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Effects on fertility

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
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
Specific details on test material used for the study:
Test material contained dry solids 46,7% and water 53.3%
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 Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood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
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
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
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
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
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
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
Reproductive effects observed:
not specified
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".

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
5 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a valid GLP certificate
Additional information

A reproduction and developmental toxicity screening test, according to OECD Guideline 422 was performed for Green liquor sludge.

The study was performed to assess and evaluate the toxic characteristics of the test substance by subacute oral administration via gavage to rats. The method comprises a reproduction and developmental toxicity screening test. GLS dose groups were 500 mg, 1580 mg, 5000 mg/kg/day (wwt.). (Dry weigtht to wet weight conversation in the test samples: dry matter 46,7%, water 53.3%)

"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.

"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).

- 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).

In the male animals the pulmonal arteries showed thickening and proliferation of the tunica media 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 No-observed-adverse-effect-level (NOAEL) of GLS was 5000 mg/2335 mg (wwt/dwt) per kg body weight for both males and females.

The No-observed-effect-level (NOEL) of GLS was 5000 /2335 mg (wwt/dwt) mg per kg body weight for females and 1580/738 mg (wwt./dwt.) per kg body weight for males.


Short description of key information:
Repeated dose toxicity of Green liquor sludge was tested using oral administration in rats (OECD 422). The results showed no effect of the test substance on the reproduction.
The No-observed-adverse-effect-level (NOAEL) of GLS was 5000 mg per kg body weight for both males and females.
The No-observed-effect-level (NOEL) of GLS was 5000 mg per kg body weight for females and 1580 mg per kg body weight for males.

Effects on developmental toxicity

Description of key information
No adverse effect observed in OECD Guideline 414 (Prenatal Developmental Toxicity Study). Based on the results the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 1000 mg/kg/day. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 2014 to 11 December 2014
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 GLP certificate.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below under "principles of method if other than guideline".
Principles of method if other than guideline:
The study was carried out according to the study plan without any Amendment, however the following deviation occurred:Due to technical reasons, temperature in the animal room was increased over the limit of 25°C (up to 25.2°C) during 4 days toward the end of the study. This deviation was considered by the Study Director to have no impact on the outcome of the study or interpretation of the results.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Species and strain: Hannover Wistar rats (CRLHan)Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colonyJustification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to experience with this strain in teratology studies. Housing condition: Standard laboratory conditions; individual housing Number of animals: 98 female animals, plus an appropriate number of spares: 24-25 mated female animals/group, 4 groups (one control group and 3 test item treated groups); 58 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were usedAge of animals: Young adult female rats, nulliparous and non-pregnant, at least 11 weeks oldBody weight at onset of treatment: Not exceeded ± 20% of the mean weight, and was in the range of 201-257 g. Acclimation period: At least 5 daysAnimal health: Only healthy animals were used for the test, as certified by the staff Veterinarian.Cage type: Type II and/or III polycarbonateBedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and Grade 5 Fibre Litter Animal Bedding, manufactured by Johannes Brandenburg GmbH & Co. KG (Arkeburger Str. 31 DE-49424 Goldenstedt, Germany) were used. Nest building material (Cotton Rolls, DC GmbH, Germany) were also added to the cages. The bedding and nesting material were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.Temperature*: 21.0 - 25.0°C Relative humidity: 30 - 65% Ventilation: 15-20 air exchanges/hourDue to technical reasons, temperature in the animal room was increased over the limit of 25°C (up to 25.2°C) during 4 days toward the end of the study. The temperature and relative humidity was recorded twice daily during the study.Diet and water supplyThe animals were provided with ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest Germany) and tap water as for human consumption, in water bottles, ad libitum. The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. 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.Animal Identification Adult animals were identified by temporary numbers written with indelible ink on the tail during the entire study. During necropsy and caesarean sections procedures each evaluated dam was given additional number (evaluation number indicating group number), and cross referenced with the numbers using during the in-life phase of the study.The cages were marked with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date. Cages were arranged to minimize any possible effects due to cage placement. The litters were identified at necropsy with litter numbers.
Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose & Humaqua (distilled water for injection)
Details on exposure:
The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd.Due to the chemical and physical nature of the test item, it is 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. Dosing formulations were made by mixing the test item with 1% aqueous Methylcellulose. The required amount (mg) of the test item was suspended in the vehicle to achieve the desired test item concentrations of 10, 30 and 100 mg/mL, of the test item for each dose level (100, 300 and 1000 mg/kg/day, respectively) and was stirred using magnetic stirrer until a homogenous dosing form was obtained.During dosing formulations 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.Pending administration to the animals, the dose formulations were stirred on a magnetic stirrer at room temperature.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 twice during the study (during the first and last weeks of treatment). Similarly, one sample was taken in duplicate on each occasion from the Group 1 (control) solution, for the detection of the test item.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimatization the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male: 1-3 females) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
From Gestation Day (GD) 6 to GD19
Frequency of treatment:
The control or test item dose formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis.
Duration of test:
20 days
No. of animals per sex per dose:
98 female animals, plus an appropriate number of spares: 24-25 mated female animals/group, 4 groups (one control group and 3 test item treated groups); 58 male animals for mating; no study-procedures were carried out on the male animals; untreated.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-selection and route of administrationThe dose levels were set by the Sponsor in consultation with the Study Director based on available data from the previous studies.According to the Sponsor’s information, results from a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Green liquor sludge (GLS) in Sprague Dawley (Crl:CD) rats, performed according to OECD Test Guideline 422 by oral administration, are available. In this study a parental No Observed Adverse Effect Level (NOAEL) was found to be 5000 mg/kg bw wet weight of the test material (Seibersdorf Labor GmbH Austria, code: SL-LT-397/10, November 2010, internal study code: POY54). Based on the above results, doses of 100, 300 and 1000 mg/kg/day were selected for the study.The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this developmental toxicology study.
Maternal examinations:
Clinical observations Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Cage-side clinical observations were made at least daily after the treatment. Detailed clinical observations were made on all animals at the onset of treatment (GD6) then weekly.The animals were monitored for any changes including pertinent behavioural changes and 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), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were noted during the study. On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).Body weight measurement Body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption measurement Food was measured with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD6-20 and GD0-20.Caesarean section and necropsy Before expected delivery, on GD20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthasol 40% inj.; Produlab Pharma B.V., Forellenweg 16, 4941 SJ Raamsdonksveer, Nederland; Lot number: 13E22 9, exp.: April 2016) administered by intraperitoneal injection, followed by exsanguination was used for euthanasia. The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.HistopathologyIn the absence of macroscopic findings at necropsy, no histopathology evaluation was performed.
Ovaries and uterine content:
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
Each live foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination. For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. For the foetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue-acetic acid-ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; additionally photographic records were made where the Study Director considered it appropriate.
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2) using the litter as the unit for data analysis. 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. Dams were excluded from statistical analysis as follows: - Sperm positive but non pregnant females (total exclusion) [164 (control), 183, 129, 219, 145 and 215 (300 mg/kg/day), 146 (1000 mg/kg/day)]- Dams with complete intrauterine loss (total exclusion) [1523 (control)]- Dams with ≤ 5 implantations (total exclusion) [4525(1000 mg/kg/day)]Although these animals were excluded from the statistical analysis the report contains all data from these animals.From the foetal body weight evaluation, body weight values of two litters were excluded: 1506 (control) and 2504 (100 mg/kg/day), due to outlying values, i.e control liter consisted of 14 retarded foetuses, with mean weight was 2.32 g, while low dose litter was small of relatively high body weight of 4.60g. The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses.
Indices:
Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software Microsoft Office Word and/or Excel, as appropriate. Data were collected to provide information on parameters including:Maternal Data:Clinical signs (by gestation day)Mortality (by gestation day), if anyBody weight and body weight gain: mean ± S.D.Corrected body weight on GD20 (body weight-gravid uterine weight) and corrected body weight gain: mean ± S.D.Net body weight change (Body weight on GD20 minus body weight on GD6 minus gravid uterine weight): mean ± S.D. Food consumption: mean ± S.D.Gross pathology findingsGravid uterine weightCaesarean Section and Necropsy Data:Number of corpora lutea: mean ± S.D.Number of implantations: mean ± S.D.Number and percent of live foetuses: mean ± S.D.Number and percent of intrauterine mortality: mean ± S.D.Classified according to time of death: Pre-implantation loss, Post-implantation mortality, Early and late embryonic, as well as foetal deathPre-implantation loss: %, group meanPost-implantation loss: %, group meanFoetal Data:Sex distribution: %, group meanFoetal body weight (accuracy 0.01 g): mean ± S.D.External abnormalities/litter: %, group meanVisceral abnormalities/litter: %, group meanSkeletal abnormalities/litter: %, group mean
Historical control data:
Historical control data provided in the full study report.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no toxicologically significant or test-item related clinical signs noted following administration oftest item.
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled mortality during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gains of dams treated at 100, 300 and 1000 mg/kg/day were comparable to the control mean during the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of dams treated at 100, 300 and 1000 mg/kg/day groups did not differ significantly from the control throughout the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathology consisted of an external examination, including identification of all clinically-recorded lesions as applicable, as well as a detailed internal examination. No test item-related macroscopic changes could be detected with the necropsy of the experimental rats.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The changes including empty stomach in 1/22 Control and 1/24 High dose females, obstipation in the cecum of 1/19 Low dose female, the thin fur on the limbs or on the trunk and crust on the neck occurred in female dosed rats were considered as incidental.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effectsDetails on maternal toxic effects:Pregnancy data of the femalesNinety eight sperm positive females were included in the study, twenty four–twenty five in each group. The number of confirmed pregnant, evaluated dams in the dose groups treated at 100, 300 and 1000 mg/kg/day was 19 in the Low dose group, 24 in Mid and High dose group and 22 in the control group.Clinical Signs and MortalityThere was no unscheduled mortality during the study. There were no toxicologically significant or test-item related clinical signs noted following administration of test item. Thin fur (focal) or focal alopecia and small skin lesions (scar) were occasionally observed in few dams in all experimental groups including control (i.e. 2/22 in control, 3/19, 2/24 and 3/25 animals in the Low, Mid and High dose groups, respectively. In one High dose female (4514) thin fur was observed during the first four days only. See overview table below. These observations were regarded as incidental and not related to the test item.Maternal Body weight and Body Weight GainBody weights and body weight gains of dams treated at 100, 300 and 1000 mg/kg/day were comparable to the control mean during the treatment period. Although slightly lower body weight gain was noted at 100 mg/kg/day between GD14-16 (p<0.05) and GD12-14 (not statistically significant), it was regarded as incidental. The “corrected” body weight gain (body weight gain between GD0-20 adjusted for the gravid uterine weight) and the net body weight gain during the treatment period (body weight gain between GD6-20 adjusted for the gravid uterine weight) of the dams were comparable with the controls in all treated groups.Food consumption The food consumption of dams treated at 100, 300 and 1000 mg/kg/day groups did not differ significantly from the control throughout the