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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:
13 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to USFDA Toxicological Principles for the Safety of Food Ingredients, Redbook 2000, updated to April, 2004, an accepted scientific standard. Also conducted using Good Laboratory Practices.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: USFDA Toxicological Principles for the Safety of Food Ingredients, Redbook 2000, updated to April, 2004; IV.C.4.a, Subchronic Toxicity Studies with Rodents
Deviations:
not specified
Principles of method if other than guideline:
USFDA Toxicological Principles for the Safety of Food Ingredients, Redbook 2000, updated to April, 2004; IV.C.4.a, Subchronic Toxicity Studies with Rodents. In addition, the study was conducted under the US FDA Good Laboratory Practice Requirements, 21 CFR, Part 28.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Name of Test Material (as cited in study report): TXIB Plasticizer
Stability under Test Conditions: Stable

Test animals

Species:
rat
Strain:
other: CD[Crl:CD(SD)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at Study Initiation: approximately 8 weeks (6 wks of age when received, with 12-day acclimation period).
- Housing: Individually housed in suspended, stainless steel wire-mesh type cages. During urine collection, animals were housed in stainless steel metabolism cages and urines collected for at least 16 hours.
- Diet: Meal Lab Diet, (Certified Rodent Diet #5002), PMI International, Inc., available ad libitum.
- Water: Tap water delivered via an automatic watering system; supply is monitored for specific contaminants at periodic levels per SOP.
- Acclimation Period: 12 days.
- Randomization procedure: standard block procedure
- Animal identification: microchip implanted with unique number


ENVIRONMENTAL CONDITIONS
- Temperature: (degrees F), monitored and recorded daily.
- Humidity: 30-60%, monitored and recorded daily.
- Photoperiod (hours dark/hours light): Fluorescent lighting provided 12 hours per day.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
For each diet, the test substance was mixed with a small amount of the commercial feed to form a premix. The premix was then mixed with additional feed to prepare the test diets. The diets were offered to groups of twenty male and twenty female rats.
The diets were prepared in concentrations sufficient to result in consumption of the test substance in daily doses of 0, 30, 150 or 750 mg/kg/day. Diets were administered for at least 13 weeks. Test diets were prepared weekly and stored at room temperature. The concentrations of the test substance in the diets were modified weekly based on animal weight and feed consumption to provide constant dosage on a mg/kg basis. Control animals were allowed ad libitum access to plain diet not containing the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each of the low- and high- concentration test diets were analyzed for homogeneity prior to study initiation. This was accomplished by taking samples from the top, middle, and bottom of each batch following completion of diet preparation, and analyzing the diets for concentration of the test substance. Samples of prepared diets analyzed for homogeneity were stored at room temperature for 14 days. After 7 and 14 days, samples of these diets were analyzed for stability. Subsequently, samples of the prepared diets at each concentration were collected weekly for the first four weeks of the study and then every fourth week thereafter to assess concentration of the test substance in the diets.
Duration of treatment / exposure:
Animals were exposed continuously via diet for 13 weeks.
Frequency of treatment:
Animals were exposed to the test substance daily throughout the 13-week length of the study.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
Doses were selected based on data from previous studies with the test substance. Each week, diet concentrations were adjusted based on feed consumption from the previous week and the weights of the animals in order to provide consistent doses. All animals were weighed and observed during acclimation with respect to general health. Detailed clinical observations and ophthalmological examinations were conducted prior to the start of the study, and at study start, animals had body weights within + or - 20% of the mean body weight for each sex. A standard block randomization procedure was used to assign animals to treatment groups, and each animal was implanted with a microchip bearing a unique number. During the study, each cage was identified by animal number, study number, group number and sex.
Positive control:
No positive control substance was run in this study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Animals were observed twice daily for morbidity, mortality, injury, and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Animals were given detailed clinical evaluations, which included neurobehavioral observations, once weekly during Weeks 1 to 12. Observations included changes in skin, fur, eyes, mucous membranes, autonomic activity, unusual respiratory patterns, changes in gait, posture, reactivity to handling, stereotypies, and bizarre behavior.

FUNCTIONAL OBSERVATIONAL BATTERY: FOB evaluations were made prior to study start and during Week 13. Evaluations consisted of observations made in the home cage, in open field, or during handling and included, but were not limited to: activity, posture, behavior, movements, gait, reflexes, and response to stimuli. Grip response, hindlimb splay, and pain perception were also evaluated.

BODY WEIGHTS: Body weights were measured and recorded upon receipt, prior to randomization, on Day -1, and weekly during the study.

FOOD CONSUMPTION: Food consumption was measured weekly.

WATER CONSUMPTION: Subjectively monitored, but no quantitative investigation was instituted.

