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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:
2012-05-29 - 2013-01-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on Sep 21, 1998
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Neopentylglycol (Lab test substance number: 10/0030-2)
- Physical state: solid/white
- Analytical purity: 99.4 area%
- Lot/batch No.: 32637316K0
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed.
- Storage condition of test material: room temperature; protect against moisture; avoid strong oxidizing agents; protect against fire
- Other: the test substance was homogeneous (visually)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han) from Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 36 ± 1 days when supplied, 42 ± 1 days at the start of the administration period
- Housing: 5 animals per cage in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
Motor activity measurements were conducted in polycarbonate cages (floor area about 800 cm2) supplied by TECNIPLAST, Hohenpeißenberg,
Germany
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water from water bottles; ad libitum


ENVIRONMENTAL CONDITIONS (fully air-conditioned rooms in which central air conditioning)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released manually. The test-substance preparations were produced at least every eight daxs.

VEHICLE
- Concentration in vehicle: 0.5, 2.5 and 10.0 g/100 ml, respectively in the 50, 250 and 1000 mg/kg bw dose groups.


The means of the nominal concentrations of the samples taken at the beginning of the study were in a range of 90-110% of the nominal concentrations. The means of the nominal concentrations of the samples from test substance preparations prepared at the end of the study were in a range of
90-110% of the nominal concentrations. These results demonstrated the correctness of the concentrations of Neopentylglycol in drinking water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability of the test substance in drinking water at room temperature for a period of 8 days was demonstrated analytically before the start of the administration period;
- Concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.
Duration of treatment / exposure:
92 (male rats) and 91 days (female rats)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the following dose levels were selected for the present study: 1000 mg/kg bw/day as highest dose, 250 mg/kg bw/day as mid dose, and 50 mg/kg bw/day as low dose.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check for moribund and dead rats was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If rats were in a moribund state, they were sacrificed and necropsied. All rats were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each rat.
- Cage side observations included: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/ arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the administration period (day o) and subsequently once a week

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period, on study day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.

FOOD CONSUMPTION
- Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per rat and day.

WATER CONSUMPTION
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior and at the end of the administration period
- Dose groups that were examined: all prior to the administration period. At the end of the administration period, i.e. study day 90, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administration period: day 0)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surving animals per test group and sex
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test; HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administration period: day 0)
- Animals fasted: Yes
- How many animals: all surving animals per test group and sex
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium (NA), potassium (K), Chloride (Cl), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

URINALYSIS: Yes
- Time schedule for collection of urine: day 90
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color (turbidity), volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: functional observation battery (FOB; including home cage observation, open field observations and sensory motor tests reflexes) and motor activity assessment.

OTHER:
-Estrous cycle determination
-Sperm parameters: sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; the animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: anesthetized animals, adrenal glands, brain, cauda epididymis, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicle with ciogulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix

HISTOPATHOLOGY: Yes; the following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides left(modified Davidsons´s solution), esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson´s solution), femur with knee joint, Harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testis left(modified Davidsons´s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
Statistics:
- Clinical observations: body weight, body weight change and estrous cyle were analyzed by a comparison of each group with the control group using DUNNETT's test (two-sided) for the hypothesis of equal means. Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test and motor activity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Clinical pathology: Blood parameters: parameters with bidirectional changes gravity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. Parameters with unidirectional changes were analysed pairwise with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians. Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.Urine pH ,volume, specific gravity ,color and turbidity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test . Spermanalysis parameters were analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment.
- Pathology: weight parameters were analyzed by Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- No rat died prematurely in the present study.
- No test substance-related, adverse findings were observed

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of body weight and body weight change were observed in any test group.

FOOD CONSUMPTION
No test substance-related effects on food consumption were obtained.

