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Description of key information

In the key subchronic gavage study according to OECD Guideline 408, the NOAEL was determined to be 1000 mg/kg bw/day.
In a supporting gavage study conducted according to OECD Guideline 422 the NOAEL in male rats was 300 mg/kg bw/day.
The NOAEL in a 36-d feeding study in young rats was 10000 ppm or 1000 mg/kg bw/day.
No valid data are available on the toxicity after repeated inhalation or dermal exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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)
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
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
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
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
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.
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.
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.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a subchronic toxicity study conducted in rats, the potential of the test substance to induce repeated dose toxicity was

evaluated according to the OECD TG408. Neopentylglycol was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; drinking water served as vehicle control; test group 0), 50 mg/kg bw/d (test group 1), 250 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of about 90 days.

Food consumption and body weights were determined weekly. The rats were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations.

Because no treatment-related, adverse findings were observed, it is concluded that the oral administration of the test substance by

gavage over a period of 3 months revealed no signs of toxicity in male and female Wistar rats up to a dose level of 1000 mg/kg bw/d.

Therefore under the conditions of the present study, the NOAEL was 1000 mg/kg bw/d.

In a study report by Eastman Kodak (1966; study comparable to OECD Guideline 407 with acceptable restrictions: only two dose levels tested, however, the high dose level is close to the recommended limit dose of 1000 mg/kg bw/day; partly limited documentation, e.g. no details about the test substance; no data about functional observation battery; limited parameters in clinical chemistry [only two enzymes measured]; haematology without haematocrit, platelet count and a measure of blood clotting time; organ weights of epididymides and thymus not measured; histopathology without spinal cord, thymus, trachea, uterus, prostate, lymph nodes, and peripheral nerve) young rats (n=5 per dose per sex) were exposed for 36 days via the diet to dose levels of 0, 1000 or 10000 ppm corresponding to 0, 100 and 1000 mg/kg bw. Food consumption, body weight gain, and behavior were recorded. Hematology, clinical chemistry, and urinalysis were performed as well as macro- and microscopic pathology at termination. Relative and absolute organ weights were determined. No toxic effects were detected in rats exposed via the diet to dose level of 1000 or 10000 ppm (100 or 1000 mg/kg bw/day) except a decrease in food consumption at the high dose resulting also in decreased weight gain at this dose level. In conclusion, the NOAEL in a 36-d feeding study in rats was 10000 ppm or 1000 mg/kg bw/day. These data fits with results in the following gavage study.

A further study in rats was conducted according to OECD Guideline 422 (Biosafety 1993). GLP standards were fulfilled. The study is documented in details therefore the data base is sufficient for evaluation (acceptable restrictions: no data were given on historical control range for hematology and clinical chemistry of this laboratory; hematology & clinical chemistry only in males; no details about test animals, application volume and concentration, mating procedure, analysis of dose, and stability in vehicle). Twelve male and 12 female Sprague-Dawley rats per dose were gavaged with 0, 100, 300, or 1000 mg/kg bw/day. The exposure period for males was 45 days; females were treated from day 14 before mating to day 3 of lactation. The premating exposure period for males was not clearly stated but presumably ca. 6 weeks. There was no post exposure observation period. Body weight and food consumption was measured in all groups. Hematology and clinical chemistry was performed in males. At termination organ weights (absolute and relative) were determined and necropsy and histopathology performed.

Concerning the repeated dose toxicity in females of the parent generation no effects were detected. At 100 mg/kg bw/day no effects were recorded in males. At >= 300 mg/kg bw/day a dose dependent increases in the levels of total protein, total bilirubin and albumin in clinical chemistry was measured and the absolute and relative liver weight was increased in males. At a dose level of 1000 mg/kg bw/day a reduced blood glucose value was observed and absolute and relative kidney weight was increased in males; histopathology of high dose males revealed hypertrophy of the liver in 2/12 males; increased severity in basophilic alteration of the renal tubular epithelium accompanied by an increased incidence in hyaline droplets and protein casts were found at the high dose.

The toxicological relevance of altered parameters in clinical chemistry is questionable and not used for final evaluation (a detailed rationale is given). The increase in liver weight was not found to be paralleled by any histpathologically detected liver lesion and furthermore no clear indication of such a liver lesion was found in other parameters investigated. Therefore, the increase in liver weight at mid and high dose level and liver hypertrophy at the high dose is considered to be an adaption to the increased metabolism of neopentyl glycol and not an adverse effect per se. However, the increased kidney weight at 1000 mg/kg bw/day accompanied by hyaline droplets and protein casts in the eliminating renal lumen plus alterations of the renal tubular epithelium (increased basophilicity) are suggested to be adverse in nature. In conclusion, at 1000 mg/kg bw/day no effects were noted in females; histopathology revealed effects in the kidney of males at this dose level accompanied by increased relative and absolute kidney weight.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study

Justification for classification or non-classification

Classification is not warranted. The criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 are not met.