LI01

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three samples of prepared dosing solution were analysed for each concentration, and it was confirmed that the dosing solutions had been prepared to the prescribed concentrations.

Duration of treatment / exposure:

from 14 days prior to the start of mating, until day 3 of lactation after delivery for the females (40-51 days), and for 44 days for the males

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control, solvent administered

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were set based on the results of a dose-setting study. In the dose-setting study, 6-week-old SD (Crj:CD(SD)) rats were assigned to 6 groups: a control group to which solvent was administered, and test item (50, 100, 200, 500 and 1000 mg/kg bw/day) -dose groups. There were 4 males and 4 females per group, and repeated oral administration was undertaken for a period of 14 days.
As a result, transient red urine excretion, urine-stained lower abdominal fur and a tendency to increased liver and adrenal weights, and the like, were observed, but there were no deaths, not even in the 1000 mg/kg bw/day group.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each animal was measured the day dosing was started (immediately before dosing was started), at 7-day intervals thereafter, on the final dosing day, and on the day the animal was killed or found dead.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately before the animal was killed in the case of animals killed in extremis, and immediately before necropsy on the day after the completion of the dosing period (45 days after starting dosing) in the case of animals killed as scheduled.
- Anaesthetic used for blood collection: Yes (ether anaesthetic)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above (same as for haematology)
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Forty-two days after starting dosing, fresh urine was sampled by compressive stimulation of the lower back of the rat.
- Animals fasted: No
- Parameters checked: pH, occult blood, protein, glucose, ketone bodies, bilirubin and urobilinogen

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table3)

Statistics:

