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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04/05 November 2014 to 03/04 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a valid certificate.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See "principles of method if other than guideline" below.
Principles of method if other than guideline:
Planned changes to the Study Plan were made at the discretion of the Study Director in agreement with the Sponsor and documented by a Study Plan Amendment that became effective at the time of Study Director's signature. The amendment was sent to the Sponsor, distributed to all recipients of the Study Plan, as presented in Appendix 8 of the report.Unplanned changes to the Study Plan were documented in the raw data, communicated to the Sponsor, and reported. The following minor deviations were noted and were considered by the Study Director to have no impact on the outcome of the study and interpretation of the results:Due to a technical issue, on 5 occasions, the target temperature in the animal room was slightly increased above the limit of 25oC up to 25.3oC.Due to technical reason, Release 300 mg/mL (as pentobarbital sodium) was used for terminal euthanasia. Due to technical reason, the absolute Reticulocyte count is reported. Due to technical reason, in animal No. 1008 the aorta abdominal was not histologically evaluated, because it was not present.Due to technical reason, for data collection and tabulation software PROVANTIS v7 and/or v9 were used.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.
EC Number:
923-511-9
IUPAC Name:
Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.
Test material form:
other: solid (similar to modelling clay when undried)
Details on test material:
Name: Green liquor sludge (GLS)Chemical name: Reach name: Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycleCAS number: EC number: 923-511-9Batch number: Green liquor sludge, slamfilter, 2014-07-14—2014-07-18Appearance: Black (undried), light grey (dried) solid (similar to modelling clay when undried)Purity: 46.5% (The remaining 53.5% is water)Manufacture date: 14 July 2014 – 18 July 2014 (sampling period; collect sample)Expiry date: 30 September 2016Storage conditions: Room temperature (15-25oC, below 70 RH%), under inert gas; In closed containerSafety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials will be applied to assure personnel health and safety.Correction factor was used at dose formulation preparation, e.g. the dose levels (mg/kg bw/day) was calculated on dry weight basis of the test substance (the paste like GLS delivered to the laboratory contains 53.5 % water).

