Bis(2,3-epoxypropyl) terephthalate

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:

13 Jan - 11 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:

Bis(2,3-epoxypropyl) terephthalate

EC Number:

230-565-0

EC Name:

Bis(2,3-epoxypropyl) terephthalate

Cas Number:

7195-44-0

Molecular formula:

C14H14O6

IUPAC Name:

1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

Constituent 2

Reference substance name:

bis(2,3-epoxypropyl)terephthalate

IUPAC Name:

bis(2,3-epoxypropyl)terephthalate

Details on test material:

- Name of test material (as cited in study report): bis(2,3-epoxypropyl)terephthalate
- Physical state: white solid
- Analytical purity: 94.3%
- Lot/batch No.: 29669168
- Expiration date of the lot/batch: August 31, 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: 209-233 g (males), 155-180 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:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was grounded to a fine powder with the help of a mortar and pestle and then weighed into a tarred plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of corn oil and homogenized using an Ultraturrax. 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.: MKBD2427 (Sigma)

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. 57780100) 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. 40, 80 and 250 mg of the test item were suspended in 5 mL corn oil.
For the sample preparation procedure the samples were homogenised. Each sample was transferred into a volumetric flask which was filled up using acetone, ultrasonificated for 5 minutes and further diluted with acetone, acetonitrile and finally with 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 – 5 mg test item/L).

The mean recovery rates for the test item were:
- in the stability specimens:
40 mg/5 mL: 23% of nominal (n = 6; SD = 6%)
240 mg/5 mL: 99% of nominal (n = 2; SD = 13%)

- in the homogeneity specimens:
40 mg/5 mL: 16% of nominal (n = 9; SD = 9%)
240 mg/5 mL: 78% of nominal (n = 3; SD = 24%)

- in the test specimens:
40 mg/5 mL: 90% of nominal (n = 3; SD = 2%)
80 mg/5 mL: 87% of nominal (n = 4; SD = 7%)
240 mg/5 mL: 92% of nominal (n = 4; SD = 3%)

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 80 and 240 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: Upon arrival as well as twice weekly daily during the treatment period and on the day of terminal sacrifice.

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):

all dose groups; slight clinical signs

Mortality:

mortality observed, treatment-related

Description (incidence):

all dose groups; slight clinical signs

Body weight and weight changes:

no effects observed

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

240 mg/kg bw/day; high protein and leucocyte level in one male

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

80, 240 mg/kg bw/day; increased weight of several organs

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and only slight clinical signs (e.g. slight piloerection in 2/5 male animals and 1/5 female animals, nasal discharge in 3/5 male animals and aggressiveness in 2/5 male animals and 3/5 female animals) were observed in the dose groups and the control group.
Male animal no. 20 of the high dose group showed moderate piloerection and slightly reduced spontaneous activity. This is possibly related to a mesenchymal tumor diagnosed macroscopically and histopathologically in one kidney of this animal and is not assumed to be test item-related.

HAEMATOLOGY
At the end of the treatment period haemoglobin was slightly – but statistically significantly lower in male animals of the high dose group than in controls. At the same time, a tendency towards a slight – however not statistically significant – increase in reticulocyte level was found, suggesting a regenerative process. A statistically significantly higher red blood cell count in male animals of the mid dose group than in controls is not assumed to be biologically relevant.

CLINICAL CHEMISTRY
Total bile acid level was markedly elevated in female animal no. 37 of the high dose group, but more prominently in female animal no. 26 of the low dose group. Apart from minimal mononuclear infiltrates, there were no associated findings indicating hepatotoxicity in these animals and TBA was also increased in female control animal no. 21. Thus, this effect is not assumed to be related to the test item.

URINALYSIS
High protein and leucocyte levels were found in the urine of male animal no. 20 of the high dose group. This was associated with a renal mesenchymal tumor in one kidney and minimal tubular degeneration and basophilic tubules in the cortex. High protein and leucocyte levels were also found in the urine of female animal no. 30 of the low dose group (not associated with any pathological signs).

ORGAN WEIGHTS
A moderately higher spleen weight in male and female animals was found in the high dose group, when compared to controls. A slightly higher kidney weight in the high dose group and - in male animals also in the mid dose group, was observed. A slightly higher heart weight in male animals is not assumed to be biologically relevant as no considerable difference was found when related to brain weight or in terms of absolute heart weights. A slightly lower liver weight was found in male animals of the high dose group.
In respect to reproductive systems, slightly to moderately higher uterus and ovary weights were found as well as a tendency towards slightly lower weight of epididymides in the high dose group and a slightly higher prostate weight in the mid dose group.
All effects were not associated with any test item-related histopathological findings.

GROSS PATHOLOGY
An enlarged kidney fused with an enlarged adrenal gland containing a large white subcapsular white mass (5x5x3.5cm) was observed in one male animal of the high dose group. Histopathologically this was diagnosed as renal mesenchymal tumor which is not assumed to be related to the test item.
Slight single or occasional findings, like red mesenteric lymph nodes, yellow areas on the epididymides, a cyst on an ovary, grey stomach or dark lung or red duodenum were observed in control animals and/or are assumed to be common background findings in this strain.
A foam-filled lung in one male animal of the mid dose group is assumed to be related to euthanisation of the animal.

HISTOPATHOLOGY: NON-NEOPLASTIC
A diffuse minimal hyperkeratosis of the nonglandular part of the stomach was found in the majority of female rats. This effect might be related to a local irritant effect of the test item formulation when administered repeatedly by oral gavage and was therefore not considered relevant for humans.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOAEL

Effect level:

>= 240 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the present study, no effects of bis (2,3-epoxypropyl) terephthalate were found at dose levels of 40 and 80 mg/kg bw. The NOEL of bis (2,3-epoxypropyl) terephthalate in this study is considered to be 80 mg/kg bw.
At a dose level of 240 mg/kg bw slight clinical symptoms occurred in few more animals than in control animals and a tendency towards an attenuated body weight gain and food intake were observed. A slightly lower heamoglobin level in male animals was associated with a slight compensatory increase in reticulocytes. These effects are not considered to be in the respective toxic range. Thus, the NOAEL in this study is considered to be 240 mg/kg bw.
Diffuse minimal hyperkeratosis of the nonglandular part of the stomach was found in the majority of female rats dosed with 240 mg/kg bw. This effect might be related to a local irritant effect of the test item formulation when administered repeatedly by oral gavage and was therefore not considered relevant for humans.