Trinonyl benzene-1,2,4-tricarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trioctyl benzene-1,2,4-tricarboxylate

EC Number:

201-877-4

EC Name:

Trioctyl benzene-1,2,4-tricarboxylate

Cas Number:

89-04-3

Molecular formula:

C33H54O6

IUPAC Name:

trioctyl benzene-1,2,4-tricarboxylate

Details on test material:

- Name of test material (as cited in study report): 1,2,4-benzenetricarboxylic acid, trioctyl ester (BCTO)
- Molecular formula (if other than submission substance): C33-H54-O6
- Molecular weight (if other than submission substance): 546.79
- Smiles notation (if other than submission substance): CCCCCCCCOC(=O)c1ccc(c(c1)C(=O)OCCCCCCCC)C(=O)OCCCCCCCC
- InChl (if other than submission substance): 1S/C33H54O6/c1-4-7-10-13-16-19-24-37-31(34)28-22-23-29(32(35)38-25-20-17-14-11-8-5-2)30(27-28)33(36)39-26-21-18-15-12-9-6-3/h22-23,27H,4-21,24-26H2,1-3H3
- Substance type: pale yellow clear liquid
- Physical state: liquid
- Analytical purity: >= 99.0%
- Lot/batch No.: no data
- Stability under test conditions: Stated to be stable
- Storage condition of test material: Ambient conditions

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan's Atsugi Breeding Center
- Age at study initiation: 10 weeks;
- Weight at study initiation: Males: 377.1-433 g; Females: 225.2-238.8 g
- Fasting period before study: No
- Housing: wire mesh cages, in pairs for mating; From 18 days gestation onwards (day of confirmed copulation = gestation day 0), females were housed in plastic rat breeding cages (w: 350w × d: 400 × h: 180 mm) with paper pulp chip bedding (Alpha-dri, Kasho Co., Ltd.).
- Diet: pelleted diet (CE-2 from Clea Japan Inc.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 50-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: May 2, 2000 To: June, 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: weekly, dissolving of required weight of the substance in corn oil, thereafter kept in a refrigerator in airtight containers in the dark until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance is poorly soluble in water
- Amount of vehicle (if gavage): 2ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of preparations were analysed and the results were in the range 98-102% of nominal. Formulated substance stable for at least 8 days.

Duration of treatment / exposure:

males: 42 days
females: 2 weeks prior mating, through mating, gestation and up to lactation days 4 (min 40 days to 53 days)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

mid dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of preliminary study (14 days repeated oral toxicity study using concentrations of 0, 500 and 1000 mg/kg bw/d - effects on body weight, increased liver weight and oedema of gastric mucosa observed in the high dose group)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were included: daily,during administration period observations were made twice or more per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health/moribund condition and response to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: days 0 (day of administration) , 7, 14, gestation days 1, 7, 14, 20 and parturition, lactation days 0 and 4 and days of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (supplied and remaining food were measured on administration days)
- Males: days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42;
- Females: days 1-2, 7-8, 14-15; days 0-1, 7-8, 14-15 and 20-21 of gestation, days 3-4 of lactation
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not applicable (no dietary study)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable (no drinking water study)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - after 42 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (sodium barbital)
- Animals fasted: Yes - 18-24 hours
- How many animals: all animals
- Parameters examined. Activated partial thromboplastin time (APTT), prothrombin time (PT), red blood cell count (RBC), platelet count and mean corpuscular volume (MCV), white blood cell count (WBC), haemoglobin, differential count, reticulocyte count
mean corpuscular haemoglobin (MCH), haematocrit, mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males - after 42 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (sodium barbital)
- Animals fasted: Yes - 18-24 hours
- How many animals: all animals
- Parameters examined: Glucose, blood urea nitrogen (BUN), total cholesterol, albumin, total protein, creatinine, alkaline phosphatase, GOT (AST) activity, GPT (ALT) activity, γ-GTP, calcium, total bilirubin, triglycerides, inorganic phosphorus, sodium, potassium and chlorine.
- Parameters calculated: A/G ratio,

URINALYSIS: Yes
- Time schedule for collection of urine: 4 hour collection from 5 males/5 females/group during Week 5 of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: pH, occult blood, protein, sugar, ketone bodies, urobilinogen, bilirubin, turbidity and colour.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Macroscopic examination of organs and tissues including organ weights: brain, heart, thymus, liver, kidneys, spleen, adrenal gland, and for males testis and epididymis. In females particular attention paid to uterus and ovaries (number of implantations number of corpus lutea and, under a stereoscopic microscope, implantation scars.)
HISTOPATHOLOGY: Yes (Testes and epididymides (all males), liver (all males and females of control, mid and high dose groups) stomach (all females and males of high dose and control groups). For other organs 5 males/5 females examined from high dose and control groups)

Statistics:

Graded data from the histopathological findings of test substance administered groups was handled by Mann-Whitney U test. Detection of significant difference between the total of positive grades and the control group was conducted by one-tailed Fisher’s exact test. For other data, uniformity of distribution for each group was first obtained by Bartlett method and, where distribution was uniform, one-way analysis of variance was conducted and, where there was significance between groups, multiple comparison testing was conducted by Dunnett method. However, where distribution was 0 for any of the groups and where distribution was not uniform, Kruskal Wallis rank test was performed, and where significance was found between groups, multiple comparison testing was conducted by Dunnett method. Significance level was set at 5% in all cases.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one female in the 500 mg/kg administration group died following administration on gestation day 23

Mortality:

mortality observed, treatment-related

Description (incidence):

one female in the 500 mg/kg administration group died following administration on gestation day 23

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant reduction in body weight increase for gestation days 7-14 in 500 mg/kg bw/d group compared to controls (only transient effect)

Food consumption and compound intake (if feeding study):

no effects 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):

Reduced RBC in females treated at 125 & 500 mg/kg/day (compared to controls minus 9% & 8%, respectively)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

at 500 mg/kg males - increased ALP, reduced total protein; in females - reduced total protein, significant decrease in creatinine concentration and significant increase in glucose concentration

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

majority of males in 500 mg/kg bw/d group showed elevated pH in urine, but group mean differences not biologically significant

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weight in females treated at 125 & 500 mg/kg/day (compared to controls plus 16% & 15%, respectively)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Hepatocellular hypertrophy centrilobular region of liver in males treated at 500 mg/kg/day

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY – 1 female in the 500 mg/kg administered group died following administration on administration day 42 (gestation day 23). In addition to salivation after dosing, which was seen intermittently from day 14, to administration, on the day of death, red discharge and soiled fur around the eyelid were observed.
In surviving animals salivation was observed immediately after administration in 11 males and 4 females in the 500 mg/kg group. Salivation was seen intermittently early on in the animals from day 9 of administration, but in all of these animals it disappeared 2-3 hours after administration and was a temporary change. Other than this, 2-5 males and females showed fur loss in each administration group, and 1 female in the control group was observed with emaciation, loose stool and soiled perianal region

BODY WEIGHT AND WEIGHT GAIN - For males, no significant difference was found for actual body weight and weight increase between control and treatment groups. For females, no significant difference was found for actual body weight, but comparison of the control group with the 500 mg/kg group showed a significant reduction in body weight increase for gestation days 7-14.

FOOD CONSUMPTION AND COMPOUND INTAKE - There were no significant difference between control and treatment groups.

URINALYSIS - A majority of males in the 500 mg/kg administered group showed elevated urine pH. No other parameters showed relevant differences.

HAEMATOLOGY - In males, there were no statistically significant difference between the control and treatment groups. In females, RBC was reduced, relative to controls, in animals treated at 125 or 500 mg/kg/day. A significant decrease in white blood cell count was observed in animals treated at 500 mg/kg/day. Females treated at 125 mg/kg/day exhibited a statistically significant reduction, relative to controls, in haematocrit and haemoglobin. A statistically significant decrease in neutrophils was apparent in animals treated at 30 mg/kg/day but these parameters showed no dose response relationship.

CLINICAL CHEMISTRY - A significant decrease in total protein concentration was observed in males and females administered 500 mg/kg/day. Males treated at this level also exhibited a significantly increased alkaline phosphatase activity. Females treated at 500 mg/kg/day also showed a significantly decreased creatinine concentration and a significantly increased concentration of glucose. Males treated at 30 mg/kg/day showed a statistically significant decrease in activity of γ-GTP although no dose-response relationship was apparent.