WATER CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings. All apparent findings were assessed as being incidental in nature since they occured in individual animals only and did not show a dose-response relationship.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed At the end of the study, in males of test group 3 (1000 mg/kg bw/d) relative reticulocyte counts were lower compared to controls. In females of the same test group red blood cell (RBC) counts, hemoglobin and hematokrit values were increased. All mentioned values besides the hematokrit in females of test group 3 (1000 mg/kg bw/d) were within historical control ranges (males: relative reticulocyte counts: 0.9-2.5%; females: RBC 7.16-8.25 Tera/L; hemoglobin 8.7-9.5 mmol/L; hematokrit 0.376-0.424 L/L). The hematokrit mean in females was the only red blood cell parameter which was marginal above the historical control range. Therefore, all mentioned red blood cell parameter changes apart from the hematokrit of females of test group 3 (1000 mg/kg bw/d) were regarded as incidental and not treatment-related. The hematokrit values in females of test group 3 (1000 mg/kg bw/d) may be regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Regarding differential blood cell counts, in males of test group 3 (1000 mg/kg bw/d) relative large unstained cell (LUC) counts were lower, and in all dosed females (test groups 1, 2 and 3; 50, 250 and 1000 mg/kg bw/d) relative basophil counts were higher compared to controls. Both parameters were within historical control ranges (males relative LUC counts 0.2-0.6%; females: relative basophil counts 0.0-1.3%). Therefore, both alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed. In males of test group 3 (1000 mg/kg bw/d) glucose values were lower and cholesterol values were higher compared to controls. Glucose mean in this test group was within the historical control range and cholesterol was marginally above this range (males: glucose 5.11-7.16 mmol/L, cholesterol 1.51-2.23 mmol/L). Cholesterol was the only altered clinical chemistry value in these individual. Therefore, the glucose change was regarded as incidental and not treatment-related and the cholesterol increase was regarded as maybe treatment-related but not adverse.

URINALYSIS
No treatment-related changes among urinalyses parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) incidences of transitional epithelial cells (grade 2: 4 of ten animals) and epithelial and granulated casts (grade 2: 3 of ten animals) in the urine sediment were higher compared to study controls. However, in historical controls of this rat strain and age higher incidences of transitional epithelial cells and casts were found (mean + 2 SD counts of transitional epithelial cells grade 2: 3.2 of 10 animals; mean + 2 SD counts of casts grade 2 1.4 of 10 animals, PART III, Supplement). These higher incidences of transitional epithelial cells and casts were regarded as a rat strain specific effect with no relevance to humans. In males of test group 2 and 3 (250 and 1000 mg/kg bw/d) pH values of the urine were lower and in females of test group 3 (1000 mg/kg bw/d) urine volume was higher compared to controls. Both alterations in the absence of other relevant findings in the urinalysis were regarded as maybe treatment-related, but not adverse.

NEUROBEHAVIOUR
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental
-Home cage observation: No test substance-related effects were observed.
-Open field observations: No test substance-related effects were observed.
-Sensorimotor tests/reflexes: No test substance-related effects were observed.
-Quantitative parameters: No test substance-related effects were observed.
- Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female rats. Single interval 5 of male animals of test group 1 (50 mg/kg bw/d was increased and single interval 5 of male animals of test group 2 (250 mg/kg bw/d) was decreased. Additionally, overall motor activity of female animals in test group 1 (50 mg/kg bw/d) was decreased. Since no dose-response effect was observed all changes were assessed as being incidental and not related to treatment.

ORGAN WEIGHTS
- Absolute organ weights: All mean absolute weight parameters did not show significant differences when compared to contral animals.
- Relative organ weights: when compared with the control group 0 (set to 100%), the mean relative kidney weight was significantly increased in male animals (112 % in the 1000 mg/kg bw group); relative liver weights (to body weight) was significantly increased in male animals (109% in the 1000 mg/kg bw/d), the mean relative spleen weight was significantly decreased in female animals ( 87% in the 250 mg/kg bw/d). The increase in relative kidney and liver weights in male animals of test group 2 and 3 (250 and 1000 mg/kg bw/d) was regarded to be related to treatment.Due to a missing histopathologic correlate this finding was regarded to be adaptive and not adverse. The decreased relative spleen weight in females of test group 2 (250 mg/kg bw/d) was regarded to be incidental and not related to treatment due to a missing dose-response relationship and no histopathologic findings in the spleen of test group 3 (1000 mg/kg bw/d) females. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

ESTROUS CYCLE
-No test substance-related effects on estrous cycle lenght and the number of cycles were obtained.

SPERM PARAMETERS
- Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

GROSS PATHOLOGY
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to test substance and treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All off them were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTORICAL CONTROL DATA (if applicable)
Historical control data were available and were used for the evaluation of the biological significance of the observed changes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse findings were observed at clinical examinations, clinical pathology and pathology up to a dose level of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion