Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-10 to 2015-11-30
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
BY29 - similar substance 01
IUPAC Name:
BY29 - similar substance 01

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy- Age at study initiation: Approximately 6 weeks- Weight at study initiation: Approximately 180 grams at the start of dosing- Fasting period before study: No fasting period- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulphone solid bottomed cages- Diet (e.g. ad libitum):standard rodent diet, ad libitum- Water (e.g. ad libitum): Drinking water, ad libitum- Acclimation period: 11 days ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 +/-2- Humidity (%): 55 +/- 15- Air changes (per hr): 15 to 20 - Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: 2014-11-17 to 2015-01-05

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous solution of carboxymethylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The required amount of the test item was dissolved/suspended in the vehicle (0.5% aqueous solution of carboxymethylcellulose). The formulations were prepared daily [concentrations of 3, 9.5 and 30 (day 1-9) or 20 mg/mL (from Day 10 of treatment)]. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations, prepared on Weeks 1 and 4, were analysed to check the homogeneity and concentration. Results of the analyses werewithin the acceptability limits for suspensions (80-120% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
Minimum of 4 consecutive weeks. No treatment was given during the recovery period.
Frequency of treatment:
All animals were dosed once a day, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:0, 30, 95 and 300 (day 1-9), 200 (from day 10 of the study) mg/kg bw/dayBasis:other: nominal in aqueous solution of carboxymethylcellulose
No. of animals per sex per dose:
Group 1 (0 mg/kg bw/day): 10 males and 10 femalesGroup 2 (30 mg/kg bw/day): 5 males and 5 femalesGroup 3 (95 mg/kg bw/day): 5 males and 5 femalesGroup 4 (300/200 mg/kg bw/day): 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose Levels and group size:Each group comprised 5 male and 5 female rats. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery. Due to toxicity observed in the high dose animals, from day 10 of the study the treatment was reduced from 300 to 200 mg/kg bw/day
Positive control:
N.A.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice a day each working day. Once a day at weekends and public holidaysDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: twice a day. Additionally, once before commencement of treatment and at least once per week from the start of the treatment, each animal was given a detailed clinical examination. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unususal respiratory pattern)BODY WEIGHT: Yes- Time schedule for examinations: each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes FOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data- Time schedule for examinations:OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data- Time schedule for examinations:- Dose groups that were examined:HAEMATOLOGY: Yes - Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period- Anaesthetic used for blood collection: Isofluorane- Animals fasted: Yes - How many animals: all animals- Parameters checked: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, platelets, prothrombin timeCLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period- Animals fasted: Yes - How many animals: all animals- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chlorideURINALYSIS: Yes - Time schedule for collection of urine: at the end of week 4 of treatment- Metabolism cages used for collection of urine: No data- Animals fasted: individual overnight urine samples were collected under conditions of food and water deprivation. Before starting urine collection, water bottles were removed from each cage and each animal received 10 ml/kg bw of drinking water by gavage. - Parameters checked: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood. The sediment, obtained from centrifugation at 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal componentsNEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: once during week 4 of treatment and once during week 2 of recovery- Dose groups that were examined: week 4: all dose groups, week 2 of recovery: control and high dose group- Battery of functions tested: sensory reactivity to stimuli of different modalities (auditory, visual and proprioceptive stimuli), grip strength, motor activity
Sacrifice and pathology:
Euthanasia:Animals in extremis and those that have completed the scheduled test period will be killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, will be subjected to necropsy, supervised by a pathologist. Necropsy:The clinical history of the animal will be studied and a detailed post mortem examination will be conducted (including examination of the external surface and orifices). Changes will be noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination. Organ weights: From all animals completing the scheduled test period, the organs indicated in the protocol will be dissected free of fat and weighed.The ratios of organ weight to body weight will be calculated for each animal. Tissues fixed and preserved:Samples of all the tissues listed in Annex of the protocol will be fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides, which will be fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).Histopathological examination: The tissues required for histopathological examination are listed in Table 1. After dehydration and embedding in paraffin wax, sections of the tissues will be cut at 5 micrometer thickness and stained with haematoxylin and eosin. In the first instance the examinations was restricted to tissues from all animals in the control and high dose group killed after 4 weeks of treatment, tissues from all animals killed or dying during the treatment period and all abnormalities in all main phase groups. Due to the high rate of mortality noted in group 4 animals, the examination was extended to male and female animals of group 3 killed after 4 weeks of treatment in order to investigate any treatment-related changes. Recovery animals were not examined.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Twelve animals belonging to the high dose group were sacrificed for humane reasons or were found dead within Day 16 of the study, with the exception of one female which was found dead on Day 24. Surviving animals of the high dose group generally showed orange/yellow staining of skin/fur in different regions of the body surface and/or piloerection and/or swollen abdomen. No clinical signs were observed in the animals of groups 1 and 2, while in group 3 animals only orange/yellow staining of skin/fur was seen. No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods.
Mortality:
mortality observed, treatment-related
Description (incidence):
Twelve animals belonging to the high dose group were sacrificed for humane reasons or were found dead within Day 16 of the study, with the exception of one female which was found dead on Day 24. Surviving animals of the high dose group generally showed orange/yellow staining of skin/fur in different regions of the body surface and/or piloerection and/or swollen abdomen. No clinical signs were observed in the animals of groups 1 and 2, while in group 3 animals only orange/yellow staining of skin/fur was seen. No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A very slight reduction in body weight gain was noted in high dose group animals, when compared to controls, starting from Day 8.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in food consumption was observed in high dose group animals, when compared to controls, during the first two weeks of treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Leucocytosis, involving mainly lymphocytes, was recorded in many treated males but was not dose-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose increased in males of medium dose and in both sexes receiving the high dose level. Decrease of albumin and globulin in the high dose males. High dose females showed increases of potassium, phosphorus and bile acids. Reversibility in the recovery
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Decreases in spleen and thymus weights were seen in the high dose animals at the end of treatment. After recovery period, a slight reduction was still recorded in group for spleen of both males and females and for thymus in males only.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in Groups 3 and 4 animals.
Histopathological findings: neoplastic:
not specified
Details on results:
-MORTALITYSeveral animals belonging to the high dose group were sacrificed for humane reasons or were found dead during the study. A total of 6 males (nos. A0156044, A0156046, A0156050, A0156052, A0156056 and A0156060) and 6 females (nos. A0156041, A0156045, A0156047, A0156051, A0156055 and A0156059) of Group 4 died within Day 16 of the study, with the exception of one female (no. A0156051) which was found dead on Day 24. Generally, clinical signs observed in these animals were pale appearance, hunched posture, piloerection, decreased activity, swollen abdomen, rales/dyspnoea, cold to touch, orange/yellow staining of skin/fur in different regions of the body surface. At post mortem examination, the following range of changes were noted: gastro-intestinal tract (stomach, duodenum, jejunum, ileum, caecum, colon): distended with gas, abnormal yellow/orange/brown, and/or red colour and mucoid contents; kidneys: red colour; lungs: red and incomplete collapse; seminal vesicles and prostate: small size; testes: dark; trachea: yellow colour; skin, tail and head: yellow colour; abdominal cavity: swollen; thymus: dark and/or red and small; cervical and mesenteric lymph nodes: yellow or red; spleen: thin; liver and pituitary: dark; ovaries: enlarged. At histopathology, the following range of changes were noted: liver: hepatocytic vacuolation consistent with fatty changes; thymus: congestion and cortical atrophy; epididymides: reduced spermatozoa; seminal vesicle and prostate: atrophy; adrenal: cortex-haemorrhage; heart: myocardium haemorrhage; spleen, cervical and mandibular lymph nodes: lymphoid atrophy; bone marrow (in the sternum and femur): atrophy; lungs, thymus, and kidneys: congestion; stomach - non glandular mucosa: hyperplasia; uterus: hydrometra. The changes observed in all organs are considered as secondary to stress, related to the very high dose, or being associated with agony (Everds et al.2013).- DAILY CLINICAL SIGNSSurviving animals of the high dose group generally showed orange/yellow staining of skin/fur in different regions of the body surface and/or piloerection and/or swollen abdomen. No clinical signs were observed in the animals of Groups 1 and 2, while in Group 3 animals only orange/yellow staining of skin/fur was seen.