The mean values and frequencies obtained were tested, using the following methods, for significant differences compared to the control group (risk rate 5% or lower)
Parametric data, such as bodyweight, food consumption, haematology and blood biochemistry data, organ weights, number of corpora lutea, number of implantation sites, gestation period, number of pups born, number of live pups born and number of dead pups born, was tested using Bartlett’s test for variances. If the variance was uniform, one-way analysis of variance was undertaken, and if a significant difference was found, the group was compared with the control group using the Dunnett’s method or Scheffé’s method (if the group size differed). If the variance was not uniform, a test method used for nonparametric data was employed.
Nonparametric data, such as the implantation index, live birth index, delivery index, viability index and qualitative data from urology tests, was tested using the Kruskal-Wallis rank test, and if a significant difference was found, the group was compared with the control group using the Dunnett’s method or Scheffé’s method (if the group size differed). Categorical data, such as parental animal viability, copulation index, conception index, gestation index, sex ratio for pups born and incidence of animals with abnormal histology test results or the like, was tested using the χ2 test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The excretion of red urine was observed in males in the 100 mg/kg bw/d and higher dose groups, and in females in the 100 and 300 mg/kg bw/d groups. Urine-stained abdominal fur and salivation were seen in males in the 100 mg/kg bw/d and higher dose groups, and in females in the 300 mg/kg bw/d and higher dose groups. In most cases, the excretion of red urine was a transitory change observed only the day after dosing was started. Salivation developed immediately after dosing or, in some cases, when the animal was restrained for dosing, and lasted a few minutes; apparent aversion to the dosing solution, and urine-stained lower abdominal fur, were observed intermittently throughout the dosing period. All of these changes were more often seen in males, with low incidence in females, with the exception of salivation in the 1000 mg/kg group. males in the 1000 mg/kg bw/d group and females in the 100 mg/kg bw/d and higher dose groups became emaciated with repeated administration or after delivery, and some also exhibited decreased locomotor activity and deep respiration. Some females also exhibited chromodacryorrhea, loose stool or rough fur.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 1000 mg/kg bw/d group, 2 males and 1 female died on dosing day 13. Also, 1 female was found moribund on dosing day 14 and 1 male was found moribund on dosing day 28: both were killed in extremis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant inhibition of body weight increase compared to the control group was observed in males in the 1000 mg/kg bw/d group throughout the dosing period. A tendency to the same was observed in females in the 1000 mg/kg bw/d group, although the change was mild compared to that seen in the males, and a statistically significant difference was seen only in the body weight measured on dosing day 15 and on day 7 of pregnancy.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption was significantly lower than in the control group in the 100 and 300 mg/kg bw/d groups on dosing day 8 and in the 1000 mg/kg bw/d group on dosing days 1 and 8. However, there was no notable difference compared to the control group thereafter. In females, no significant difference compared to the control group was observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg bw/d group, a significant increase in the mean corpuscular haemoglobin concentration was observed. This was because the erythrocyte counts and haematocrit values were slightly lower than in the control group, although these changes were not statistically significant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No statistically significant changes in any of the items tested were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in any of the items tested were observed in any dose group. However, in the 1000 mg/kg bw/day group, urine tended to be slightly acidic, and the occult blood reaction was mildly positive in 1 animal in the 300 mg/kg bw/day group and in 1 animal in the 1000 mg/kg bw/day group.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant increases in the absolute and relative weights of the liver were observed in females in the 1000 mg/kg bw/d group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The main findings were changes to the digestive tract. Specifically, in the forestomach, mucosal thickening was observed in males and females in the 300 mg/kg bw/d and higher dose groups. In the 1000 mg/kg bw/d group in particular, membranoid formation was observed on the surface of the thickened mucosa, and detached tissue was observed in the lumen. Males in the 1000 mg/kg bw/d group also exhibited distended caecum with very watery contents. Furthermore, in the 1000 mg/kg group, the males and females that died or were killed in extremis and the females whose pups all died exhibited scattered black spots on the mucosa of the glandular portion of the stomach and distension of the stomach and intestine due to the accumulation of gas.
In addition to these changes to the digestive tract, enlargement of the liver was seen, albeit at low incidence, in females in the 1000 mg/kg bw/d group. Also, atrophy of the thymus and spleen were observed in animals that died or were killed in extremis, and in females whose pups all died in all dose groups; hypertrophy and/or decolouration of adrenals was observed in females whose pups all died; and dark red colouration of the lungs, darkening of the liver and kidneys, and red colouration of the adrenals were observed in animals that died or were killed in extremis.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males: In the 100 mg/kg bw/day and higher dose groups, epithelial hyperplasia with degeneration and ulceration were observed in the bladder mucosa. In the 300 mg/kg bw/day and higher dose groups, thickening of the mucosa due to epithelial hyperkeratosis and hyperplasia were observed in the forestomach, and there were also cases of erosion and ulceration of the glandular stomach mucosa and of the thickened forestomach mucosa. In the 1000 mg/kg bw/day group, distended caecum with epithelial degeneration was observed.
Females: in the 100, 300 mg/kg bw/day and higher dose groups there was damage to the bladder and gastric mucosa, in the 1000 mg/kg bw/day group there was hepatocyte enlargement and increased liver weight.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Necropsy findings
The main findings were changes to the digestive tract. Specifically, in the forestomach, mucosal thickening was observed in males and females in the 300 mg/kg bw/day and higher dose groups. In the 1000 mg/kg bw/day group in particular, membranoid formation was observed on the surface of the thickened mucosa, and detached tissue was observed in the lumen. Males in the 1000 mg/kg bw/day group also exhibited distended caecum with very watery contents. Furthermore, in the 1000 mg/kg bw/day group, the males and females that died or were killed in extremis and the females whose pups all died exhibited scattered black spots on the mucosa of the glandular portion of the stomach and distension of the stomach and intestine due to the accumulation of gas.
In addition to these changes to the digestive tract, enlargement of the liver was seen, albeit at low incidence, in females in the 1000 mg/kg bw/day group. Also, atrophy of the thymus and spleen were observed in animals that died or were killed in extremis, and in females whose pups all died in all dose groups; hypertrophy and/or decolouration of adrenals was observed in females whose pups all died; and dark red colouration of the lungs, darkening of the liver and kidneys, and red colouration of the adrenals were observed in animals that died or were killed in extremis.
It should be noted that no particular abnormalities of the reproductive organs were noted in the infertile breeding pairs or in the females whose pups all died.

