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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2010 to 17 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully guideline and GLP compliant study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
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.
Details on test material:
Name: Green liquor sludge.Chemical name: Green liquor sludge.Physical form: Paste (alkaline, with water).Molecular formula: UVCB.Appearance: Black solid.Solubility in water: In water: washed substance is not solubleIn other solvents: not available.Conditions of storage: Room temperature. Storage in the dark but may be used under light.Date of receipt: 02 December 2009.Label on the shipping container: GLS2, GREEN LIQUOR SLUDGE, Slamfilter, 2009-11-16-2009-11-20, Södra Cell Mörrum, Johnny Petersson.
Specific details on test material used for the study:
Test material contained dry solids 46,7% and water 53.3%

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Germany- Age at study initiation: About 8-9 weeks- Weight at study initiation (first administration of the test substance: mean body weights between 351 and 356 g (males) and between 227 and 228 g (females)- Fasting period before study: none- Housing:Dose Range Finding Study: Group CagingMakrolon cages Type IV Type IV (33 cm x 55 cm area, 20 cm height), wire mesh lids.Sanitation of cages once a week. Main Study:Single caging (in general, except for the mating period for and for dams with offspring). Double caging (for the mating period, 1 male + 1 female per cage).Group caging (1 dam plus her offspring per cage, for the post-partum period).Makrolon cages Type III, high version (39 cm x 23 cm ground area, 18 cm height).Sanitation of cages once a week. - Diet (e.g. ad libitum):Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany. Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood sampling.Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).- Water:Tap water, acidified with HCl to pH >=3, from an automatic watering system, ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).- Acclimation period: one weekENVIRONMENTAL CONDITIONS- Temperature: Mean 21.3 °C.- Humidity: Mean 52.5 %.- Air changes (per hr): 12- Photoperiod: 12 (hrs dark / 12 hrs light)IN-LIFE DATES:Dose Range FInding Study: From: 4 Apriul 2010 To: 20 April 2010Main Study: From: 28 April 2010 To: 13 June 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Water: Water pro analysis, Merck item No. 1.16754.5000.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:An oral administration was performed by stomach intubations using a metal gavage once a day in the morning on 7 days per week.The individual dose volumes were calculated using the last determined body weights.VEHICLE- Justification for use and choice of vehicle: Aqueous solutions are the first choice for oral administrations.- Concentration in vehicle: nominal 50, 158 and 500 g per L- Amount of vehicle (if gavage): 10 mL/kg body weight- Preparation of vehicle: not appropriate
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation:until proven mating or until the end of the mating period of 14 days- Proof of pregnancy: During the mating period, starting with the day after the commencement, all females were subjected to a daily examination (once in the morning,) for the presence of a vaginal plug. In case of absence of a vaginal plug, the females were subjected also once a day to a vaginal smear. The unstained vaginal smears were examined microscopically for the presence of sperm.Presence of a vaginal plug or of sperm in the smears was taken as an evidence for successful mating."Day 0 of pregnancy" (of the given individual) was the day of proven mating."Day 0 post-partum" (of the given individual) was the day of birth (when parturition was complete).- Further matings after two unsuccessful attempts: no- After successful mating each pregnant female was caged individually-- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
"Day 1" (of the entire experiment) was the day of the 1st administration of the test substance. Commencement of dosing was at the beginning of the pre-mating period (see 2.6)."Day 0 of pregnancy" (of the given individual) was the day of proven mating."Day 0 post-partum" (of the given individual) was the day of birth (when parturition was complete).All adult animals were treated with the test substance solutions or with the vehicle once a day from Day 1 onwards until the day prior to their sacrifice (maximum duration: 54 days).
Frequency of treatment:
Once daily, 7 days/week
Details on study schedule:
not appropriate
Doses / concentrations
Remarks:
Doses / Concentrations:500, 1580 and 5000 mg per kg body weight and dayBasis:nominal conc. test material wet weight.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The doses chosen were derived from and based on the results of the Dose Range Finding Study.Summarised results of the Dose Range Finding Study: All animals survived until their scheduled sacrifice. No indications for test substance related effects were found. In the Main Study, the high dose shall induce a clear toxicity, but no or at most isolated mortality. Based on this, the doses of 500 mg and 1580 mg and 5000 mg per kg body weight were selected for the Main Study. The low dose was set at 10% of the high dose. The mid dose was interpolated geometrically.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: once daily, plus viability check once dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: prior to first test substance administration, then once a week, all adult animals.Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.BODY WEIGHT: Yes- Time schedule for examinations:Adult animals (except for pregnant females): Determined on Day 1, then once a week, and at termination.Pregnant females: On Days 0, 7, 14 and 20 of pregnancy and within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.Offspring: The total weight of the litter is determined within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.FOOD CONSUMPTION:- Food consumption for each animal determined: YesDetermined for weekly periods (except for changes in caging, e.g. at the beginning of mating or at proven mating; forming an interim endpoint), all animals (or per cage during mating period or with offspring).- Mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD 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: NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.- Anaesthetic used for blood collection: Yes (slight ether anaethesia)- Animals fasted: Yes, overnight- How many animals: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.- Parameters examined: Red blood cell count (RBC)Haemoglobin concentration (HGB)Haematocrit (HCT)Mean corpuscular haemoglobin (MCH)Mean corpuscular haemoglobin concentration (MCHC)White blood cell count (WBC)Mean cell volume (MCV)Platelet count (PLT)Differential white blood cell count (% of the different cell species) Prothrombin time (Quick) as an indicator of blood clotting capacity.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.- Anaesthetic used for blood collection: Yes (slight ehter anaethesia)- Animals fasted: Yes, overnight- How many animals: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.- Parameters examined:Alanin aminotransferase (ALT, GPT)Albumin Alkaline phosphatase (AP)Aspartate aminotransferase (AST, GOT)Bile acidsCholesterol Creatinine Gamma glutamyl transferase (GGT)Glucose Potassium (K+)Sodium (Na+)Total protein UreaURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations:Males: Once in the last week of the dosing period.Females: Once during the lactation period (i.e. on Days 1-4 post partum).Offspring: Not examined.- Dose groups that were examined: all- Battery of functions tested: Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) and grip strength.OTHER: Sacrifice and pathology GROSS PATHOLOGY: YesAdult animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death onMales: On the day after the end of the mating period.Females: On Day 4 post partum or Day 54 (i.e. 25 days after the end of the mating period).The following organs/tissues (if appropriate) were fixed in 4 % buffered formaldehyde, with the exception of the eyes (fixed in Davidson's fixative) and the skin and the testes (both fixed in Bouin's solution, the testes were punctured on both poles with a needle before immersion in the fixative). Organs/tissues marked with an "*" were not included in routine histopathology. gross lesions, tissue masses or tumoursadrenal glandsaorta *brain including cerebrum, cerebellum and pons)caecum *coagulating glandsepididymideseyesheartkidneyslacrimal glands *large intestine (colon)liverlungslymph nodes (mandibular, mesenteric)oesophagus *ovariespancreas *pituitary gland *prostaterectum *salivary glands*sciatic nerveseminal vesiclesskeletal muscle (thigh)skin, mammary glands *small intestine (duodenum, ileum, jejunum; including Peyer's patches), prepared as "Swiss Roll"spinal cord (cervical, thoracal, lumbar)spleensternum with bone marrowstomachtestesthymusthyroid glandstrachea.urinary bladderuterusvagina with cervixAdditional examinations were performed in the females: Counting of corpora lutea per ovary (visual). Counting of implantation sites (visual, no staining).The pups were killed by overdosed chloroform anaesthesia at the same time as their mothers and subjected to an external examination for gross abnormalities. No organs are weighed or preserved in the pups.ORGAN WEIGHTS: YesFresh weights of the following organs were determined of all adult animals at necropsy:adrenal glands (both together)brainepididymides (both together)heartliverkidneys (both together)spleentestes (both together)thymusRelative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.HISTOPATHOLOGY: YesGroups K and C:Histopathological examination was performed in the first 5 adult animals of groups K and C, of all fixed organs and tissues listed above, except those, labelled with a "*". Groups A and B:Lung: the first 5 males.Justification: Indication for test substance related effects in group C.The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:- Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons were included in these sections.- Spinal cord (three sections, a cervical, a thoracal and a lumbar).- Liver (two sections).The small intestine (duodenum, jejunum and ileum) was fixed and trimmed to form two "Swiss Rolls" (one of the cranial and one of the caudal part), according to "Moolenbeek and Ruitenberg: The Swiss Roll, a simple technique for histological studies of rodent intestine"; Lab.Animals 15 (1981), p.57-59.The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 µm were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB. To describe the severity of lesions, the following grades were applied, if appropriate: minimal (1), mild (2), moderate (3), marked (4), severe (5).The term "focal" together with a higher degree of severity also stands for "multifocal".
Oestrous cyclicity (parental animals):
not recorded
Sperm parameters (parental animals):
not applicable
Litter observations:
STANDARDISATION OF LITTERSnot appropriatePARAMETERS EXAMINEDThe following parameters were examined in F1 offspring:•Runts: Pups, that are substantially smaller than corresponding control pups.•Abnormal pups: Pups showing any abnormalities or malformations.•Sex ratio: Number of males / number of females.•Litter weight: Sum of the body weights of all live pups.•Pup weight: Litter weight / number of pups.•Pre-natal, post-implantation loss of offspring: Implants minus live births.•Post-natal loss of offspring: Live births minus pups alive on Day 4 post partum.GROSS EXAMINATION OF DEAD PUPS:yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