OPHTHALMIC EXAMINATION: Ophthalmic examinations were conducted on all animals pretest and prior to necropsy.

HEMATOLOGY:
- Collection Schedule: Days 15 and 45 and prior to scheduled necropsy on 10 animals/sex/group.
- Method: via orbital sinus following anesthesia on Days 15 and 45; via cardiac puncture following CO2 inhalation prior to necropsy. Blood was collected into tubes containing EDTA or citrate (for coagulation parameters).
- Anesthetic used for blood collection: yes
- Fasted: Animals fasted overnight but had access to drinking water.
- Parameters Measured: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, differential leukocyte count, absolute reticulocytes, percent reticulocytes, activated partial thromboplastin time (APTT), and prothrombin time (PT).

CLINICAL CHEMISTRY:
- Collection Schedule: Days 15 and 45 and prior to scheduled necropsy on 10 animals/group.
- Method: Samples (2 to 3 mL) via orbital sinus following anesthesia on Days 15 and 45; via cardiac puncture following CO2 inhalation prior to necropsy. No coagulant was used.
- Parameters Measured: Sodium, potassium, chloride, calcium, phosphorus, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, glucose, gamma glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH).

URINALYSIS:
- Collection Schedule: On the day of or just prior to termination.
- Method: animals were housed in stainless steel metabolism cages and urines were collected for at least 16 hours.
- Parameters Measured: Color and appearance, volume, specific gravity, microscopic elements, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen.
Sacrifice and pathology:
EUTHANASIA: Carbon dioxide inhalation followed by exsanguination via vena cava.

GROSS PATHOLOGY: Animals were examined for external abnormalities including masses. Abdominal, thoracic, and cranial cavities were examined; organs were collected and fixed in neutral buffered formalin except eyes and testes which were fixed using a modified Davidson's fixative. Lungs and bladder were infused to inflate. Tissues were collected from all animals.

TISSUES COLLECTED: Adrenals, aorta, bone with marrow (femur), bone with marrow (sternum), bone marrow smear, brain (cerebrum, midbrain, cerebellum, medulla/pons), epididymides, esophagus, eye including optic nerve, gastrointestinal tract, including stomach [glandular and non-glandular], duodenum, jejunum, ileum, cecum, colon, rectum, gonads, including ovaries, oviducts, testes, gross lesions, harderian glands, heart, kidneys, exorbital lachrymal glands, liver, lung, mandibular lymph nodes, mesenteric lymph node, mammary gland, nose, pancreas, pituitary, prostate, mandibular salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (biceps, femoris), skin, spinal cord (cervical, thoracic, and lumbar), spleen, thymus, thyroids/parathyroids, tongue, trachea, urinary bladder, uterus with cervix, and vagina.

TISSUES EVALUATED MICROSCOPICALLY: Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on sections of tissues designated by protocol.
Other examinations:
ORGAN WEIGHTS:
- Organs Weighed: Adrenals, brain, epididymides, gonads (ovaries, testes), heart, kidneys, liver, spleen, thymus, thyroids/parathyroids, uterus with cervix.
Statistics:
Data for each sex were analyzed separately.
- Group Pair-wise Comparisons, including Levine's/ANOVA-Dunnet's/Welch's: Body weight, food consumption, hematology except leukocyte counts, clinical chemistry, organ weights (absolute weights and relative to brain weights), FOB continuous data, including body weight, body temperature, defecation, urination, rearing, thermal response, forelimb grip strength, hindlimb grip strength, hindlimb splay.
- All pair-wise endpoints were analyzed using two-tailed tests.
- Chi-Square Test for Homogeneity: FOB (except continuous data).
- Log Transformation/Group Pair-wise Comparison: Total leukocyte counts, differential leukocyte counts.
- Rank Transformation with Dunnett's Test: Urine volume, specific gravity, and pH.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
There were no deaths related to the test substance in this study. One control male died on Day 27; one 750 mg/kg/day male was euthanatized in extremis on Day 64 following observations of mouth abrasion, decreased activity, and an unkempt appearance. One other 750 mg/kg/day male had clinical signs of difficult breathing and malocclusion during Weeks 4 and 6, but survived. One 150 mg/kg/day female had increased activity, rapid breathing and tremors in week 1 only. There were no other significant clinical findings.

BODY WEIGHT AND WEIGHT GAIN:
At 750 mg/kg/day, there were 5 to 7% reductions in body weight gain in males and females beginning in weeks 8 to 10. Only the mean body weight in males in Week 9 was significantly decreased. All other body weights remained in the same range as the controls during the course of the study.

