Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

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:

19 Jan - 17 Feb 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

EC Number:

230-638-7

EC Name:

Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

Cas Number:

7237-83-4

Molecular formula:

C18H18O9

IUPAC Name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate

Details on test material:

- Name of test material (as cited in study report): tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate
- Physical state: light yellow liquid
- Analytical purity: 81.8%
- Batch No.: Baj Hj 04
- Expiry Date: Jan 06, 2012
- Storage condition of test material: at RT in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 192-226 g (males), 148-170 g (females)
- Housing: individually in IVC cages, type lll H, polysulphone cages on Altromin saw fibre bedding (lot no. 150910)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1013), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 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

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tarred plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of PEG 300 and were mixed using a vortex machine until a homogenous suspension was obtained. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a vortex machine. The test item formulations were prepared freshly on each administration day before the administration procedure.

VEHICLE
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 9R001812 (AppliChem)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose verification was performed at IBACON GmbH (Rossdorf, Germany) by quantification of the test item using HPLC method (Project No. 57800100) in accordance with GLP.
In order to determine the concentration of the test item in the dosing formulations, samples were retained from all groups once weekly during the treatment period and stored between -15 and -35 °C. The stability of the dosing formulations was tested once at the beginning of the treatment period. From all dose groups samples of dosing formulations were frozen at 0 h and 6 h after preparation and stored at -15 and -35 °C. Stability was tested in samples of dosing formulations of the low and high dose groups only. In the first week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, mid and low dose formulations and stored between -15 and -35 °C. Homogeneity was tested in samples of dosing formulations of the low and high dose groups only. At the end of the treatment period all samples of dosing formulations were shipped on dry ice and protected from light to IBACON GmbH.
To fortify samples at different concentrations approx. 50, 150 and 500 mg of the test item were suspended in 5 mL PEG 300.
For the sample preparation procedure the samples were homogenised. Each sample was transferred into a volumetric flask which was filled up using acetone and further diluted with acetone and a mixture (50:50 v/v) of acetonitrile and pure water. Measured samples were quantified by measuring the peak area with reference to the calibration curve (calibration range 0.05 – 2.5 mg test item/L).

The mean recovery rates for the test item were:
- in the stability specimens:
50 mg/5 mL: 92% of nominal (n = 2; SD = 0%)
500 mg/5 mL: 94% of nominal (n = 2; SD = 10%)

- in the homogeneity specimens:
50 mg/5 mL: 97% of nominal (n = 3; SD = 9%)
500 mg/5 mL: 106% of nominal (n = 3; SD = 1%)

- in the test specimens:
50 mg/5 mL: 88% of nominal (n = 4; SD = 10%)
150 mg/5 mL: 96% of nominal (n = 4; SD = 4%)
500 mg/5 mL: 101% of nominal (n = 4; SD = 8%)

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150 and 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
- Parameters examined: Spontaneous activity, lethargy, recumbent position, convulsions, tremors, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: Once before assignment to the experimental groups, on the first day of administration and weekly during the treatment period. To achieve a more accurate individual dosing volume, one additional body weight measurement was performed during each week of the treatment period. Body weight was also measured at necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly during the treatment period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Together with detailed clinical observations.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All surviving animals
- Parameters examined: haematocrit value (HCT), haemoglobin content (Hb), red blood cell count (RBC), MCV, MCH, MCHC, reticulocytes (RE), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All surviving animals
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: At terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity, nitrite, pH, protein, glucose, ketones, urobilinogen (ubg), bilirubin, blood, leucocytes, colour/appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure, as well as once in the fourth week of exposure.
- Dose groups that were examined: All groups
- Battery of functions tested: not indicated in the report

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organs weighed: adrenals, brain, epididymides (males), heart, kidneys, liver, pituitary gland, ovaries (females), prostate with seminal vesicles (males), spleen, testes (males), thymus, uterus with cervix (females)

HISTOPATHOLOGY: Yes
- Tissues examined: adrenal glands, aorta, bone with bone marrow (sternum), brain (cerebrum, cerebellum and pons), epididymides (males), eyes, gross lesions, heart, kidneys, liver, lung, lymph nodes (mesenteric and axillary), mammary gland, oesophagus, ovaries (females), pancreas, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, pituitary, prostate with seminal vesicles (males), salivary glands, skin, small and large intestines (including Peyer´s patches), spinal cord, spleen, stomach, testes (males), thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus with cervix (females), vagina (females)