- WEEKLY DETAILED CLINICAL SIGNSNo changes of toxicological significance were found at the weekly clinical examination, which included an evaluation of neurotoxicity.-NEUROTOXICITYNo differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods. Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.-BODY WEIGHTA very slight reduction in body weight gain (ranging from -12% to -3% in males and -8% to -3% in females), statistically significant in males only on Day 8, was noted in the high dose group, when compared to controls, starting from Day 8. The body weights remained lower than controls throughout the study, with a slight tendency of recovery at the end of treatment. During the recovery period, the body weights remained lower than controls but the changes observed were in the normal range.-FOOD CONSUMPTIONMild reduction in food consumption (-23% to -36% in males and -14% to -29% in females) was observed in high dose group animals, when compared to controls. This decrease was seen during the first two weeks of treatment. Starting from the third week of study, following reduction of the high dose level, a slight increase in food consumption was noted in comparison with the previous week.-HAEMATOLOGYDosing Phase:Increase of white blood cells was recorded in many treated males (approximately 29%). Leucocytosis involved mainly lymphocytes and was not dose related. In addition, a slight reduction of reticulocytes was recorded in some males dosed with 95 mg/kg body weight/day (22%). Due to the absence of related changes of the other red cell parameters and dose-relation, this finding was considered incidental. Slight increase of platelets was also observed in one male dosed with 95 mg/kg body weight/day (animal no. A0156038) and in one female receiving 300/200 mg/kg body weight/day (animal no. A0156049). This finding was of slight incidence and severity (approximately 25%), therefore it was considered of no toxicological significance.Recovery Phase:No relevant differences between control and treated animals were recorded.-COAGULATIONDosing PhaseProthrombin time was significantly increased, at statistical analysis, in males receiving 95 mg/kg body weight/day (11%). Due to the absence of dose relation, this finding was considered unrelated to treatment.-CLINICAL CHEMISTRYDosing Phase:Glucose was increased in males dosed with 95 mg/kg body weight/day (30%) and in animals of both sexes receiving 300/200 mg/kg body weight/day (44% in males and 21% in females). In males, individual changes did not show dose-relation, even though mean group data seemed to be dose-related. In addition, males dosed with 300/200 mg/kg body weight/day showed decrease of protein, albumin and globulin (approximately 14%) and females from the same group showed increases of potassium (28%), phosphorus (24%) and bile acids (148%). Two females dosed with 95 mg/kg body weight/day showed high bile acids levels. One female dosed with 300/200 mg/kg body weight/day showed slight increase of alkaline phosphatase and alanine aminotransferase (109% and 100%, respectively). Due to the low incidence, this finding was not attributed to treatment. The other statistically significant changes (increase of calcium recorded in males receiving 95 mg/kg body weight/day and increase of cholesterol in females dosed with 30 mg/kg body weight/day) were not dose-related, therefore considered unrelated to treatment.Recovery Phase:Changes observed during the dosing phase showed full reversibility, with the exception of bile acids, which were still higher than controls in treated females. Due to the absence of other related changes, this finding was considered of no toxicological importance. In addition, the same parameter was also increased in males. This was considered unrelated to treatment since it was not observed during the dosing phase.-URINALYSISNo relevant changes were observed.-TERMINAL BODY WEIGHTS and ORGAN WEIGHTSTerminal body weight:No significant differences in terminal body weights were noted between treated animals and controls at the end of treatment and recovery periods.Absolute and relative organ weights:A decrease in absolute and relative weights was seen in the spleen (absolute: 11% for males and 21% for females; relative: 8% for males and 20% for females) and thymus (absolute: 13% for males and 26% for females; relative: 10% for males and 25% for females) of the high dose animals at the end of treatment. In addition, the weights of spleen still remained lower than control animals also at the end of the recovery period (absolute: 3% for males and 11% for females) and in males only for thymus (22%).-MACROSCOPIC OBSERVATIONSFinal sacrifice:At post mortem examination, no treatment-related changes were noted in males and females sacrificed at the end of treatment. A range of lesions were seen, all considered as incidental findings, characteristically seen in Sprague Dawley rats of the same age.Recovery sacrifice:At post mortem examination, no treatment-related changes were noted in males and females sacrificed at the end of the recovery period. A range of lesions were seen, all considered as incidental findings, characteristically seen in Sprague Dawley rats of the same age.-MICROSCOPIC OBSERVATIONSFinal sacrifice:At microscopic examination, no treatment-related changes were noted in groups 3 and 4 animals. A minor range of changes were seen in control and treated animals, having a relatively comparable incidence, or are characteristically seen in untreated Sprague Dawley rats of the same age.