see above (Parental animals: Observations and examinations)
Postmortem examinations (offspring):
none performed
Statistics:
Analysis of variance followed by the Scheffé-test: all data with means and standard deviations determined, comparison of more than two groupst-test: all data with means and standard deviations determined, for comparison of two groups onlyH-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilledChi2-test: counted eventsFisher's exact test: counted events, if the Chi2-Test was not applicableResults were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used.Numerical data have been rounded for presentation; a manual recalculation therefore may yield slightly different results to those given in the tables.Please note: Whenever the term "significant" is used in this report, it stands for "statistically significant".
Reproductive indices:
•Pairs started: Number of pairs at the beginning of the mating period.•Females showing evidence of copulation: Females with a vaginal plug or with sperm in the vaginal smear.•Females achieving pregnancy: Number of females giving birth to live or dead pups.•Duration of pregnancy: Days between proven mating and parturition.•Corpora lutea per dam: Sum of corpora lutea of both ovaries.•Implants per dam: Number of implantation sites in the uterus, found at necropsy on Day 4 post partum.
Offspring viability indices:
Runts: Pups, that are substantially smaller than corresponding control pups.Abnormal pups: Pups showing any abnormalities or malformations.Sex ratio: Number of males / number of females.Litter weight: Sum of the body weights of all live pups.Pup weight: Litter weight / number of pups.Pre-implantation loss of offspring: Corpora lutea minus implants.Pre-natal, post-implantation loss of offspring: Implants minus live births.Post-natal loss of offspring: Live births minus pups alive on Day 4 post partum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
yes, not relevant
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
yes, not relevant
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITYOne animal died in the course of taking blood samples on Day 0 and was replaced by a spare animal. Another animal died in the course of taking blood samples on day 15. All other animals survived until their scheduled sacrifice.OBSERVATIONS IN LIFEBlack discoloured faeces were observed in 20 animals due to the black coloured test substance. One animal showed chromodacryorrhea on one day. Another animal had diarrhoea on three consecutive days. One male animal had a wound on its nose from day 16 on. Alopecia which is known as a spontaneous alteration of the strain of rats used, was noted in several individuals. Its presence is not related to the test substance.FUNCTIONAL OBSERVATIONS AND GRIP STRENGTH DETERMINATIONNo test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.There was no significant group difference in the grip strengths of both sexes.BODY WEIGHTS AND BODY WEIGHT GAINSignificant lowered body weight gain was seen in groups B and C between Days 0 and 7 of pregnancy and may be a typically event at the beginning of pregnancy. This event is no longer significant in the later pregnancy. Since no further findings were found, also for example no changed (reduced) feed consumption, this in this study is regarded as not toxicological relevantly.Beside from that no significant difference in body weight and body weight gain was noted between the groups. In the body weights of the females before mating and of the non-inseminated dams no significant differences were found.FEED CONSUMPTIONThere were no noteworthy differences or dose related trends noted in the feed consumption of both sexes. HEAMATOLOGYThere were no significant differences nor dose related trends in the haematological parameters of both blood sampling terms, on Day -2 and on Day 15.CLINICAL BIOCHCEMISTRYElevated sodium levels in the low dosed females on Day 15 are not given toxicological relevance, as there was no dose-respojnse relationship.GROSS PATHOLOGYNo findings were made at the gross examination at necropsy which gave an indication for a specific mode of action of the test substance.ORGAN WEIGHTSNo significant differences in organ weights between the groups could be found. HISTOPATHOLOGYThe livers of both the high dosed group showed minimal to moderate vacuolar changes in the centrilobular hepatocytes. The same alteration was found in the control group. Hepatocytes with cytoplasmatic vacuoles, as found in both control and high dosed animals in about the same incidence, may have undergone a non-degenerative fatty change. This is occasionally observed in control rats and not related to the test substance. In the high dosed group the livers exhibited lymphohistiocytic and apoptotic foci to a minimal extent. Neither of these alterations was given a toxicological significance.Extramedullary haematopoiesis in the spleens of some females is a sequel of the blood loss at birth, 4 days before sacrifice.The pulmonal arteries showed thickening and proliferation of the tunica media in the male animals of the high dosed group. This finding was not associated with any clinical signs and may be based on an uncommonly pronounced, but normal, occurrence of the thick muscular layers in the acute angle arterial branches of the rat lungs or a test substance related change with equivocal toxicological relevance.Some other isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence. None of them is related to the test substance.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effectsclinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

