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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
Repeated Dose 90-Day Oral Toxicity Study in Rodents
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st May 2016 to 15th September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was undertaken in accordance with OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998 and to Good Laboratory Practice. All procedures aheared too with no deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methoxy-3-methylbutan-1-ol
EC Number:
260-252-4
EC Name:
3-methoxy-3-methylbutan-1-ol
Cas Number:
56539-66-3
Molecular formula:
C6H14O2
IUPAC Name:
3-methoxy-3-methylbutan-1-ol
Test material form:
liquid
Details on test material:
see analytical report as attached to the study report
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 72528
- Expiration date of the lot/batch: 15 February 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Stability for test formulation with vehicle under test conditions was investigated and proved for a 10 day retention time.
- Solubility and stability of the test substance in the solvent/vehicle: Stability for test formulation with vehicle under test conditions was investigated and proved for a 10 day retention time.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of aqua ad iniectabilia (sterile water) and further vortexing it for 2-3 minutes.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and due to previous toxicological tests also using sterile water as vehicle. The test item formulation was prepared once in 10 days.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The vehicle was also used as control item.
- Preliminary purification step (if any): None

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Species/strain: Sprague-Dawley rats, CD rats Crl:CD(SD) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species/strain: Sprague-Dawley rats, CD rats Crl:CD(SD) (Full Barrier)
- Number and sex of animals: 80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: males: 191 – 232 g (mean: 210 g, ± 20% = 168 – 252 g); females: 146 – 177 g (mean: 160 g, ± 20% = 128 – 192 g)
- Fasting period before study: None
- Housing: Full barrier in an air-conditioned room. The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least 5 days)

DETAILS OF FOOD AND WATER QUALITY:
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours light, 12 hours dark

IN-LIFE DATES: 7 June 2016 - 15 September 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item or vehicle was administered as a single daily dose by oral gavage at a volume of 5 ml/kg body weight. The individual dosing volume for each animal was calculated based on the most recent body weight measured.
Vehicle:
water
Remarks:
aqua ad iniectabilia (sterile water)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of aqua ad iniectabilia (sterile water) and further vortexing it for 2-3 minutes.
The test item formulation was prepared once in 10 days.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The vehicle was also used as control item.

VEHICLE
- Concentration in vehicle:
Control: 0 mg/ml
Low dose: 10 mg/ml
Mid dose: 50 mg/ml
High dose: 200 mg/ml
- Amount of vehicle (if gavage): The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
- Lot/batch no. (if required): 605070
- Purity: Not reported

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation samples from all dose groups were retained in weeks 1, 5, 9 and 13 of the treatment period. Approximately 5 ml of each sample was retained in duplicate (sample A and sample B). Samples A were analysed on the same day (samples from week 1, 5 and last) or after 6 days (samples from week 9). Samples B were retained as backup until the analysis of samples A had been performed. Samples were kept at -15 to -35°C for three months following the date on which the final phase report was audited by the Quality Assurance Unit.

Stability was investigated and proven for 10 days retention time.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Single dose via gavage daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
No. of animals per sex per dose:
80 animals (40 males and 40 females; 10 male and 10 female animals per group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Justification for the Selection of the Test System:
The rat is a widely used rodent species in toxicological studies and acceptable to regular authorities.
This study provides information on the possible health hazards which could arise from repeated exposure over a period of 90 days.

- Dose selection rationale: Doses were selected based on previous results from a 28-day repeat dose study. 3 dose groups were selected (LD = low dose, MD = medium dose, HD = high dose). The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): Before the first administration all animals were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 9.4.0 software).
- Section schedule rationale (if not random): The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before the first administration and once a week thereafter.
- Cage side observations checked in table [No.?] were included. Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs during the entire treatment period of 90 days. General clinical observations were made once a day, approximately at the same time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also be presented as figures.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was measured weekly during the treatment period.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also be presented as figures.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.
- Dose groups that were examined: All animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), and large unstained cells (Luc).

BLOOD COAGULATION
- Time schedule for collection of blood: Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: prothrombin time (PT); activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: 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: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked: The following parameters were measured using qualitative indicators
(Urine, Stripes, Henry Schein).
specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: These tests were conducted in all animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
One day after the last administration (study day 91) all surviving animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded.

Following organs were weighed: liver; uterus with cervix; kidneys; thymus; adrenals; thyroid/ parathyroid glands; testes; spleen; epididymides; brain; prostate, seminal vesicles and coagulating glands; pituitary gland; ovaries; and heart.