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Crl:WI ratsSource: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633Housing conditions: SPF at the supplier, standard laboratory conditions during the studyJustification of species/strain: The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studiesNumber of groups: 4 groups; one control and 3 test item-treated groupsNumber of animals: 10 animals/group/sex: 40 male and 40 female ratsAge of animals: Young adult rats, approximately 7 weeks old at onset of treatmentBody weight: Not exceeding ± 20% of the mean weight for each onDay -1: males: 189-231 g, females: 175-196 gAcclimation period: 5/6 days (males/females)Animal health: Only healthy animals were used for the test. The Veterinarian certified the health status prior to acceptance for use in the study.Animal room: 523Cage type: Type III polycarbonateBedding: Laboratory bedding, Lignocel 3/4-S (produced by J. Rettenmaier & Söhne GmbH+CO.KG, D-73494 Rosenberg, Germany) and GRADE 5 (produced by Johannes Brandenburg GmbH & Co. KG; Arkeburger Str. 31; DE-49424 Goldenstedt) were available to animals during the study. Details of bedding quality are archived with the study raw dataIllumination: Artificial light, from 6 a.m. to 6 p.m.Temperature: 20.0 – 25.3°CRelative humidity: 30 – 66 %Ventilation: 15-20 air exchanges per hourHousing/Enrichment: Rodents were group-housed (2/3 animals/sex/cage), to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.The temperature and relative humidity were checked and recorded twice daily during the study.Diet and water supply: The animals were provided with ssniff® SM R/M-Z "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 bottles, ad libitum.The supplier provided analytical certificates for the batches used (Batch No.: 190 1786Expiry date: January 2015; Batch No.: 680 2237 Expiry date: March 2015) which are archived with the study raw data.The quality control analysis of the water 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). Copies of the relevant Certificates of Analysis are retained in the archive at CiToxLAB Hungary Ltd.The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.Randomisation: During the acclimation period, 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.Identification: Each animal were identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB HungaryLtd., as described in the Experimental Design.The cages were identified by cards holding information at least about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose, Humaqua (distilled water for injection) and 10% HCl solution used for pH adjustments of the formulations
Details on oral exposure:
Based on the results of the preliminary formulation trial, aqueous 1 % (w/v) Methylcellulose solution was selected as vehicle in agreement with the Sponsor.Vehicle components1. Name: MethylcelluloseLot/Batch number: 1D30012N13 / 2H20012N11Manufacturer: Dow ChemicalsExpiry Date: April 2016 / 25 February 2017Storage: Room temperature2. Name: Humaqua (distilled water for injection)Lot No.: 9321113 / 2171213Manufacturer: TEVA Co.Expiry Date: November 2016 / December 2016Storage: Room temperature10% HCl solution used for pH adjustments of the formulationsHydrochloric acid 37%Lot/Batch number: 13F030514Manufacturer: VWR InternationalExpiry Date: June 2018Storage: Room temperatureHumaqua (distilled water for injection)Lot No.: 9321113 (as above)Formulation The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd.Due to the chemical and physical nature of the test item, it was not technically feasible to perform a real stability assessment, but since the substance is an aqueous mixture of inorganic materials, hence dilutions in aqueous vehicle are considered to be stable. Therefore the formulations are considered to be adequately stable for use in the study as described below.Dose formulations were prepared by mixing the test item with 1% Methylcellulose. The required amount (mg) of the test item was suspended in the vehicle to achieve the desired test item concentrations of 6.25, 25 and 100 mg/mL, of the test item for each dose level (62.5, 250 and 1000 mg/kg bw/day, respectively) and was stirred using magnetic stirrer until a homogenous dosing form was obtained.During dose formulation preparation, the correction for water content of the test item (53.5%) was applied.The pH of the formulations at all dose levels was reduced to approximately 10.5 using 10% HCl solution to prevent animal welfare issues of excessive pH test item.Formulations were prepared fresh prior to administration to animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd., by a flame photometry method, measuring the marker element of Calcium (the most prevalent element in the material according to the Certificate of Analysis). Top, middle and bottom duplicate samples were taken from the test item formulations and analysed from each formulation at least five times during the study. Similarly, one sample was taken in duplicate on each occasion from the Group 1 (control) solution, for the detection of the test item.Principle of the Analytical MethodConcentration of Green liquor sludge (GLS) in the test solution was determined using the calcium content during the test.Two samples were taken from the top, middle and bottom of the test solutions, and two samples were taken from the control solution.The samples were analysed by a flame photometric method Calcium detection.Equipment and ChemicalsApparatusFlame photometer: BWB XP digital flame photometerBalance: Sartorius, BP221S, BP210SUltrasonic bath: ELMA, Elmasonic S300HDensity meter: Anton Paar, DMA500Water purification system: MILLIPORE, DIRECT Q 8 UVMaterialsSubstance name: Green liquor sludge (GLS)Batch number: Green liquor sludge, slamfilter, 2014-07-14—2014-07-18Purity: 46.5 % (The remaining 53.5 % is water)Description of the test item: Black (undied), light grey (dried) solid (similar to modelling clay when undried)Manufacture date: 14-07-2014 – 18-07-2014 (sampling period; collect sample)Expiry date: 30 September 2016Storage condition: Controlled room temperature, in closed container under inert gasOther materials:Ultra pure water (ASTM Type I): prepared by Direct-Q 8 UV system, MilliporeCalcium chloride: reagent, VWR, Batch: 14D070004Hydrochloric acid 0.5 mol/l: Merck, Batch: HC389253Method for the analysis of Sodium Sulphate contentFormulation samples were acidified with hydrochloride acid and diluted into the calibrated range with water, filtered with 0.22 μm membrane filter and analysed by the flame photometric method.SamplingTwo samples were taken from the top, middle and bottom of the test solutions.Results of the AnalysisThe test solutions proved to be homogeneous at receipt as evidenced by the small deviation of the replicate samples taken from different areas of the test solutions.
Duration of treatment / exposure:
Dose formulations were administered from Day 0 for 91 consecutive days
Frequency of treatment:
Dose formulations were administered daily.
Doses / concentrations
Remarks:
Doses / Concentrations:6.25, 25 and 100 mg/mL, of the test item for each dose level (62.5, 250 and 1000 mg/kg bw/day, respectively)Basis:actual ingested
No. of animals per sex per dose:
10 males & 10 females per test group (80 in total)
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of the intended dose levels and route of administrationThe dose levels were selected by the Sponsor based on previous data available, including the results of an OECD 422 performed by the Sponsor (in a separate laboratory in Crl:CD(SD) rats where the NOAEL was determined to be 5000 mg/kg bw wet weight of the test material), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.The oral route was selected as it is a possible route of exposure to the test item in humans.Dosing procedureDose formulations were administered daily starting from Day 0 for 91 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 10 mL/kg bw was administered to all animals/in all groups.The actual volume to be administered was calculated and adjusted based on most recent individual body weight. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of necropsy.
Positive control:
Not required for this study.