treatment period. Necropsy findings of dams At necropsy of dams (GD20), no test item related macroscopic changes were found.Minor changes, including empty stomach in 1/22 Control and 1/24 High dose females, obstipation in the caecum of 1/19 Low dose female, the thin fur on the limbs or on the trunk and crust on the neck occurred in female dosed rats were considered as incidental.Caesaren SectionIntrauterine mortality, corpora lutea and implantation sitesThe mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups.The late embryonic loss was slightly higher, than control in all test item treated groups. The differences were not statistically significant. The incidence of the observation was within the historical control range.There was no foetal death at any of the dose group.The post implantation loss and total intrauterine mortality was comparable with the control at 300 and 1000 mg/kg/day and slightly higher at 100 mg/kg/day. The differences were not statistically significant. As the differences were of low magnitude and showed no coherent dose-relationship therefore were considered incidental. The pre-implantation loss was also slightly higher, than control at 100 mg/kg/day. The differences were not statistically significant.It should be noted, in the control group, there was one female (1523) with total intrauterine death, all classified as early embryonic loss. This female was excluded from the statistical analysis.Viable foetuses and their sex distributionThe mean number of viable foetuses was comparable with the control mean at 300 and 1000 mg/kg/day and slightly lower at 100 mg/kg/day. The difference attained statistical significance (p<0.05). Although the mean group value was lower than the historical control mean, there is no evidence for an adverse treatment related effect on embryo-foetal survival at this dose level and the differences were considered incidental and attributed to the relatively small number of the dams in this group. The sex distribution of foetuses did not differ significantly between the control and treatment groups.Evaluation of placentaeThere were no toxicological significant differences in the placenta at evaluation of the treated groups compared to controls. One altered placenta was observed in the Mid dose group (300 mg/kg/day, 3508/8&9) in the form of fused placental disk. One of foetuses belonging to this placenta was intact (subjected to visceral examination), another one was of low body weight (2.61 g) with delayed ossification of sternal bodies classified as variation.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Incidence of malformed fetuses, one (1/225) -mandible short,fused
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations, incidence one (1/110) mandible short
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations: incidence one (1/113) -absent kidney, unilateral
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:Evaluation of foetusesNo adverse effect of the treatment on the foetal body weight was noted at any of the dose level. The mean weight of foetuses at 100, 300 and 1000 mg/kg/day, both litter means and mean foetus weights were slightly higher than the control mean, by approximately 4-7% and the differences were regarded as incidental. No significant differences in body weight were observed between male and female foetuses at the same dose level. External examinationThere was no foetal external malformation ascribed to the test item administration. One external malformed foetus was found in the control group in the form of short mandible (1512/9 male foetus). This malformation was confirmed during the skeletal examination.Visceral examinationThere were no foetal visceral abnormalities ascribed to the test item administration. The number and percentages of the minor structural changes (variations) recorded in the test item treated groups did not differ significantly from the control group. The variations included for example short brachiocephalic trunk, small renal papilla and unilaterally malpositioned kidney.These variations occurred with low incidence across the groups and were similar to the laboratory Historical control data and were considered incidental, unrelated to test item administration. Three malformed foetuses were found at the visceral examination, one/group in Mid, High dose and in the control groups. At 300 mg/kg/day total situs inversus was found (3515/3 male foetus), at 1000 mg/kg/day malpositioned stomach, pancreas, spleen and oesophagus (4518/9 male foetus) were noted, while in the control absent kidney, unilateral (1502/11 female foetus) was found. Based on the isolated occurrence, observations on transposed/malpositioned organs were considered incidental, ascribed to individual variability and not related to treatment. Transposition of organs spontaneously occurs in Wistar rats and total situs inversus is listed in Historical control data of this laboratory. Skeletal examinationNo skeletal abnormalities that could be correlated with test item administration under the conditions of this study were noted at any of dose levels. The number and percentages of the abnormalities recorded in the test item treated groups was comparable to the control group. Four skeletally malformed foetuses were found during the skeletal examination, two in the Mid dose, one in the High dose and one in the control group. At 1000 mg/kg bw/day, in one foetus (4502/8 female) malformation of vertebra (split TH XIII vertebral body with branched 13th rib at right) was noted. At 300 mg/kg bw/day, in one foetus (3518/2 female, developmentally retarded, of low body weight 2.59g) malformation of vertebra (TH XI hemivertebra and misaligned TH X, XII) was noted and split sternum in was found in another litter (3517/9 female). These findings were not considered test item related, since both type of malformations spontaneously occur in Wistar rats. Split sternum and malformation of vertebra (hemivertebra) are listed in Historical control data of Wistar rats in this laboratory.Short, fused mandible was observed in one control foetus (1512/9) already classified as malformed at external examination. At 100 mg/kg bw/day, no skeletally malformed foetus was found. Minor changes (variations) occurred, including for example in skull bones or ossification of sternal bodies, dumbbell shaped and/or asymmetric, bipartite vertebral bodies, observed with low incidence throughout all groups including controls, or with lower incidence in the treated groups than in the controls.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Summary of analytical results of the dosing formulations