FEED CONSUMPTION AND COMPOUND INTAKE:
Feed consumption was slightly (7 to 8%) reduced in 750 mg/kg/day males from Week 8 to the end of the study; the reduction was statistically reduced only during Week 10. In 750 mg/kg/day females, feed consumption was reduced 6 to 9% periodically; the reduction was significant in weeks 1, 6, and 13 only.
Mean weekly compound intake was calculated for males at 30.28, 151.34, and 751.59 mg/kg/day and for females at 30.84, 153.03, and 754.81 mg/kg/day.

OPHTHALMOSCOPIC EXAMINATION: No abnormalities noted

HEMATOLOGY:
There were no adverse test substance-related effects on hematology parameters. Changes that were observed included decreased hematocrit (4%) at Day 15 in 750 mg/kg/day males and at termination (5-6%) in 150 and 750 mg/kg/day females. Decreased absolute (20-24%) and relative (17-22%) reticulocyte values were noted in all groups of treated males at termination, though there was no dose-dependency and no effect on circulating red cells. Other statistical changes (32% increased basophils at termination in 150 mg/kg/day males, and 2% increased MCHC at termination in 750 mg/kg/day females) were considered spurious.

CLINICAL CHEMISTRY:
-Cholesterol (mg/dL) was statistically increased over control 1.29 to 1.42- fold on Day 15 (58.4 vs 44.5), Day 45 (69.1 vs 53.5), and at termination (81.4 vs 57.3) in 750 mg/kg/day males and 1.30 to 1.37- fold over control on Day 45 (71.2 vs 54.9) and at termination 81.2 vs 59.1 in 750 mg/kg/day females.
-Total Bilirubin (mg/dL) was statistically increased 1.24- fold over control (0.26 vs 0.21) at termination in 750 mg/kg/day males.
-Creatinine (mg/dL) was statistically increased 1.24- fold over controls at termination in 750 mg/kg/day males (0.46 vs 0.37).
-Gamma Glutamyltransferase was slightly increased on Day 45 (3.1 U/L) and at termination ( 3.2 U/L) in 750 mg/kg/day males vs control values of 3.0 U/L.

Additional statistically significant differences in clinical chemistry parameters were not considered toxicologically relevant by the study director; they included:
-Sodium (mEq/L): 147.6 (150 mg/kg/day males, Day 15) vs 146.5 (control). Also, 146.8 (30 mg/kg/day females, Day 45) vs 144.6 (control).
-Chloride (mEq/L): 97.4 and 98.1 (750 mg/kg/day males, Day 15 and Day 45, respectively) vs 100.7 and 100.4 (control).
-Calcium (mg/dL): 11.73 (750 mg/kg/day males, terminal) vs 11.38 (control).
-Phosphorus (mg/dL): 9.93 and 9.68, (30 and 750 mg/kg/day males, respectively, Day 15) vs 10.73 (control).
-AST (U/L): 74.0 and 69.1 (150 and 750 mg/kg/day males, respectively, Day 15) vs 86.9 (control) and 65.4 (750 mg/kg/day males, Day 45) vs 79.2 (control).
-Alkaline Phosphatase (U/L): 750 mg/kg/day females, Day 15: 96.3 vs 132.4 (control), Day 45: 58.6 vs 81.9 (control), and at termination: 38.2 vs 46.6 (control).
-Sorbitol Dehydrogenase: 17.45 (750 mg/kg/day males, Day 45) vs 21.78 (control).
-Total Protein (g/dL): 6.82 (750 mg/kg/day males, Day 15) vs 6.47 (control).
-Albumin (g/dL): 3.54 (30 mg/kg/day females, Day 15) vs 3.79 (control).
-Globulin (g/dL): 3.38 (750 mg/kg/day males, terminal) vs 3.09 (control).
-Glucose (mg/dL): 85.2 and 84.2 (150 and 750 mg/kg/day males, respectively, Day 15) vs 99.2 (control). Also, 102.5 and 97.1 (30 and 750 mg/kg/day females, respectively, Day 45) vs 117.3 (control).
The authors suggested that the clinical chemistry findings suggested an alteration of lipid metabolism at 750 mg/kg/day.

URINALYSIS: No abnormalities noted

NEUROBEHAVIOR: No abnormalities noted

ORGAN WEIGHTS:
Organ weight changes included: increased kidney weights relative to controls with respect to body weight (0.8568 vs 0.7546, [14%]) and brain weight (2.0740 vs 1.8935[10%]) in 750 mg/kg/day males. The increased weights were not considered adverse as weight changes correlated to an increase in hyaline droplets, a condition to which male rats are predisposed.

Possible test article-related changes were present in both male and female liver weights. Increased absolute liver weights and liver weights relative to controls in 750 mg/kg/day males were as follows: Absolute values (g) were 18.562 vs 15.908 (17%); weights with respect to body weight (%) were 3.8604 vs 3.0794 (25%) and relative to brain weight were 9.3937 vs 7.8198 (20%). Also, increased absolute liver weights and liver weights relative to controls were present in 750 mg/kg/day females: Absolute values (g) were 10.286 vs 8.851 (16%); weights with respect to body weight (%) were 3.8117 vs 3.1675 (20%) and relative to brain weight were 5.5239 vs 4.7538 (16%).