Examinations

Observations and examinations performed and frequency:
Mortality and clinical observationsAnimals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). General clinical observations were made at least once a day at approximately the same time. Detailed clinical observations were made on all animals outside the home cage at randomisation (Day -1), on Day 6 and at least once a week afterwards.Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, etc., aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. Hunchback posture, etc), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Neurological assessment (Functional Observation Battery)Towards the end of the treatment period, during Week 13, each animal was subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and motor activity assessment.Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.A detailed assessment for neurotoxicity effects was made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.Motor activity assessment was conducted using the Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour during which a DVD recording of movement was made. Recording was made for a duration of 60 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software.Ophthalmology evaluationOphthalmoscopy was conducted in all animals before treatment (Day -1), and in the Control Group 1 and High dose Group 4 animals, during Week 13 (Day 86/84, males/females). Mydriasis was produced after instillation of eye drops "Humapent" (5 mg/mL cyclopentolate hydrochloride; Batch No.: 2730614, exp.: June 2016) into the conjunctival sac. The evaluation was performed using a Gowlland ophthalmoscope.Body weight measurementBody weight was recorded with a precision of 1 g at randomisation (Day -1), then at least weekly, including on Day 90 (last treatment day) and prior to necropsy (fasted, on Day 91).Food consumption measurementThe determination of food consumption was performed once a week. The remaining, non-consumed food was weighed at least weekly from Day 6 with a precision of 1 g.Weekly food consumption was calculated.CLINICAL PATHOLOGYAt the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals.After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.UrinalysisUrine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which were placed in metabolic cages.The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable.
Sacrifice and pathology:
Terminal proceduresNecropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Days 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.Macroscopic evaluationAfter exsanguinations, the external appearance of each rat was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as applicable.In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed. The smears will be stored/archived at CiToxLAB Hungary Ltd.Organ weight measurementOrgans were trimmed of fat and weighed in all animals (detailed in table form – See Any other information).Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights was calculated and reported.HistopathologyOn completion of the macroscopic examination tissues and organs were retained from all animals. (detailed in table form – See Any other information). The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.Full histopathology was performed in Groups 1 (Control) and 4 (High dose).
Other examinations:
Examination of vaginal smearsPrior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution.The smears were examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Statistics:
Data were collected using the software PROVANTIS (Instem LSS Ltd. UK) or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software PROVANTIS, Microsoft Office Word and/or Excel, as appropriate.The statistical evaluation of the numerical data were performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). Evaluation was made by comparing the data for each of the Groups 2 to 4, respectively, against the Control Group 1. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a oneway analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of abnormal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The mean and standard deviations values, the frequency of clinical observations, macroscopic and microscopic findings were calculated as applicable.