Analytical occasion

Nominal concentration mg/mL

Measured concentrations with the 95% confidence intervals, mg/mL

Measured concentration in percentage of the nominal

17 November 2014

10

9.07±0.26

91%

30

31.1± 2.05

104%

100

96± 3.29

96%

08 December 2014

10

9.63± 0.51

96%

30

32.9± 0.38

110%

100

105± 1.02

105%

 

Summary of pregnancy data

Parameters

Dose Groups (mg/kg/day)

Vehicle Control

100

300

1000

Treated females

24

24

25

25

Non-pregnant

1

5

1

0

Females with complete intrauterine loss

1

0

0

0

Dams with≤5 implantations

0

0

0

1

Dams evaluated

22

19

24

24

 

Incidence of skin lesions through experimental groups, (in bracket, duration of the observation is presented)

 

Dose Groups (mg/kg/day)

Vehicle Control

100

300

1000

Evaluated animals

Incidence

2/22

3/19

2/24

3/24

Thin fur/alopecia (focal)

1503 (GD 19-20)

1518 (GD 0-20)

2503 (GD 16-20)

2512 (GD 4-20)

2517 (GD 3-24)

3508 (GD 6-20)

3521 (GD 8-14)

4508 (GD 3-13)

4509 (GD 3-20)

4514 (GD 0-4)

Isolated scar/crust

-

2517 (GD 0-3)

-

4503 (GD 20)

Excluded animals

Incidence

0/2

1/5

0/1

0/1

Alopecia (focal)

-

145 (D 5-20)

-

-

 

Mean values of intrauterine mortality (litter means) and mean number of viable fetuses

 

Dose Groups (mg/kg/day)

 

Vehicle Control (n=22)

100 (n=19)

300 (n=24)

1000 (n=24)

Corpora lutea (Mean)

12.2

11.7

12.0

12.5

NS

Implantation (Mean)

11.0

10.0

11.1

11.0

NS

Pre-implantation loss (%)

9.6

14.1

7.0

11.9

NS

Post-implantation loss (%)

7.1

11.4

7.2

4.9

NS

Early embryotic loss (%)

6.6

9.8

4.0

3.8

NS

Late embryotic loss (%)

0.4

1.6

0.9

1.1

NS

Total intrauterine mortality (%)

16.1

23.9

11.7

15.9

NS

Mean number of fetuses

10.2

8.8*

10.6

10.5

DN

NS = not significant

 

Incidence of malformed fetuses and type of malformations

 

Dose Groups (mg/kg bw/day)

Vehicle Control

100

300

1000

External malformations:

1/225

0/168

0/254

0/253

-mandible short

1*

0

0

0

Visceral malformations:

1/113

0/85

1/126

1/126

-absent kidney, unilateral

1

0

0

0

-situs inversus total

0

0

1

0

-malpositioned stomach, pancreas, spleen and oesophagus

0

0

0

1

Skeletal malformations:

1/110

0/83

2/128

1/127

-malformed vertebra (split)

0

0

0

1

-malformed vertebrae, retarded

0

0

1

0

-split sternum

0

0

1

0

-mandible short, fused

1*

0

0

0

*Malformation observed in one fetus.

Conclusions:
In conclusion, administration of Green liquor sludge (GLS) to pregnant Hannover Wistar rats by oral gavage daily from gestation days GD 6 to 19 at dose levels of 100, 300 and 1000 mg/kg/day was not associated with maternal or developmental toxicity.Based on the results the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 1000 mg/kg/day.
Executive summary:

The objective of the study was to assess the effects of the test item on pregnant females and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism. The study was performed in the rat, which is the preferred rodent species. One control and 3 test item-treated groups of Hannover Wistar rats were treated daily by oral (gavage) administration. The study was performed in accordance with OECD Guidelines for Testing Chemicals, No.: 414, Prenatal Developmental Toxicity Study, adopted 22ndJanuary 2001.