Effect levels

Dose descriptor:
NOAEL
Effect level:
95 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Severe toxicity occurred in the animals when administered the test item at the highest dose level (300 mg/kg body weight/day). Reducing the high dose to 200 mg/kg body weight/day, the surviving animals showed a tendency of recovery.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Mortality
   
Animal No Sex Group Type of death Phase name Day of phase
 
A0156044 m 4 found dead dosing 8
A0156046 m 4 found dead dosing 9
A0156050 m 4 found dead dosing 13
A0156052 m 4 found dead dosing 10
A0156056 m 4 found dead dosing 5
A0156060 m 4 humane kill dosing 5
A0156041 f 4 found dead dosing 13
A0156045 f 4 found dead dosing 8
A0156047 f 4 found dead dosing 6
A0156051 f 4 found dead dosing 24
A0156055 f 4 found dead dosing 5
A0156059 f 4 found dead dosing 16

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained by a subacute repeated dose toxicity study with an additional 2 weeks recovery period, severe toxicity occurred in the animals when administered the test item at the highest dose level (300 mg/kg body weight/day). Reducing the high level to 200 mg/kg body weight/day, the surviving animals showed a tendency of recovery. No changes which could be considered to be adverse were observed in male and female rats following dosing with the test item, when administered by oral gavage for 4 consecutive weeks at the dosages of 30 and 95 mg/kg body weight/day. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 95 mg/kg bw/day.
Executive summary:
The toxicity of the test item (94.3 % purity) in rats, following daily oral administration by gavage for four consecutive weeks and recovery from any treatment related effects during a period of two weeks, were investigated in this study (according to OECD 407). Each group comprised five male and five female Sprague-Dawley rats. Control and high dose groups included five additional animals per sex to be sacrificed after two weeks of recovery. The dose levels orally administered were 0, 30, 95 and 300 mg/kg bw/day. Due to severe toxicity observed in the high dose group, the dose in this group was reduced to 200 mg/kg bw beginning with day 10 after starting of treatment. Pathologogical investigation showed severe stress to be the origin of the adverse effects at 300 mg/kg bw/day. The NOAEL was 95 mg/kg bw/day based on the effects on mortality, body weight, food consumption, certain clinical chemistry parameters and organ weights seen in the high dose group. No treatment-related effects were observed at the histopathological evaluation in any dose group. No changes which could be considered to be adverse were observed in male and female rats following dosing with the test item, when administered by oral gavage for four consecutive weeks at the dosages of 30 and 95 mg/kg body weight/day. The subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral OECD 407 study.