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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
other: dose range finding study for OECD 422
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2014 september 24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study
Qualifier:
no guideline required
Principles of method if other than guideline:
As a preliminary study, this study did not follow a specific guideline and was designed to allow selection of appropriate dose levels for use in the subsequent studies.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals were provided with ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum.
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are archived with the raw data at CiToxLAB Hungary Ltd.

During the acclimation period, one day prior to initiation of treatment, the animals were assigned to their respective dose groups by stratified allocation based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Cage type: Type IV polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality (Batch number: 03018131007, Expiry date: October 2016) are archived with the raw data.
Artificial light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1-23.1°C
Relative humidity: 37 - 70 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: The animals were housed by 5 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.
The temperature and relative humidity in the animal room were recorded twice daily during the study.

Start of treatment: 27 May 2014
End of treatment: 09 June 2014
End of experiment: 10 June 2014 (last necropsy)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Animals were inspected for signs of morbidity and mortality and clinical signs were recorded twice daily (at the beginning and end of each working day).
The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.
The body weight of each animal was recorded with a precision of 1 g at randomization, then on Day 0 and Days 3, 6, 10 and 13 (prior to necropsy, on Day 14).
Food consumption was recorded with precision of 1 g on Day 0 and Days 3, 6, 10 and 13.
At the end of treatment period, on Day 14, blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia. After an overnight period of food deprivation of animals, 2 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the dose formulations was determined once during the treatment period (during the second week of treatment). No test item was detected in the control solution samples. The dose formulations were found to be in the range of 97 to 102% of nominal concentrations. Based on these results, test item formulations were considered suitable for the study purposes
Analytical occasion: 3 June 2014 (Secondweek) - Nominal concentration (mg/mL) : 20, Measured concentrations with SD (mg/mL): 19.4 ± 0.56, Measured concentration as a percentage of the nominal: 96.9% - Nominal concentration(mg/mL) : 60, Measured concentrations with SD (mg/mL): 59.7 ± 0.97, Measured concentration as a percentage of the nominal: 99.5%. - Nominal concentration (mg/mL) : 200, Measured concentrations with SD (mg/mL): 204.3 ±3.89, Measured concentration as a percentage of the nominal: 102.1%