 

This developmental toxicity study was performed to assess the effects of the test item Green liquor sludge (GLS) on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control group and 3 treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The dose levels were 100, 300 and 1000 mg/kg/day and were set by the Sponsor in consultation with the Study Director based on available data including the results of a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Control dams were treated with vehicle aqueous 1 % Methylcellulose solution only.

Treatment solutions were analyzed for test item concentration twice during the treatment period (during the first and last weeks of treatment), using a flame photometry method. All formulations were within the range of 91 to 110% of nominal concentration and were homogenous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes.

Parameters monitored during the study were mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically.

The number of confirmed pregnant, evaluated dams in the dose groups treated at 100 mg/kg/day was 19, at 300 and 1000 mg/kg/day was 24 in each of group and 22 in the control group.

Results

There was no mortality during the study.

There were no toxicologically significant or test-item related clinical signs noted following administration of test item.

No effect on body weight was noted in any of the test item treated groups.

No effect was observed on food consumption.

No test item related macroscopic observations were made at necropsy on GD20.

There were no toxicological significant differences in intrauterine mortality between test item treated and control females.

The mean number of corpora lutea and implantation sites was comparable with the controls in all treated groups.

The mean number of viable foetuses was comparable with the control mean.

There were no toxicological significant differences in the placenta at evaluation of the treated groups compared to controls.

No adverse effect was noted on weight of foetuses in any of the test item treated groups. The incidence of retarded foetuses was comparable to control.

There was no foetal external malformation ascribed to the test item administration. One external malformed foetus was found in the control group (short mandible).

Three malformed foetuses were found at visceral examination: one in high dose (malpositioned stomach, pancreas, spleen and oesophagus), one in mid dose level (total situs inversus) and one in the control group (unilateral absence of kidney). The incidence of malformations was within the expected range based on historic data. Based on the isolated occurrence, these observations were considered incidental, ascribed to individual variability and not related to treatment.

There were no foetal skeletal abnormalities ascribed to the test item administration.

Four skeletally malformed foetuses were found: one in high dose (split TH XIII vertebra), two in mid dose (one retarded foetus with malformation of vertebrae and one with split sternum) and one in control group (short, fused mandible, already listed during external examination). These findings were considered not to be treatment related, since these types of malformation have commonly been observed in Wistar rats and the incidence is within the expected range based on historic data.

 

Conclusion 

In conclusion, administration of Green liquor sludge (GLS) to pregnant Hannover Wistar rats by oral gavage daily from gestation days GD 6 to 19 at dose levels of 100, 300 and 1000 mg/kg/day was not associated with maternal or developmental toxicity.

There were no external, visceral or skeletal malformations ascribed to treatment with test item.

At 1000 mg/kg/day, two malformed foetuses were found (one with malformed vertebra and one with malpositioned stomach, pancreas, spleen and oesophagus). As the incidence of malformations was similar to the control and Historic control data, this finding was regarded as incidental.  

At 300 mg/kg/day, three malformed foetuses were noted (one retarded foetus with malformation of vertebrae, one with split sternum and one showing total situs inversus). These findings were not considered test item related, since both type of malformations spontaneously occur in Wistar rats and are consistent with Historical control data in this laboratory. 

At 100 mg/kg bw/day, no malformed foetus was found.

Based on the above results the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Additional information

The objective of the study was to assess the effects of the test item on pregnant females and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism. The study was performed in the rat, which is the preferred rodent species.

One control and 3 test item-treated groups of Hannover Wistar rats were treated daily by oral (gavage) administration. The study was performed in accordance with OECD Guidelines for Testing Chemicals, No.: 414, Prenatal Developmental Toxicity Study, adopted 22ndJanuary 2001. This developmental toxicity study was performed to assess the effects of the test item Green liquor sludge (GLS) on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy.

The dams (one control group and 3 treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20. The dose levels were 100, 300 and 1000 mg/kg/day and were set by the Sponsor in consultation with the Study Director based on available data including the results of a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Control dams were treated with vehicle aqueous 1 % Methylcellulose solution only. Treatment solutions were analyzed for test item concentration twice during the treatment period (during the first and last weeks of treatment), using a flame photometry method. All formulations were within the range of 91 to 110% of nominal concentration and were homogenous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes. Parameters monitored during the study were mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically. The number of confirmed pregnant, evaluated dams in the dose groups treated at 100 mg/kg/day was 19, at 300 and 1000 mg/kg/day was 24 in each of group and 22 in the control group.