Results and discussion

Results of examinations

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITYNo animal mortality occurred during this study.CLINICAL OBSERVATIONSAll animals were clinically normal and there were no toxicologically significant or test item related findings during the study.NEUROLOGICAL ASSESSMENT (FUNCTIONAL OBSERVATION BATTERY)There were no treatment related effects.There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.During evaluation of motor activity, the total travelled distance was comparable to the control in both sexes. In low dose females, increased activity was seen between 0-5, 10-15 and 25-30 minutes with attaining statistical significance (p<0.05) which resulted a statistically higher overall value (0-60 min.; p<0.05). In high dose females, decreased activity was seen between 40-45 minutes with attaining statistical significance (p<0.05). However these differences were considered to be incidental or individual findings, which were not related to treatment, or were with no toxicological significance. The profile of the locomotor behaviour over one hour was normal in all groups, the sporadic statistical differences were not considered to represent an effect of treatment.EXAMINATION OF VAGINAL SMEARSThere were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.OPHTHALMOLOGYNo test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.BODY WEIGHT MEASUREMENTThere were no effects on bodyweight at any dose levels.The mean body weights of males and females were comparable to the control throughout the treatment period. The overall body weight gains (Days -1-90) of males and females were similar to the controls.Minor differences to control were recorded in body weight or in body weight gain, e.g. a lower body weight in mid dose females on Day 20 (p<0.05) or a lower body weight gain value in high dose males between Days 76-83 (p<0.01), which were without any biological significance.FOOD CONSUMPTION MEASUREMENTThe 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. Slightly higher mean values were recorded in females at mid dose group.CLINICAL PATHOLOGYHaematologyWhen compared to the controls, there were no differences that were considered toxicologically significant.Minor variations were noted in a few parameters, on occasion attaining statistical significance, including statistically higher Mean Corpuscular (erythrocyte) Haemoglobin (MCH) (p<0.01) and Mean Corpuscular (erythrocyte) Haemoglobin Concentration (MCHC) (p<0.05) in low dose males or in female animals statistically higher than control absolute Basophil (BASO) value at mid dose level (p<0.05). Although the differences were statistically significant, they were not dose-related, showed no consistent gender response and/or were within the normal historical control ranges. Therefore they were considered to be of no toxicological significance.Coagulation parametersThere was no effect of treatment on coagulation parameters investigated, i.e. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). In high dose males, the Prothrombin Time (PT) was statistically lower than the control value (p<0.01). Although this difference observed between the control and the high dose group was considered to be incidental or individual findings, which were not related to treatment, was generally comparable with the expected physiological range or were with no toxicological significance.Clinical chemistryWhen compared to the controls, following 91 days of treatment, there were no adverse effects that could be ascribed to the test item administration under the conditions of this study.Occasional variations were noted in females at all dose levels or in males at the high dose level, but were not considered to be toxicologically significant. These variations included slightly lower Urea concentration (Urea) in high dose males (p<0.05) or in females slightly higher Triglycerides (Trig) at low dose (p<0.05), Chloride concentration (Cl-) at low and mid dose (p<0.05) or Potassium concentration (K+) at high dose and slightly lower Urea concentration (Urea) at low dose (p<0.05). These differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.UrinalysisNo effects were noted, which could be related to the test item.The urine volume in low dose in males was lower than the control value, although this variation was unrelated to treatment and within the normal range. Results of sediment analysis revealed no significant differences between control and treated animals.PATHOLOGY EVALUATION AND ORGAN WEIGHTSNo test item-related macroscopic changes were observed up to dose level of 1000 mg/kg bw/day.A few black foci of the glandular stomach mucosa in 1/10 control female, red diffuse discoloration of the thymus in 1/10 high dose female, were considered to be incidental changes.Organ weightsThere were no toxicologically significant effects on organ weights.Compared to controls, weight difference attaining statistical significance was noted in seminal vesicle weight for males at 1000 mg/kg bw/day. The difference attained statistical significance for absolute and relative mean values. As the changes had low magnitude, showed no consistent dose response, and/or were not correlated with pathological findings, they were considered incidental and not related to treatment.HistopathologyThere were no test item-related microscopic findings at a dose level of 1000 mg/kg bw/day.Findings included minimal adrenal cyst in 1/10 control female, minimal mixed mononuclear infiltrate of the right epidydimis/left Harderian’s gland in 1/10 high dose male, mild focal myocardial degeneration of the heart in 1/10 high dose male, minimal tubular basophilia/cast/mixed mononuclear infiltrate of the kidney in 3/10 high dose males, minimal neutrophilic infiltrate in 1/10 control male and mild cell debris of the prostate in 2/10 high dose males, minimal to mild extramedullary hematopoiesis of the spleen in 7/10 control males, 8/10 high dose males, 2/10 control females and 8/10 high dose females, mild diffuse haemorrhage of the thymus in 1/10 high dose female.These changes were regarded as incidental or a common background based on the incidence and distribution cross control and treated animals.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No overt adverse effects that could be ascribed to test item administration.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Summary of Clinical Signs data