GROSS PATHOLOGY - In males, a single animal treated at 125 mg/kg/day exhibited a small and oedematous right testis. Nodules on the left epididymis were noted in a single male treated at 500 mg/kg/day. Both of these findnings were on one side only. One animal of the control group exhibited an elevated area on the seroa of the ileum. Single animals treated at 500 mg/kg/day exhibited an enlarged spleen and/or a thickening of the gastric mucosa of the fore-stomach together with a white cloudy mucosa. One female treated at 500 mg/kg/day died on Day 23 of pregnancy. The kidneys appeared dark, the spleen and liver pale. The thymus appeared small and the adrenals enlarged. The glandular mucosa of the fore-stomach was yellowed. In surviving females, black or dark red points of colouration of the glandular stomach mucosa were observed in two animals treated at 500 mg/kg/day and a single animal treated at 30 mg/kg/day. One animal treated at 125 mg/kg/day exhibited an enlarged liver, spleen and mesenteric lymph nodes, together with an area of yellow/white adhesion to the kidney, ovary, stomach and adipose tissue. The colouration of the bone marrow also appeared pale. The thymus of a second animal treated at 125 mg/kg/day and two control animals appeared small. The spleen of these control animals appeared small and the kidney and liver appeared pale.

ORGAN WEIGHTS – Testis weight of males treated at 125 mg/kg/day was statistically significantly reduced, but no dose-response relationship was observed. No other significant differences between control and treatment groups were noted. In females, a statistically significant increase in liver weight was observed, relative to controls, in animals treated at 125 mg/kg/day and higher (but no increase inbetween 125 and 500 mg/kg bw/d group). The relative weight of the heart, relative to controls, was significantly reduced in all treated groups. This significance was not apparent in absolute weights of the organ.

HISTOPATHOLOGY: NON-NEOPLASTIC – Decedent female (500 mg/kg/day) - Expansion of the lumen in the cortex accompanied by cell debris vacuolar degeneration and necrosis of proximal and distal tubule of the kidney was observed. Deposition of pigment in the proximal tubule and eosinophilic bodies were observed in many cortical tubules and the medulla. In the spleen extramedullary haematopoiesis and brown pigmentation was observed in the white pulp and the red pulp was atrophic. The thymus was atrophied, there was erosion of the gastric mucosa, an increase of lipid droplets in the zona fasciculata of the adrenal gland and peri-portal fatty change of the liver.

Surviving animals – Sex organs - In one animal each of the control group and those treated at 30 mg/kg/day, in two animals treated at 125 mg/kg/day and one animal treated at 500 mg/kg/day atrophy of seminiferous tubules was observed. In the epididymis, cell debris in the lumen and reduced epididymal sperm were observed in single animals of the control group and groups treated at 30 and 500 mg/kg/day. The animal treated at 500 mg/kg/day also exhibited spermatic granuloma. Interstitial lymphocytic infiltration was observed in each of two control animals and those treated at 30 mg/kg/day and in single animals of the groups treated at 125 or 500 mg/kg/day. Cellular infiltration of lymphocytes and plasma cells was observed in the interstitium/epithelium of the prostate of two animals treated at 500 mg/kg/day and four control animals. No ovarian abnormalities were observed in females.

Other organs - Mild hypertrophy of hepatocytes the centrilobular region of the liver was observed in five males treated at 500 mg/kg/day. Peri-portal fatty change was observed in male animals of all groups, including controls, although this was reduced in severity in animals treated at 500 mg/kg/day. Micro-granulomas were noted in individual animals of all groups. Similar findings were noted in female animals, one female treated at 500 mg/kg/day also exhibiting focal necrosis. Mild squamous cell hyperplasia of the fore-stomach was seen in three control females, four females treated at 30 mg/kg/day, two females treated at 125 mg/kg/day and five females treated at 500 mg/kg/day. Erosion of the gastric mucosa was seen in one female treated at 500 mg/kg/day and ulceration in one female treated at 30 mg/kg/day. No remarkable changes were noted in the stomach of male animals. The kidney of males of the control group and those treated at 500 mg/kg/day exhibited eosinophilic bodies and basophilic tubules of the cortex. Similar findings were noted in females and, in addition, females treated at 500 mg/kg/day showed vacuolar degeneration of the proximal tubule in one animal and mineral deposits in two animals. Extended necrosis and vacuolar degeneration of the proximal tubule was noted in two control females. No treatment-related effects were apparent. In one female animal treated at 125 mg/kg/day, enlargement of the spleen was observed at autopsy. Deposition of brown pigment, extramedullary haematopoiesis, fibroblasts and infiltration of macrophages and neutrophils and localised necrotic areas were observed microscopically. The liver of this animal showed proliferation of Kupffer cells with sinusoidal swelling, infiltration of lymphocytes and neutrophils, hyperplasia, granulomatous small bile duct was observed. In the mesenteric lymph nodes proliferation of plasma cells in the medullary cord and macrophages in the sinus was noted. Increased haematopoiesis of granulocyte lineage cells was observed in the bone marrow. In addition to the female treated at 125 mg/kg/day, atrophy of the thymus was noted in a female of the control group and two females treated at 500 mg/kg/day. An accumulation of foam was noted in the lungs of individual animals of both sexes from both the control group and those treated at 500 mg/kg/day. Mineral deposition was observed in the lungs of one male treated at 500 mg/kg/day and one control female. Degeneration and fibrosis of the myocardium was seen in one control male and lymphocyte infiltration was observed in the lamina propria of the urinary bladder of two control males.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects in clinical chemistry at 500 mg/kg bw/day