BIRTH, PERI- AND POSTNATAL PERIODA pregnant female (no.140, group C) gave birth to two pups (male and female), one of which was a stillbirth (female). The following day the male pup had been cannibalised (empty cage). This single case is not related to the test substance.Again, no test substance related group differences were noted.OFFSPRINGThere was no indication for a test substance related effect on the offspring

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 2 344 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
dryweight.
Sex:
male/female
Basis for effect level:
other: reproduction
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 5 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
wwt.
Sex:
male/female
Basis for effect level:
other: reproduction

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No-observed-adverse-effect-level (NOAEL) of "Green liqor sludge" was at 5000 mg per kg body weight for both males and females.The No-observed-effect-level (NOEL) of "Green liqor sludge" was at 5000 mg per kg body weight for females and at 1580 mg per kg body weight for males.According to EU-Directive 2001/59/EC, a classification with "R48" is not considered as necessary for "Green liquor sludge", as no severe toxic effects were noted at doses of up to 5000 mg/kg.According to EU-Directive 2001/59/EC, a classification with "R60" is not considered as necessary for "Green liquor sludge", as no toxic effects on reproduction were noted.According to Regulation (EC) No. 1272/2008 (CLP), "Green liquor sludge" needs not to be classified for "Reproductive Toxicity" and also not to be classified for "Specific Target Organ Toxicity after Repeated Exposure".
Executive summary:

Aim of the study

The study was performed to assess and evaluate the toxic characteristics of the test substance, resulting from a subacute oral administration via gavage to rats and comprises a reproduction and developmental toxicity screening test, according to OECD-Guideline 422,.