 

Male

Female

0 mg/kg bw/day

62.5 mg/kg bw/day

250 mg/kg bw/day

1000 mg/kg bw/day

0 mg/kg bw/day

62.5 mg/kg bw/day

250 mg/kg bw/day

1000 mg/kg bw/day

Observation Type: General

From Day 0 (Start Date) to 90

Normal

10

10

10

10

10

10

10

10

Observation Type: Detailed

From Day -1 to 91

Normal

10

10

10

10

10

10

10

10

Observation Type: Ophthalmoscopy

Day -1 and Day 86/Day 84

Normal

10

10

10

10

10

10

10

10

Values = Number of Animal Affected

 

Summary of Bodyweight data

Bodyweight (g)

 

Day numbers relative to Start Date

Group

Sex

 

-1

6

13

20

27

34

41

48

55

62

69

76

83

90

1CD

M

Mean

209.9

269.0

322.0

357.1

399.4

430.9

453.4

478.6

499.3

515.0

530.5

545.8

560.4

566.3

S.D.

12.2

16.5

24.2

29.3

36.6

41.2

45.0

51.2

56.2

57.8

58.6

58.6

64.8

68.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

2LD

M

Mean

210.2

265.5

321.7

361.6

400.4

431.6

457.7

478.6

496.2

515.7

529.3

541.4

554.5

563.9

S.D.

11.0

16.6

19.4

23.9

24.9

27.9

31.7

35.8

37.0

37.7

39.0

43.9

47.2

50.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

3MD

M

Mean

210.4

267.2

320.5

360.1

402.3

431.7

455.3

476.0

494.8

508.6

523.9

535.1

548.4

555.3

S.D.

12.2

16.1

20.2

26.0

30.7

32.4

37.5

41.1

42.9

43.7

43.5

47.9

48.7

50.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

4HD

M

Mean

210.7

269.5

326.5

369.4

409.3

440.1

460.9

486.3

502.9

519.5

538.2

552.6

560.6

568.6

S.D.

11.7

11.6

13.2

18.3

25.2

25.2

30.1

31.7

30.9

33.7

34.6

34.0

35.5

35.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

1CD

F

Mean

186.9

208.2

223.2

235.7

244.5

252.8

258.8

267.2

271.7

274.5

277.3

282.3

285.8

284.3

S.D.

6.8

7.9

11.5

12.3

10.5

13.3

15.5

15.2

13.7

14.5

15.3

13.7

13.6

15.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

2LD

F

Mean

187.0

208.3

223.1

240.4

251.2

260.9

265.6

269.7

276.8

278.3

282.9

288.0

291.7

292.9

S.D.

6.4

7.2

13.6

15.3

18.2

17.6

21.1

18.4

20.8

21.8

22.4

23.4

23.2

25.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

3MD

F

Mean

187.1

203.2

211.1

220.9*

231.7

238.0

245.2

253.7

260.7

262.6

262.6

269.1

274.1

279.1

S.D.

6.4

8.9

10.0

14.4

18.0

19.9

16.8

17.2

16.5

17.5

20.5

21.4

18.4

16.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

4HD

F

Mean

186.9

208.1

222.0

238.8

248.2

256.4

262.7

270.1

276.7

283.1

287.6

289.0

295.0

297.9

S.D.