HISTOPATHOLOGY: Yes

Tissues included: adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum , kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin , spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina.
Statistics:
Body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.
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 was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The weekly detailed clinical examination showed no significant differences in test-item groups compared to the corresponding control group. Effects such as alopecia were observed in all dose groups including the controls which indicates that this is not a treatment related effect. Effects such as moving bedding and salivation were also observed. Salivation just before the dose administration was considered to be a conditional reflex which persisted even after the dose administration. The moving bedding immediately after the dose administration was considered to be due to the local effect caused by test-item gavage.
Mortality:
no mortality observed
Description (incidence):
There was no mortality due to test-item toxicity. Two males and one female died to gavaging error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females, there were no effects on body weight in test-item groups compared to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males and females, the food consumption from day 1 to day 90 was not affected in test-item groups compared to the control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The high dose group (males) showed a statistically significant increased mean lymphocyte count and lower mean neutrophil count when compared to controls. In the high dose female group statistically significantly lower mean reticulocyte and higher prothrombin time were reported when compared to controls. The differences between the highest dose groups and the controls are only marginally different for these parameters. Considering no other adverse effects in the study, the findings were considered to be of minor toxicological relevance

Other effects such as significantly lower mean haemoglobin levels in high and mid dose males (16.21 g/dl and 16.36 g/dl, respectively), lower red blood cell counts in mid dose males and lower basophil levels in the low dose males were observed. However there were no clear dose-response relationships for these effects and therefore are not considered treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose female group, statistically significant higher mean glucose and potassium levels were observed when compared to controls. The differences between the highest dose groups and the controls are only marginally different for these parameters. Considering no other adverse effects in the study, the findings were considered to be of minor toxicological relevance

Other effects such as significantly lower mean aspartate-aminotransferase (ASAT) in all dose groups in females and lower alanine aminotransferase (ALAT) in females from the low and mid dose groups were observed but are not toxicologically relevant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine parameters showed no significant effects in the animals of the test-item groups compared to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological assessment revealed no significant differences in functional neurobehavioural parameters between the controls and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In males and females, there were no statistically significant differences in the absolute and relative (to brain and body weight) organ weights measured at necropsy.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross lesions recorded at necropsy were considered to be within the range of normal historical control values.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mid and high dose females showed an increase of inflammatory cell foci in the liver. In the absence of necrosis, Kupffer’s cell proliferation, apoptosis, fibrosis, alteration in liver function and no significant increase in alanine aminotransferase (ALAT) or aspartate-aminotransferase (ASAT), this finding is deemed to be of a non-adverse nature.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: blood parameters
Organ:
other: blood system
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
A NOAEL of 250 mg/kg bw/day was derived based on statistically significant increased mean lymphocyte count and lower mean neutrophil count in high dose males, lower mean reticulocyte, higher prothrombin time, and higher glucose and potassium levels in high dose females.
Executive summary:

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with MMB (3-Methoxy-3-Methyl-1-Butanol) in male and femaleSprague-Dawleyrats, with dose levels of 0, 50, 250, and 1000 mg/kg body weight/ day the following conclusions can be made:

Treatment with the test item MMB (Methoxy-3-Methyl-1-Butanol) induced an increase of inflammatory cell foci in the liver of medium and high dose females. However, in the absence of necrosis, Kupffer’s cell proliferation, apoptosis, fibrosis, alteration in liver function and no significant increase in alanine aminotransferase (ALAT) or aspartate-aminotransferase (ASAT), this finding is deemed to be of a non-adverse nature.

The haematological data showed a statistically significantly higher mean lymphocyte in the male high dose group (85.47%) compared to the control (79.59%), statistically significantly lower mean neutrophil in the male high dose group (10.69%) compared to control (15.95%), a statistically significantly lower mean reticulocyte in the female high dose group (1.63%) compared to the control (2.24 %) and a statistically significantly higher mean prothrombin time value in the female high dose group (22.87 Sec) compared to the control (19.82 Sec). The clinical biochemistry data showed a statistically significantly higher mean glucose in female high dose (10.309 mmol/L) compared to the control (8.676mmol/L) and statistically significantly higher mean potassium in female high dose group (4.514 mmol/L) compared to the control (3.843 mmol/L). The differences between the highest dose groups and the controls are only marginally different for these parameters, but they are statistically significant and indicate a toxicological response to the test item and therefore are considered significant effects.

Based on the results of haematological and clinical biochemistry parameters, the LOAEL for the systemic toxicity is 1000 mg/kg bw/ day and the NOAEL is 250 mg/kg bw/ day.