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p < 0.05 was considered as statistical significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

150, 500 mg/kg bw/day; slight clinical signs

Mortality:

mortality observed, treatment-related

Description (incidence):

150, 500 mg/kg bw/day; slight clinical signs

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day; decreased body weight (gain) in males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day; decreased food consumption in males

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day; increased weight of several organs

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

150, 500 mg/kg bw/day; several enlarged organs in some animals

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day; changes in testis end epididymis

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and only slight clinical signs were observed.
Piloerection was observed in all groups, the degree of severity, however, was higher in some animals of the high dose group. In 3/5 male and 1/5 female animals of this group this was associated with a slightly reduced spontaneous activity. Moreover, 3/5 or 4/5 female animals of the mid and high dose group, respectively, appeared anxious.
Salivation, nasal discharge and moving through the bedding with the snout also occurred in all groups but are assumed to be related to the vehicle PEG 300. Besides, animals appeared aggressive in all groups, which might also be related to the vehicle.
Single transient findings that are not assumed to be related to the test item were relieving and dragging the left hindleg in one control animal, slight respiratory sounds in one animal of the low, mid and high dose groups, respectively, dyspnoe and gasping in one animal of the control and low dose groups, respectively, tremor during handling of one animal of the mid dose group and slight alopecia in animal of the low dose group.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was significantly affected in treated male – but not treated female animals. Body weight gain during the treatment period was statistically significantly lower in male animals of the high dose group when compared to controls.
Overall, no effect on body weight gain during the treatment period was found in male animals of the mid and low dose groups and in dosed female animals. A statistically significantly lower body weight gain in female animals of the high dose group in the 3rd treatment week was compensated by statistically significantly higher body weight gain in the 4th treatment week, when compared to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In accordance to the lower body weight gain of male animals, food consumption was slightly but statistically significantly lower during the treatment period in the high dose group. The difference to the control group was statistically significant during the first three treatment weeks (between 81 and 85% of controls). In female animals of the high dose group, slightly but statistically significantly lower food consumption was found in the first and third treatment week (between 83 and 84% of controls, respectively).
Food consumption was not affected in male animals of the mid and low dose groups and in dosed female animals of this study.

HAEMATOLOGY
In male animals of the high dose group, red blood cell parameters, i.e. haematocrit and haemoglobin, were very slightly but statistically significantly lower, when compared to controls. In female animals of the high dose group a very slightly but significantly lower haemoglobin level was noted. Both, in male and female animals, reticulocyte level was slightly and statistically significantly higher than controls, suggesting a compensatory mechanism. As the decrease in red blood cell parameters was very slight and values were at the lower border of the respective normal range of variation, this effect is not assumed to be toxicologically relevant. Red blood cell parameters of the mid and low dose groups were not affected. A slight but significantly elevated level of red blood cells in the mid dose group of the female animals and number of platelets in the male animals of the high dose group are not considered to be biologically relevant.

CLINICAL CHEMISTRY
At the end of the treatment period, all parameters of clinical biochemistry were in the normal range of variation for this strain. A slight but statistically significantly higher Cholesterol level in female animals of the high dose group is not considered to be biologically relevant.

ORGAN WEIGHTS
Statistically significantly higher brain weight related to body weight was found in the male animals of the high dose group, when compared to controls. As absolute brain weights did not differ considerably between these groups, this finding is not assumed to be biologically significant.

Absolute and relative kidney weight was slightly to moderately and statistically significantly increased in female animals of the high dose group when compared to controls (between approx. 30 and 45% higher than in controls). When related to body weight, a slight increase in kidney weight was also found in male animals of the high dose group (33% higher than controls). A slight – but also statistically significantly higher kidney weight was also observed in female animals of the mid and low dose groups when related to body weight (15% and 11% above controls, respectively).

When related to body weight, also a statistically higher adrenal weight was noted in female animals of the high dose group, when compared to controls (27% above controls). A tendency was also found in male animals of the mid and high dose groups (15 and 21%, respectively above controls) and female animals of the mid dose group (10% above controls). In terms of absolute adrenal weight and in adrenal weight when related to brain weight also an increase was observed (16% and 18% above controls, respectively).

Slightly but statistically significantly lower heart weight (absolute and in relation to brain weight was found in male animals of the high dose group, when compared to the control group (approx. 20% lower). The difference between those groups, however, was only small, when compared to body weight.