Results

There was no mortality during the study. There were no toxicologically significant or test-item related clinical signs noted following administration of test item. No effect on body weight was noted in any of the test item treated groups. No effect was observed on food consumption. No test item related macroscopic observations were made at necropsy on GD20. There were no toxicological significant differences in intrauterine mortality between test item treated and control females. The mean number of corpora lutea and implantation sites was comparable with the controls in all treated groups. The mean number of viable foetuses was comparable with the control mean. There were no toxicological significant differences in the placenta at evaluation of the treated groups compared to controls. No adverse effect was noted on weight of foetuses in any of the test item treated groups. The incidence of retarded foetuses was comparable to control. There was no foetal external malformation ascribed to the test item administration. One external malformed foetus was found in the control group (short mandible). Three malformed foetuses were found at visceral examination: one in high dose (malpositioned stomach, pancreas, spleen and oesophagus), one in mid dose level (total situs inversus) and one in the control group (unilateral absence of kidney). The incidence of malformations was within the expected range based on historic data. Based on the isolated occurrence, these observations were considered incidental, ascribed to individual variability and not related to treatment. There were no foetal skeletal abnormalities ascribed to the test item administration. Four skeletally malformed foetuses were found: one in high dose (split TH XIII vertebra), two in mid dose (one retarded foetus with malformation of vertebrae and one with split sternum) and one in control group (short, fused mandible, already listed during external examination). These findings were considered not to be treatment related, since these types of malformation have commonly been observed in Wistar rats and the incidence is within the expected range based on historic data.

Conclusion

In conclusion, administration of Green liquor sludge (GLS) to pregnant Hannover Wistar rats by oral gavage daily from gestation days GD 6 to 19 at dose levels of 100, 300 and 1000 mg/kg/day was not associated with maternal or developmental toxicity. There were no external, visceral or skeletal malformations ascribed to treatment with test item. At 1000 mg/kg/day, two malformed foetuses were found (one with malformed vertebra and one with malpositioned stomach, pancreas, spleen and oesophagus). As the incidence of malformations was similar to the control and Historic control data, this finding was regarded as incidental. At 300 mg/kg/day, three malformed foetuses were noted (one retarded foetus with malformation of vertebrae, one with split sternum and one showing total situs inversus). These findings were not considered test item related, since both type of malformations spontaneously occur in Wistar rats and are consistent with Historical control data in this laboratory. At 100 mg/kg bw/day, no malformed foetus was found. Based on the above results the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 1000 (dwt.) mg/kg/day.

Justification for classification or non-classification

Results obtained from Screening Tests (e.g. OECD 422 Combined Repeated Dose Toxicity Study with Reproduction/Development Toxicity Screening Test) can also be used to justify classification, although it is recognised that the quality of this evidence is less reliable than that obtained through full studies.

In OECD 422 test, rat pregnancy, birth, peri- and postnatal period were studied for a Green liquor sludge sample.  Repeated dose toxicity test using oral administration were performed in rats. The results showed no indication for an effect of the test substance on the reproduction.

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

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

Toxicity to reproduction

As no toxic effects on reproduction in OECD 422 study was seen, according to CLP Regulation 1272/2008, GLS needs not to be classified for Reproductive Toxicity. Conclusion: No classification for reproductive toxicity

Developmental toxicity

In the valid experiment (OECD 422) there was no indication of an effect of the test substance on development of the offsprings and there was no pronounced sex difference in the response to the test substance exposure.

The valid experiment (OECD 414) developmental toxicity study no effects were noted within the study at NOAEL = 1000 mg/kg/day; the highest dose tested.

The above results triggered no classification under the CLP Regulation (EC No 1272/2008).

Conclusion: No classification for developmental toxicity

Additional information

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