6.4

10.0

15.8

18.5

19.3

23.1

26.3

30.2

27.1

32.4

34.9

35.2

35.3

35.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

NS

NS

NS

DN

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

Group 1CD – 0 mg/kg bw/day          Group 2LD – 62.5 mg/kg bw/day     Group 3MD – 250 mg/kg bw/day    Group 4HD – 1000 mg/kg bw/day

Remarks:

NS = Not significant             NA = Not Applicable

* = p<0.05                            U = Mann-Whitney U-Test

** = p<0.01                         DN = Duncan’s Multiple Range Test

Applicant's summary and conclusion

Conclusions:
The NOAEL of Green liquor sludge (GLS) is considered to be the high-dose level of 1000 mg/kg bw/day.
Executive summary:

The purpose of the study was to obtain information on the toxicity of Green liquor sludge (GLS) when administered daily for 91 days by oral gavage to the rat at 3 dose levels of 62.5, 250 and 1000 mg/kg bw/day. The doses were selected by the Sponsor based on previous data available, including the results of an OECD 422 performed by the Sponsor (in a separate laboratory in Crl:CD(SD) rats where the NOAEL was determined to be 5000 mg/kg bw wet weight of the test material), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

BASIS OF STUDY: OECD No.: 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents” (1998).

 

The 90-day study was performed to obtain information on the toxicity of Green liquor sludge (GLS) when administered by oral gavage to Wistar rats of both sexes at dose levels of 62.5, 250 and 1000 mg/kg body weight, daily for at least 90 days.

Male and female Wistar rats were treated according to the following experimental design:

 

Gr. No.

Group Designation

Dose Level (mg/kg bw/day)

Concentration (mg/mL)

Dose volume (mL/kg bw)

Animal Number

Males

Females

1

Control

0

0

10

1001-1010

1501-1510

2

Low Dose

62.5

6.25

2001-2010

2501-2510

3

Mid Dose

250

25

3001-3010

3501-3510

4

High Dose

1000

100

4001-4010

4501-4510

 

The control group was treated concurrently with the vehicle only (aqueous 1 % (w/v) Methylcellulose solution, abbreviation: 1% Methylcellulose).

Animals were euthanized after 91 days of daily treatment, the first day of treatment was Day 0.

Analysis of test item formulations for concentration and homogeneity was performed at least five times during the treatment period using a validated flame photometry method.

Parameters monitored during the study included mortality/morbidity, clinical signs, body weights and body weight gain, food consumption. Blood for haematology and clinical chemistry measurements was collected on Day 91. Urinalysis was also performed at the end of the treatment period. Animals were subjected to neurological assessments during the week 13. Examinations included functional observation battery (FOB), landing foot splay and grip strength measurements, and automated locomotor activity measurements (SMART). Ophthalmology was performed pre-treatment and at the last week of the treatment period. Necropsy and macroscopic examination was performed on all animals at the end of treatment period on Day 91, selected organs were weighed and preserved. Tissues from control and high dose groups were processed to slides and examined histopathologically.

 

Results

All formulations were found to be in the range of 78 to 108% of nominal concentration and were homogeneous. No test item was detected in the control samples. Based on the results, test item formulations were considered suitable for the study purposes.

There was no test item related mortality observed during the study.

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

There were no signs of toxicity observed on body weights, body weight gains or food consumption.

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on last week of the treatment.

There were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

No test item related changes were noted at ophthalmoscopy examination.

When compared to the controls, there were no differences noted in the treated animals in haematology or blood clotting parameters which could be related to the test item.

There were no toxicologically relevant differences between the controls and any treated groups in the clinical chemistry parameters.

No effects were noted at urinalysis which could be related to the test item.

There were no macroscopic findings considered related to test item administration under the conditions of this study.

No notable effects were seen for organ weights.

No test item related microscopic changes were seen at histopathology.

 

Conclusion

Based on the results, Green liquor sludge (GLS) administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 91 consecutive days, in 1% Methylcellulose at a dose volume of 10 mL/kg bw, was not associated with any overt adverse effects that could be ascribed to test item administration.

In conclusion, the NOAEL of Green liquor sludge (GLS) is considered to be the high-dose level of 1000 mg/kg bw/day.

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