Duration of treatment / exposure:
14 days
Frequency of treatment:
Five males and 5 females/group were treated daily for 14 days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at 3 dose levels. A control group was treated concurrently with the vehicle only.
The first day of dosing of each animal was regarded as Day 0. The individual volume of the treatment solution was based on the most recent individual body weight of the animals.
The dosing solutions were administered daily starting from Day 0 for 14 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe, at a constant dose volume of 5 mL/kg bw.
Remarks:
Doses / Concentrations:
100
Basis:
other: mg/kg b.w./day
Remarks:
Doses / Concentrations:
300
Basis:
other: mg/kg b.w./day
Remarks:
Doses / Concentrations:
1000
Basis:
other: mg/kg b.w./day
No. of animals per sex per dose:
5 males and 5 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
Five males and 5 females/group were treated daily for 14 days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at 3 dose levels. A control group was treated concurrently with the vehicle only.
The first day of dosing of each animal was regarded as Day 0. The individual volume of the treatment solution was based on the most recent individual body weight of the animals.
The dosing solutions were administered daily starting from Day 0 for 14 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe, at a constant dose volume of 5 mL/kg bw.
Positive control:
no
Observations and examinations performed and frequency:
Clinical observations and mortality
Animals were inspected for signs of morbidity and mortality and clinical signs were recorded twice daily (at the beginning and end of each working day).
The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.
The body weight of each animal was recorded with a precision of 1 g at randomization, then on Day 0 and Days 3, 6, 10 and 13 (prior to necropsy, on Day 14).
Food consumption was recorded with precision of 1 g on Day 0 and Days 3, 6, 10 and 13.
At the end of treatment period, on Day 14, blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia. After an overnight period of food deprivation of animals, 2 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.
Haematology parameters (units) checked: RBCRed Blood Cell (erythrocyte), WBCWhite Blood Cell (leukocyte) count, HgbHaemoglobin concentration, (g/dL), HctHaematocrit(relative volume of erythrocytes) (%), MCVMean Corpuscular (erythrocyte) Volume (fL), MCHMean Corpuscular (erythrocyte)Haemoglobin, (pg), MCHCMean Corpuscular (erythrocyte)Haemoglobin Concentration, (g/dL), RDWRed Cell (erythrocyte) volume (%), PltPlatelet (thrombocyte) count(109/L), MPVMean Platelet Thrombocyte volume (fL), RETIC %Reticulocyte count (%), NE %Neutrophil (%), LY %Lymphocyte (%), MO %Monocyte (%), BA %Basophil (%), EO %Eosinophil (%), LUC %Large Unstained Cells (%).
Clinical chemistry parameters (units): Glucose Blood sugar concentration (mmol/L), T-BIL Total Bilirubinconcentration(μmol/L), UreaUrea concentration (mmol/L), Chol.Cholesterol concentration (mmol/L), Creat.Creatinineconcentration(μmol/L), Phos.Phosphorus concentration (mmol/L), Na+Sodium concentration (mmol/L), K+Potassium concentration (mmol/L), Ca++Calcium concentration (mmol/L), Cl-Chloride concentration (mmol/L), Tot. Prot.Total Protein concentration (g/L), Alb.Albumin concentration (g/L), A/GAlb/glob ration, AST/GOT Aspartate Aminotransferase activity (U/L), ALT/GPT Alanine Aminotransferase activity(U/L), ALKPAlkaline. Phosphatase –activity (U/L), GGTGamma Glutamyltransferase -activity(U/L), Bile acids(µmol/L).
Sacrifice and pathology:
Gross necropsy was performed on each animal. Terminally on Day 14, all animals were euthanised under pentobarbital anaesthesia by exsanguination. The external appearance was examined, the cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details.
Other examinations:
The following organs were trimmed of fat and weighed in all animals:
With precision of 0.01g: Brain, Seminal vesicles with Epididymides, coagulating glands, Heart, Spleen, Kidneys, Testes, Liver, Thymus, Prostate, Uterus including cervix
On completion of the macroscopic examination only gross lesions and weighed organs were retained from all animals. Testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution
No histopathology evaluation was performed.
With precision of 0.001g : Adrenals, Ovaries
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.
Statistics:
The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
There was no mortality observed during the study. The test item administration did not cause any clinical signs. All animals were symptom-free during the study.
The mean body weight and body weight gain of males was comparable to the control throughout the treatment period in all treated groups. In females at 1000 mg/kg, body weight and body weight gain values during the treatment period were comparable to the control, except weight gain values between Days 0-3, when slight body weight loss was noticed in 3 of 5 females (4502, 4503 and 4504). However, slight body weight loss was observed in single females in each dose group including control during the first week of the treatment period (e.g. control group: 1503 and 1505; low and mid dose: 2505 and 3504, respectively). The mean overall body weight gain (Days 0-13) at 1000 mg/kg bw/day was lower than control mean by approximately 23%, and the difference was attributed mostly to one female (4504). The overall body weight values of other females in this dose group were in the control range, therefore this finding was considered to be incidental.
The group mean differences were not statistically significant.
Females at 300 and 100 mg/kg bw/day had body weights and body weight gains comparable to the controls throughout the entire treatment period.
The food consumption of the animals was not affected by the test item administration. No toxicologically significant variations were recorded during the treatment period in any of the groups.
There were no test item related adverse effects in the haematology parameters at any dose level.
Minor variations on occasion attaining statistical significance were noted in few parameters at Mid dose i.e. slightly lower haemoglobin concentration and mean corpuscular haemoglobin concentration (MCHC) in males, higher mean corpuscular volume (MCV) in females. The differences were of low magnitude and the individual values were in the normal range. In addition, these changes were experienced at the Mid dose level only, without any dose dependence and were considered to be of no toxicological significance.
There were no test item related adverse effects in the clinical chemistry parameters at any dose level.
Compared to controls, slightly higher serum urea concentrations were recorded for males at 1000 and 100 mg/kg bw/day. The differences attained statistical significance (p<0.01 and p<0.05 for High and Low dose, respectively). No changes were observed in creatinine concentration. It should be noted, that urea concentration measured in control animals were in the low range.
Slight differences were noted in serum electrolytes concentration. In males slightly higher chloride concentrations were measured at 1000 and 300 mg/kg bw/day and in females higher sodium and calcium concentrations were found at 1000 mg/kg bw/day. The differences attained statistical significance (p<0.05).
However, no consistent dose or gender response was noted, the differences remained minor, were within the expected physiological range and were therefore not considered toxicologically significant.
There were no macroscopic findings considered related to test item administration under the conditions of this study.
Dilated uterine horns and body were observed in 2 of 5 female in each experimental group including control. These findings are common observation during necropsy in Wistar rats therefore were considered to be incidental.
There were no toxicologically significant differences among groups in the organ weights. Statistical significance was noted for brain relative values of testes at 300 mg/kg bw/day (i.e. lower value). As the difference was minor, all individual values were within physiological range, therefore this finding was not considered to reflect an adverse effect.
Dose descriptor:
NOAEL
Effect level:
ca. 1 000
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Mean values of body weight on day 13 and overall body weight gain (Days 0-13) of males and females