A Dose Range Finding Study preceded this study.

Methods

All details given here refer to the Main Study; a survey of the methods of the Dose Range Finding Study is included into the Results of the Dose Range Finding Study (see below).

Test substance:Greenliquor sludge.

Test substance preparation for administration:Freshly dissolved with the vehicle.

Vehicle:Water.

Route of test substance administration:Oral via gavage.

Dosing regimen:1/day for 28 consecutive days in males. 1/day until Day 4 post partum or for 54 Days in females. No test substance administration in the offspring.

Dose volume:10 mL test substance preparation or vehicle per kg body weight.

Test system (animals):Rats, Crl:CD(SD). 10 males and 10 females per group.

Groups, doses:

·                    K (negative control group)                vehicle only,

·                    A (low dose group)                                        500 mgper kg body weight,

·                    B (mid dose group)                                      1580 mg per kg body weight,

·                    C (high dose group)                                     5000 mg per kg body weight,

The doses are derived from and based on the results of the dose range finding study.

For a survey thereof, see below.

Experimental schedule:

·        2 weeks pre-mating period;

·        2 weeks mating period; then sacrifice of the males;

·        maintenance of the successfully mated females throughout their pregnancy, birth, until Day 4 post partum, then sacrifice of the dams and their offspring;

·        maintenance of the non-successfully mated females for 26 days after the end of the mating period (was not necessary),

           "Day 1"(of the entire experiment) was the day of the 1stadministration of the test
                       substance.

           "Day 0 of pregnancy"(of the given individual) was the day of proven mating.

           "Day 0 post-partum"(of the given individual) was the day of birth.

 

Investigations:

·      Animal observations:
All animals, once a day, plus a daily check for viability.

·      Detailed clinical observations:
All parental animals, once a week.

·      Functional observations:
All parental animals, once, prior to sacrifice.

·      Body weights:
All males and all females prior to successful mating once a week and at termination. All successfully mated females on Days 0, 7, 14, 20 of pregnancy and on Days 0 and 4 post partum. Pups as total litter weight on Days 0 and 4 post partum.

·      Food consumption:
All animals, for weekly periods.

·      Haematology:
The first 5 parental animals of all groups, prior to first dosing (Day 0) and on Day 15.

·      Clinical biochemistry:
The first 5 parental animals of all groups, prior to first dosing (Day 0) and on Day 15.

·      Necropsy with gross pathological examination:
All males on Day 30, all successfully mated females on Day 4 post partum, and all non-successfully mated females on Day 55.
No necropsy in pups.

·      Organ weight determination:
Selected organs in all parental animals at necropsy. Not determined in pups.

·      Histopathological examination:
Selected organs or tissues in the first 5 parental animals of groups K and C. Organs and tissues with suspected test substance related alterations also in groups A and B.
No histopathology in pups.

·      Data on reproduction and developmental performance:
Results of vaginal smear examinations; duration of pregnancy; number, sex and viability of offspring. Number of corpora lutea and implantation sites in the dams. Several parameters were additionally derived from the primary data above.

 

 

Results

DoseFinding Study:Summarized results:

All animals survived until their scheduled termination and were found to be normal at the daily observations. Body weights, body weight gain, feed consumption did not differ significantly or notably between control and test substance exposed animals. All animals of the high-dosed group showed black discoloured faeces. All animals were normal at gross examination during the terminal necropsy except of multifocal brownish-black discoloration of the kidneys of animals in the mid-dosed and high-dosed groups.Spotted kidneys are interpreted as the results of unequal hemoperfusion, also commonly seen in control animals, without toxicological significance. This finding was only seen in the dose range finding study and not in the main study.

