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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 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

Test material

Constituent 1
Reference substance name:
Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.
EC Number:
923-511-9
IUPAC Name:
Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.
Test material form:
other: solid (similar to modeling clay when un-dried)
Details on test material:
Name: Green liquor sludge (GLS)REACH name: Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycleEC number: 923-511-9Batch number: Green liquor sludge, slamfilter, 2014-07-14—2014-07-18Appearance: Black (un-dried), light grey (dried) solid (similar to modeling clay when un-dried)Purity: 46.5% (The remaining 53.5% is water)Manufacturer: Södra Cell AB, Skogsudden, SwedenManufacture date: 14 July 2014 – 18 July 2014 (sampling period; collect sample)Expiry/Retest date: 30 September 2016Substance classification: Industrial chemicalComposition/Ingredients: Green liquor sludge is an inorganic UVCB (Unknown or Variable composition, Complex reaction products or Biological materials) substance composed of ash from the pulp mill's recovery boiler in combination with lime mud and contains carbon residues from black liquor incineration and minerals from cooking liquor and cellulosic raw material.Storage condition: Controlled room temperature (15-25oC, below 70 RH%), In closed container, under inert gas; Safety Precautions: Standard safety precautions for unknown materials (lab coat, safety glasses, mask and gloves) to ensure personnel health and safety.

Test animals

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.

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

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