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Hepatocellular hypertrophy in centrilobular region of liver observed in males treated at 500 mg/kg.

3

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased liver weight and reduced red blood cell count observed in females treated at 125 and 500 mg/kg/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening according to OECD testing guideline 422 and performed according to GLP rats were treated via oral gavage with 0, 30, 125 or 500 mg 1,2,4 benzenetricarboxylic acid, trioctyl ester (C8TM)/kg bw/day for at least 42 days (males) up to 54 days (females). Mild anaemia and increased liver weight were observed in females treated at levels of 125 mg/kg bw/day or greater. However for the above noted effects no increase of response could be observed inbetween the 125 and 500 mg/kg bw/day groups. Centrilobular hypertrophy of hepatocytes in the liver was observed in males treated at 500 mg/kg/day. Moreover at the 500 mg/kg bw/day group some clinical chemical parameters were significantly different from control groups. In males increased ALP and reduced total protein was observed. In females, also reduced total protein levels were found and in addition a decrease in creatinine concentration and increase in glucose concentration. Overall the no-observed-effect level (NOEL) for repeated-dose toxicity under the conditions of this study was therefore 30 mg/kg bw/day in females and 125 mg/kg bw/day in males. In a conservative approach changes in clinical chemistry observed in both sexes at the highest dose group were used to establish the the no-observed-adverse-effect level (NOAEL). Thus the NOAEL of this study is judged to be 125 mg/kg bw/day.

Executive summary:

A Combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted for 1,2, 4 benzenetricarboxlyic acid, trioctyl ester (C8TM) at 0 (carrier), 30, 125 and 500 mg/kg for both female and male animals, with repeated administration in Sprague Dawley female and male rats (13 animals each/group) from 2 weeks before mating up until mating, then for 42 days for males and for pregnant females throughout gestation until day 4 of lactation. This GLP conform study was performed according to OECD testing guideline 422.

Other than 1 female death in the 500 mg/kg group at 23 days gestation, no deaths were recorded. In terms of changes in general condition, salivation was observed in both males and females directly following administration in the 500 mg/kg group. In females in the 500 mg/kg group, body weight increase values were low at 7-14 days gestation, but there was no influence of the test substance on male body weight or male and female food intake. In females, there was a reduction in red blood cell count and an increase in liver weight in the 125 mg/kg group, and decreased plasma protein concentration and increased glucose concentration in the 500 mg/kg group. Males in the 500 mg/kg group showed decreased serum protein and elevated alkaline phosphatase activity. Histological findings showed centrilobular liver hypertrophy in males in the 500 mg/kg group, but no reproductive system abnormalities thought to be caused by administration of the test substance were noted.

From the above test results and under the test conditions in this study, the no-effect dose of 1,2, 4 benzenetricarboxlyic acid, trioctyl ester for repeat dose toxicity was determined at 125 mg/kg day for males, 30 mg/kg day for females and the no-effect dose for reproduction/developmental toxicity was determined at 500 mg/kg day for parent animals and pups. In a conservative approach changes in clinical chemistry observed in both sexes at the highest dose group were used to establish the the no-observed-adverse-effect level (NOAEL). Thus the NOAEL of this study is judged to be 125 mg/kg bw/day.