Incidental organ weight changes included:
-Decreased absolute heart weights relative to controls in 30 and 750 mg/kg/day males (1.626 and 1.574, respectively) vs 1.756; decreased heart to brain weight ratio weights relative to controls in 30, 150, and 750 mg/kg/day males (0.7891, 0.7959, and 0.7958, respectively) vs 0.8612.
-Decreased thyroid/parathyroid gland weights relative to controls in 150 mg/kg/day males; absolute weight(g): 0.0229 vs 0.0269; weight relative to body weight (%), 0.0045 vs 0.0053. weight relative to brain weight: 0.0111 vs 0.0132.
-Increased thyroid/parathyroid gland weights relative to controls in 150 and 750 mg/kg/day females; absolute weight(g): 0.0197 and 0.0194, respectively, vs 0.0164 in controls; weight relative to body weight (%): 0.0073 and 0.0073, respectively, vs 0.0059 in controls; weight relative to brain weight: 0.0107 and 0.0105, respectively, vs 0.0088 in controls.

GROSS PATHOLOGY:
There were no test substance-related macroscopic observations noted in either sex. Macroscopic observations were of the type commonly seen in rats of this age and strain, and were considered incidental to exposure to the test substance.

HISTOPATHOLOGY, NON-NEOPLASTIC:
Test article microscopic changes were present in the kidneys of male rats.
Increased prevalence of hyaline droplets was present in all three groups of males administered the test substance. Of 20 animals per group, increases in hyaline droplets were seen in 4, 11, and 19 animals in the 30, 150, and 750 mg/kg/day groups, respectively. This finding was characterized by a minimal to mild increase in the number of eosinophilic hyaline droplets in the cytoplasm of cortical epithelial tubule cells. The severity was rated as mild for 4 of the 750 mg/kg/day animals and as minimal for all other animals. The finding was related to the test substance in all three groups of treated males, but was not considered an adverse finding by the study authors as male rats are predisposed to this condition.
Minimal chronic progressive nephropathy was also seen in male rats. The frequency was 9, 9, 12, and 17 of 20 animals in the 0, 30, 150, and 750 mg/kg/day dose groups, respectively; hence, the effect overlaid the spontaneous appearance of this condition in untreated animals, and increases in incidence were limited to the 150 and 750 mg/kg/day groups. The authors suggested that this lesion was test-article related, however, in the 750 mg/kg/day male group, as the incidence in that group was significantly increased over that seen in the control group.
No kidney effects were noted in females in any group.
The authors noted specifically that there were no test substance-related effects in the reproductive organs of either sex.
Other microscopic changes noted in animals in this study were considered incidental to consumption of the test substance. The changes were few, isolated, and not related to the test substance. Additionally, they were all of the type commonly seen in rats of the age and strain used in this study.

Effect levels

open allclose all
Dose descriptor:
LOEL
Effect level:
750 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the authors concluded that the lowest effect level (LOEL) in males in this study was 750 mg/kg/day. This effect level was based on increased numbers of hyaline droplets, taken together with an increased incidence of progressive nephropathy noted in the group. Setting this level was based on the increased incidence, but not the severity, of progressive nephropathy seen in that group.The NOAEL in males was determined to be 150 mg/kg/day, as that dose did not augment nephropathy, while causing only minimal increases in hyaline droplet formation. In females, there were no kidney effects. At 750 mg/kg/day in females, effects were limited to slight decreases in body weight, increased liver weights, and increased cholesterol levels. The authors concluded that the minor nature of these effects in females allowed for assignment of a NOAEL of 750 mg/kg/day.
Executive summary:

In a subchronic toxicity study, 2,2,4-Trimethyl-1,3-Pentanediol Diisobutyrate (Texanol Isobutyrate) was fed to groups of 20 CD[Crl:CD(SD)] rats/sex/group via diets providing doses of 0, 30, 150 or 750 mg/kg/day. There were no test substance-related mortalities, clinical signs or neurobehavioral abnormalities. In high-dose males, kidney weights were increased along with an increased presence of hyaline droplets and an increased incidence of chronic progressive nephropathy. Effects noted but not considered to be adverse included hyaline droplets in all groups of treated males, minimal decreases in body weight gain, and clinical chemistries indicative of a possible effect on the liver in both high-dose males and females, though these were not correlated with microscopic findings.

In male rats, the LOEL was 750 mg/kg/day, and  the NOAEL was 150 mg/kg/day. The NOAEL in female rats was 750 mg/kg/day.