Histopathology findings
The main effects of test item administration were observed in the liver, stomach and intestines, and in the bladder; changes were also observed in the adrenals, spleen, thymus, etc. No particular changes relating to reproductive toxicity were observed.
a. Liver (examined for all males in the control group and 1000 mg/kg bw/day group, infertile males in other groups, and all females in all groups): Centrilobular hepatocellular swelling was observed in females in the 1000 mg/kg bw/day group. Centrilobular to intermediate zone hepatocellular fatty changes were observed in the 100 mg/kg bw/day and higher dose groups, mainly in females whose pups all died. Congestion was observed in animals that died or were killed in extremis.
b. Stomach (examined for all males and females in all groups): Thickening of the forestomach mucosa due to hyperkeratosis and hyperplasia of the stratified squamous epithelium was observed in males and females in the 300 mg/kg bw/day and higher dose groups. In the 100 mg/kg bw/day and lower dose groups, these changes were only observed in females, and in the control group and 100 mg/kg bw/day group, these changes were only observed in 1 animal, respectively. Erosion and ulceration of the glandular stomach mucosa and of the thickened mucosa of the forestomach were observed in animals that died or were killed in extremis and in females whose pups all died, and ulceration of the forestomach mucosa was also observed in females killed as scheduled.
c. Intestines (examined for all males and females in the control group and 1000 mg/kg bw/day group, infertile animals in other groups; caecum examined for all males in the other groups): Mild degeneration of the surface epithelium was observed in the caecum of males in the 300 mg/kg bw/day and higher dose groups. No particular abnormalities were observed in the other parts of the male intestine, or in the females.
d. Bladder (examined for all males and females in all groups): The main findings were hyperplasia of the bladder epithelium and degeneration of the surface epithelium, observed in males and females in the 100 mg/kg bw/day and higher dose groups; endothelial necrosis and ulceration was also observed in females in the 300 and 1000 mg/kg bw/day groups.
e. Adrenals (examined for all males in the control group and 1000 mg/kg bw/day group, infertile males in other groups, and all females in all groups): In the adrenal cortex, particularly the fascicular cells, vacuolation attributed to increased lipid content was observed in the 100 mg/kg bw/day and higher dose groups. Almost all of the animals that exhibited these changes were females whose pups all died. Also, congestion was observed in animals that died or were killed in extremis.
f. Spleen and thymus (examined for all males in the control group and 1000 mg/kg bw/day group, infertile males in other groups, and all females in all groups): Atrophy of the spleen and thymus was observed in animals that died or were killed in extremis and in females whose pups all died. Brown pigment deposition in the atrophied spleen was seen in males and females, and in some females there were also changes such as decreased foci of extramedullary haematopoiesis.
g. Lungs (examined for all males and females in the control group and 1000 mg/kg bw/day group, infertile animals in other groups, and females whose pups all died): Males and females that died in the 1000 mg/kg bw/day group also exhibited congestion or congestive oedema of the lungs.
h. Brain, heart, kidneys, testes or ovaries (examined for all males and females in the control group and 1000 mg/kg bw/day group, infertile animals in other groups, and females whose pups all died): No changes attributable to the effects of the test item administration were observed in any of these organs.
i. Pituitary, and epididymis, seminal vesicles and prostate or uterus and vagina (infertile males and females, and females whose pups all died): No abnormalities were observed in any of these organs.
In addition to the findings described above, other changes were observed in the various organs examined, but these other changes were all thought to have been spontaneous pathological changes unrelated to test item administration.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: damgae to digestive tract mucosa, bladder mucosa and effects in liver

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity was determined to be 30 mg/kg bw/day.

Executive summary:

In order to investigate the toxicity of the test substance after repeated administration a combined repeated dose toxicity and reproductive toxicity study was undertaken using SD (Crj:CD(SD)) rats. There were 10 male and 10 female rats per group, and the test item doses were 0 (control: solvent administered), 30, 100, 300 and 1000 mg/kg bw/day, administered by forced oral administration every day from 14 days prior to the start of mating, until day 3 of lactation after delivery for the females (40-51 days), and for 44 days for the males. The results obtained were as follows: For the parental males, no effects of test item administration were observed in the 30 mg/kg bw/day group. In the 100 mg/kg bw/day and higher dose groups, epithelial hyperplasia with degeneration and ulceration were observed in the bladder mucosa, and red urine excretion and urine-stained lower abdominal fur were also observed. Food consumption decreased in the early stage of dosing. In the 300 mg/kg bw/day and higher dose groups, thickening of the mucosa due to epithelial hyperkeratosis and hyperplasia were observed in the forestomach, and there were also cases of erosion and ulceration of the glandular stomach mucosa and of the thickened forestomach mucosa. In the 1000 mg/kg bw/day group, distended caecum with epithelial degeneration was observed, bodyweight increase was inhibited, and there were deaths.In the parental females (in the 100, 300 mg/kg bw/day and higher dose groups) there was damage to the bladder and gastric mucosa as seen in the parental males; in the 1000 mg/kg bw/day group there were also deaths, and in addition, in the 1000 mg/kg bw/day group there was hepatocyte enlargement and increased liver weight. Also, in the 100 mg/kg bw/day and higher dose groups there were parental females whose pups all died during or after delivery. These parental females also exhibited erosion and ulceration of the gastric mucosa, and fatty hepatocytes, adrenocortical cell vacuolation, etc., were also observed.

From these results it was concluded that the main repeated dose toxicity in the parental animals was damage to the digestive tract, particularly the gastric mucosa and bladder mucosa, and the liver was also affected. The no-effect dose for general toxicological effects was determined to be 30 mg/kg bw/day, for males and females. This value was also determined to be the no-observed-adverse-effect level (NOAEL).