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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April to July 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl naphthalene-2,6-dicarboxylate
EC Number:
212-661-4
EC Name:
Dimethyl naphthalene-2,6-dicarboxylate
Cas Number:
840-65-3
Molecular formula:
C14H12O4
IUPAC Name:
dimethyl naphthalene-2,6-dicarboxylate
Test material form:
solid: flakes
Details on test material:
CAS number: 840-65-3

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 Breeding Laboratories, Inc., Portage, M
- Age at study initiation: approximately 4 weeks of age
- Housing: The rats were individually housed in stainless steel cages measuring 24.0 x 17.8 x 17.6 cm during the quarantine, feeding and recovery periods. The cages were suspended over excrement pans fitted with deotized cage boards.
- Diet (e.g. ad libitum): Certified Ground Purina Rodent Chow 5002 was provided ad libitum.
- Water (e.g. ad libitum): Water supplied from a reverse-osmosis (RO) purifier by an automatic watering system was provided ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 22°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for 12 hours followed by 12 hours of darkness.

No additional data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): All three levels of test diet were obtained by first preparing a premix (100,000 ppm) and then diluting aliquots of the premix with control feed to achieve the desired concentration of test diet. The premix and all diets were mixed using a 16-quart (11 kg) capacity Patterson-Kelly V-blender containing a high speed intensifier bar.
- Storage temperature of food: Diets were stored in sealed plastic bags inside plastic bins at room temperature prior to and during use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed using a Varian 5560 High Performance Liquid Chromatograph (HPLC) with a UV200 detector and a Varian 402 Data Station, which was calibrated against a standard calibration curve covering the range of concentrations expected in the sample analyses. The HPLC conditions were as follows:
Column: Vydac, C18, 201TP, 4.6 mm diameter x 250 mm length, 5 μm particle size
Mobile Phase: Methanol:Water (75:25)
Injection Volume: 10 μL sample loop, filled with 2.5 times volume (i.e., 25 μL injection)
Flow: 1.5 mL/min
Retention Time: approximately 4.5 minutes
Zero Offset: 5%
Chart Speed: 0.5 cm/mm
Stop Time: 8.0 minutes
Plot Attenuation: 32
Detector: Varian UV 200 at 348 nm
Time Constant: 0.5 seconds
Absorbance Range: 0.05 au/mv
Duration of treatment / exposure:
13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
120 mg/kg (2,000 ppm), 600 mg/kg (10,000 ppm) and 3,000 mg/kg (50,000 ppm)
Basis:
nominal in diet
No. of animals per sex per dose:
20/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels used in this study were selected on the basis of results obtained from a two-week dose range-finding/palatability study in which two rats of each sex were fed diets containing DM-2,6-NDC at levels of 0, 100, 1000, 2500, 5000, 10,000, 25,000 and 50,000 ppm (the maximum allowable concentration of DM-2,6-NDC for a feeding study) for two weeks. No change in body weight or food consumption was seen in any animal at the 50,000 ppm (5%) level, thus 50,000 ppm was selected as the highest feeding level for the 90-day study.
Positive control:
None stated

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Rats were observed once daily for morbidity and mortality during the 3-week quarantine period. Following initiation of feeding, all rats were observed twice daily on weekdays and once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Physical examinations for adverse clinical symptoms were performed on each animal once prior to study initiation to ensure suitability for use as a test animal and daily during the 13-week feeding period. Recovery rats were examined daily during the recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured prior to initiation of the study, weekly during the feeding period and at the termination of the study immediately prior to sacrifice (fasted weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of the study and after 13 weeks of feeding
- Dose groups that were examined: 2000, 10000 and 50000 ppm

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at their scheduled time of sacrifice
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes
- How many animals: 160

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at their scheduled time of sacrifice
- Animals fasted: Yes
- How many animals: 160

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the 13-week feeding period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Necropsies were performed on all rats and the following tissues were collected and fixed in 10% neutral buffered formalin: adrenals, brain, epididymides, eyes, esophagus, femur and bone marrow (smear), gonads, heart, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, liver, lungs, lymph nodes (mandibular, mediastinal and mesenteric), mammary gland, nasal turbinates, exorbital lachrymal glands, Harderian glands, pancreas, parathyroids, aorta, pituitary, prostate and seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, sternum (bone marrow), stomach, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, gross lesions and ear with attached tag. The adrenal glands, brain, gonads, heart, kidneys, liver and spleen were weighed at necropsy and the organ weights relative to body weights were calculated.

HISTOPATHOLOGY: Yes. Microscopic examination was performed on the complete set of collected tissues (with the exception of the femur, ear, nasal turbinates, exorbital lachyrmal glands and Harderian glands) from the control and high dose (50,000 ppm) animals sacrificed at the end of the 13-week feeding period. In addition, the lungs, liver, kidneys and gross lesions from the low and medium dose animals sacrificed after 13 weeks were also examined microscopically.
Other examinations:
None stated
Statistics:
None stated

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No signs of DM-2,6-NDC-related toxicity were seen in any animal during the 13-week feeding period. None of the rats died during the 13-week feeding or 4-week recovery periods.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in mean body weight, cumulative body weight gain or food consumption were noted among the treated and control groups during the 13-week feeding period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption data for all males and 6 of 10 females in the 2,000 ppm group was not available during the first week of the recovery period due to a technician error.
No statistically significant differences in food consumption were noted among the treated and control groups during the 13-week feeding period.

OPHTHALMOSCOPIC EXAMINATION
No ocular manifestations of a toxic nature could be attributed to feeding of DM-2,6-NDC.

HAEMATOLOGY
No statistically significant differences were noted between any DM-2,6-NDC-treated group and the respective control group for any of the hematology or differential white blood cell parameters examined after 13 weeks of feeding and/or 4 weeks of recovery.

CLINICAL CHEMISTRY
No statistically significant differences were noted between any treatment group and the respective control group for any of the parameters examined after 13 weeks of feeding, with the exception of significantly decreased BUN in the 50,000 ppm females. Since the BUN level was decreased rather than increased, as would be expected in cases of renal toxicity, the decrease in these animals was not considered toxicologically significant. Creatine kinase activity was significantly decreased in the low and high dose recovery females, however, decreased enzyme activity has no toxicologic significance.

URINALYSIS
No effects related to administration of DM-2,6-NDC were noted on any of the parameters examined after 13 weeks of feeding.

ORGAN WEIGHTS
No statistically significant differences in either the absolute or relative weight of any organ were noted between the groups fed DM-2,6-NDC and the respective control group after 13 weeks of feeding

GROSS PATHOLOGY
The most common gross lesions seen in animals sacrificed after the 13-week feeding and/or 4-week recovery periods included red and/or enlarged mandibular lymph nodes, lung foci and urinary bladder calculi (males only). Since there was no significant differences in the incidence of these observations between the treated and control groups, they were not considered to be treatment-related. Other gross observations were of a minor, incidental nature.

HISTOPATHOLOGY: NON-NEOPLASTIC
No DM-2,6-NDC-related microscopic abnormalities were seen in any organ or tissue from any animal examined at the end of the feeding period. Due to the lack of DM-2,6-NDC-related abnormalities in the high dose animals after 13 weeks of feeding, the tissues collected from the recovery rats were not processed or examined.

Effect levels

Dose descriptor:
NOEL
Effect level:
3 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (50,000 ppm) No statistically significant differences in either the absolute or relative weight of any organ were noted between the groups fed DM-2,6-NDC and the respective control group after thirteen weeks of feeding.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD 408 90-day repeated dose oral toxicity study, conducted according to GLP, dietary administration of DM-2,6-NDC to Sprague-Dawley rats for 13 consecutive weeks at levels of 2,000, 10,000 and 50,000 ppm resulted in no toxicological or pathological effects related to DM-2,6-NDC, therefore, the no-observed-effect level (NOEL) is 50,000 ppm.