Main Study:

·      Mortality

1 animal died in the course of taking blood samples on day 0 and was replaced by a spare animal. Another animal died in the course of taking blood samples on day 15. A gross pathological examination was performed on that animal. To ensure five animals per group for evaluation a histopathological examination was performed on another animal of the same group.

·      Observations in life, clinical and functional observations, grip strength determination:
All animals of the high dosed group showed black discoloured faeces, several animals had loss of hair, in one animal chromodakryorrhoe and in one further animal diarrhoe were found.

·      Body weights and feed consumption:
There were no significant differences in body weights between the groups.

·      Haematology:
No significant group differences were noted at both blood sampling terms.

·      Clinical biochemistry, organ weight determination:
Only, but all, parameters with significant differences to the negative control group (indicated by a black background) are given in this survey:

Day 15:

parameter (sex)

low dose
500 mg/kg

(% of the negative controls)

mid dose
1580 mg/kg

(% of the negative controls)

high dose
5000 mg/kg

(% of the negative controls)

Sodium (females)

101.3

   100.6

100.7

The elevated Sodium level in the low dosed group is not given a toxicological relevance due to the lack of a dose response.   ·      Organ weight determination:
There were no significant group differences in organ weights.


 

·      Necropsy with gross pathological examination and histopathology:

Histopathologically there were slight vacuolar changes in the liver of the high dose and control group. Additionally there were lymphohistiocytic foci in the high dose group but without statistically significance. Both alterations are not interpreted as a test substance related effect. Extramedullary haematopoiesis was found in the spleen of some female animals and is taken as an adaptive response after giving birth.

The pulmonal arteries showed thickening and proliferation of the tunica media in the male animals of the high dosed group. This finding was not associated with any clinical signs and may be based on an uncommonly pronounced, but normal, occurrence of the thick muscular layers in the acute angle arterial branches of the rat lungs or a test substance related change with equivocal toxicological relevance.

 

·      Reproduction data

No indications for a test substance related effect was made with any of the reproduction parameters.



Discussion and Conclusion

The livers of the high dosed group showed minimal to moderate vacuolar changes in the centrilobular hepatocytes. The same alteration was found in the control group.Hepatocytes with cytoplasmatic vacuoles, as found in both control and high dosed animals in about the same incidence, may have undergone a non-degenerative fatty change. This is occasionally observed in control rats and not related to the test substance.In the high dosed group the livers exhibited lymphohistiocytic and apoptotic foci to a minimal extent. Neither of these alterations was given a toxicological significance.

The pulmonal arteries showed thickening and proliferation of the tunica media in the male animals of the high dosed group. This finding was not associated with any clinical signs and may be based on an uncommonly pronounced, but normal, occurrence of the thick muscular layers in the acute angle arterial branches of the rat lungs or a test substance related change with equivocal toxicological relevance.

There was no indication for an effect of the test substance on the reproduction.

There was no pronounced sex difference in the response to the test substance.

The effects noted were minmal to mild.

The No-observed-adverse-effect-level (NOAEL) of "Green liqor sludge" was at 5000 mg per kg body weight for both males and females.

The No-observed-effect-level (NOEL) of "Green liqor sludge" was at 5000 mg per kg body weight for females and at 1580 mg per kg body weight for males.

According to EU-Directive 2001/59/EC, a classification with "R48" is not considered as necessary for "Green liquor sludge", as no severe toxic effects were noted at doses of up to 5000 mg/kg.

According to EU-Directive 2001/59/EC, a classification with "R60" is not considered as necessary for "Green liquor sludge", as no toxic effects on reproduction were noted.

According to Regulation (EC) No. 1272/2008 (CLP), "Green liquor sludge" needsnotto be classified for "Reproductive Toxicity" and alsonotto be classified for "Specific Target Organ Toxicity after Repeated Exposure".

 

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