When related to body weight, liver weight was slightly but statistically significantly higher in male and female animals of the high dose group, than in controls (11% and 17% above controls, respectively). When related to brain weight or in respect to absolute weight, only a tendency was noted in female but not male animals of this group. A considerable difference was not observed in any of the other groups.

Absolute spleen weight was slightly but not statistically significantly increased in female animals of all groups. When related to body weight, differences were statistically significant in all groups (22%, 27% and 27% in the low, mid and high dose groups, respectively). In male animals only a slight but not statistically significant increase of 16% was noted in the high dose group.

A tendency towards a lower thymus weight, when compared to controls was observed in the male animals of the high dose group and in the female animals of the mid and high dose groups.

Epididymides weight was moderately higher in the high dose group when compared to controls. The difference was statistically significant, when related to brain weight (23% above controls) and to body weight (41% above controls).

The effect on epididymides in the high dose group was associated with a significantly lower prostate weight, when compared to controls (absolute weight 24% below controls; in relation to brain weight 23% below controls). A tendency towards lower prostate weight was also noted in the mid dose group. When related to body weight also a statistically significantly higher testis weight was found in the high dose group (22% above controls).

Although not statistically significantly, uterus weight was slightly increased in the low and mid dose groups and moderately in the high dose group.

GROSS PATHOLOGY
In the high dose group kidneys were noted as enlarged in 2/5 male and female animals, respectively. One male animal additionally, had enlarged adrenal glands. In 2/5 animals epididymides and in 1/5 male animals of this group testis were enlarged unilaterally. Yellow spots or tissue embedded in the epididymides were observed in 2/5 animals of the low dose group, 1/5 animals of the mid dose group and 3/5 animals of the high dose group. It should be noted that, although only dosed animals were affected, the latter findings are of high spontaneous occurrence in this strain. All above-mentioned findings were not noted in any of the other groups.
Single or occasional findings that are not assumed be treatment-related were a discolored grey or white stomach, dark or red spots on the lung, a discolored red thymus, a cyst on an ovary, a cyst on the uterus.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes considered to be clearly test item-related were seen in the testis and epididymis, in the group treated at 500 mg/kg bw/day. Epididymal spermatic granuloma(s) were seen in all males of this group and were associated with inflammatory edema, increased incidence and degree of interstitial mononuclear cell infiltrates, condensed luminal content, bilateral oligospermia and/or bilateral intraluminal cellular debris in the epididymis and with tubular dilation of the testis in the same dose group.
Minimal diffuse epithelial hyperkeratosis of the nonglandular part of the stomach was noted in a proportion of females treated at 500 mg/kg/day and considered to be possibly related to a local irritant effect of the test item formulation when administered repeatedly by oral gavage and therefore not to be relevant for humans. No other test item-related pathological lesions were observed.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Slightly decreased body weight, food consumption and histopathological findings in reproductive organs.

3

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the present study, no signs of toxicity were found at dose levels of 50 and 150 mg/kg bw/day. At a dose level of 500 mg/kg bw/day the following signs of toxicity were observed: slight clinical findings, slightly decreased body weight gain and food consumption intermittently during the treatment period in male animals. Increased spleen, kidney and adrenal weight and slightly decreased heart and prostate weight were without associated histopathological finding. Histopathological changes were seen in the testis and epididymis. Epididymal spermatic granuloma(s) were seen in all males of this group and were associated with inflammatory edema, increased incidence and degree of interstitial mononuclear cell infiltrates, condensed luminal content, bilateral oligospermia and/or bilateral intraluminal cellular debris in the epididymis and with tubular dilation of the testes in the same dose group.
Due to gender-specific signs of toxicity at the high dose level, separate NO(A)ELs can be placed for male and female animals of this study.
Male animals:
Signs of toxicity observed in male animals at the dose level of 500 mg/kg bw/day were: slightly decreased body weight, food consumption and the above-mentioned histopathological findings in reproductive organs. As no signs of toxicity were observed in male animals at the dose levels of the low and mid dose groups, the dose level of 150 mg/kg bw/day marks the NOAEL for male animals in this study.
Female animals:
No signs of toxicity were observed in female animals at dose levels of 50, 150 or 500 mg/kg bw/day. Thus, the dose level of 500 mg/kg bw/day marks the NOAEL for female animals in this study.
Minimal diffuse epithelial hyperkeratosis of the nonglandular part of the stomach was noted in a proportion of females treated at 500 mg/kg bw/day and considered to be possibly related to a local irritant effect of the test item formulation when administered repeatedly by oral gavage and therefore not to be relevant for humans.