 

Dose (mg/kg bw/d)

 

control

100

300

1000

 

Males

 

Body weight (g)

409

407

401

400

NS

Body weight gain (g) das 0-13

75.6

75.4

69.4

69.2

NS

Females

 

Body weight (g)

245

244

251

244

NS

Body weight gain (g) das 0-13

22.2

23.8

27.4

17.0

NS

N.S. = not significant

Mean values of selected clinical chemistry parameters

 

Dose(mg/kg b.w./d

 

 

Control

100

300

1000

 

Males

6.50

7.49*

6.72

7.99**

DN

Urea (mmol/L)

100.3

100.3

103.3

103.1*

DN

Chloride (mmol/L)

143.2

144.5

144.9

145.7

NS

Sodium concentration (mmol/L)

 

 

 

 

NS

Females

6.79

6.72

7.43

6.96

NS

Urea (mmol/L)

105.9

106.7

107.6

107.3

NS

Chloride (mmol/L)

145.9

145.6

147.6

148.6*

DN

Sodium concentration (mmol/L)

2.43

2.45

2.47

2.53*

DN

N.S. = not significant DN. = Duncan’s multiple Range test * = p<0.05; **=p<0.01

Conclusions:
Administration of Carbohydrates and Sugars, hexitols, anhydro - LAB 4623 to Wistar rats by oral gavage, daily for 14 days, was not associated with any adverse effect.
In consultation with the Sponsor, the dose levels selected for a main toxicity study OECD422 in the rat are 150, 400 and 1000 mg/kg bw/day.
Executive summary:

The study was designed to obtain preliminary information on the toxicity of the test item administered by oral gavage to Wistar rats at three dose levels for 14 consecutive days and to allow selection of appropriate dose levels for use in a subsequent Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422).

The test item was formulated in distilled water and the control group was treated with the vehicle only.

The dose formulations were of adequate concentrations (20, 60 and 200 mg/mL, respectively) to meet the study objectives. The measured concentrations were within the 97-102% of nominal concentrations.

Male and female Wistar rats were treated at doses : 0, 100, 300, 1000 mg/kg b.w./d.

The parameters investigated daily during the study included mortality/morbidity, and clinical signs. Body weight and food consumption were measured on Days 0, 3, 6 and 13 with a fasted body weight recorded prior to necropsy on Day 14. One day after the last treatment, blood samples were collected and evaluated for hematology and clinical chemistry parameters. Gross macroscopic examination was performed at necropsy. Selected organs were weighed. No histopathology examination was performed.

There were no clinical signs or unscheduled mortality during the study.

There were no adverse effects on bodyweight or body weight gain at any dose levels.

The food consumption was not affected by the test item administration.

There were no significant treatment related changes observed on investigated hematology or clinical chemistry parameters.

No test item-related macroscopic findings were observed at necropsy.

There were no toxicologically significant effects in organ weight at any of dose levels. treatment, blood samples were collected and evaluated for hematology and clinical chemistry parameters. Gross macroscopic examination was performed at necropsy. Selected organs were weighed. No histopathology examination was performed.

Administration of Carbohydrates and Sugars, hexitols, anhydro - LAB 4623 to Wistar rats by oral gavage, daily for 14 days, was not associated with any adverse effect.

NOAEL = 1000 mg/kg b.w./d

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
As a preliminary study, this study did not follow a specific guideline and was designed to allow selection of appropriate dose levels for use in the subsequent studies. This study is a 14 days dose range finding study for an OECD 422 test.

Additional information

Justification for classification or non-classification