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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 January 2020 - 24 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Remarks:
GLP compliant, according to the Hungarian Good Laboratory Practice Regulations of 42/2014. (VIII. 19.) EMMI decree of the Ministry of Human Capacities

Test material

Constituent 1
Reference substance name:
The product from the burning of a combination of carbonaceous materials.
EC Number:
931-597-4
Molecular formula:
UVCB substance, not available. View remarks field.
IUPAC Name:
The product from the burning of a combination of carbonaceous materials.
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Mixture of fly and bottom ashes
- Physical state: solid, fine powder
- Composition range of test material (% DW): Aluminium (Al) 4.8, Calcium (Ca) 28.4, Iron (Fe) 2.1, Potassium (K) 1, Magnesium (Mg) 1.1, Sodium (Na) 0.58, Phosphorus (P) 0.42 , Silicon (Si) 11, Titanium (Ti) 0.26. Sulphur (S) 9250 mg/kg DW.
- The critical minor components examined (mg/kg d.w.): Arsenic, As 25, Cadmium, Cd 4,5, Cobalt, Co 13, Chromium, Cr 69, Copper, Cu 430, Manganese, Mn 1600, Molybdenum, Mo 6,0, Nickel, Ni 55, Lead, Pb 140, Antimony, Sb 15, Selenium, Se <3, Vanadium, V 47, Zinc, Zn 840, Barium, Ba 650
- Purity test date: the substance is UVCB substance
- Lot/batch No.: Mixed ash 11122018
- Other: Mixed Ash is named in dossier as Ash. Mixed in the substance name has meant that there has been several (mixed) fuels when producing ash.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Batch number 11122018
- Purity, including information on contaminants, isomers, etc.: 100%

RADIOLABELLING INFORMATION (if applicable): Not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C), protected from air (under argon or nitrogen)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
- Reactivity of the test material with the incubation material used (e.g. plastic ware):

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanWistar, Crl:WI (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, 11-12 weeks old at mating.
- Weight at study initiation: 191 - 254 g (the variation did not exceed ± 20% of the mean weight)
- Fasting period before study: None
- Housing: Standard laboratory conditions; individual housing
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM R/M-Z+H Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum.
- Water (e.g. ad libitum): The animals were provided with ssniff® SM R/M-Z+H Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum.
- Acclimation period: at least 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-24.0°C (target: 22 ± 3°C)
- Humidity (%): 26-66% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To: From 02 Jan 2020 (start of acclimation) to 13 Feb 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (1% aqueous methylcellulose solution) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of Charles River Laboratories Hungary Kft.
Formulations were be prepared fresh prior to administration to animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, 1% aqueous methylcellulose solution was selected as vehicle for this study in agreement with the Sponsor.
- Concentration in vehicle: 1%
- Lot/batch no. (if required): Dispense codes: S11172 / S11173
Details on analytical verification of doses or concentrations:
Analysis of the formulations for homogeneity and concentration of test item were performed using a validated gravimetric analysis method (Charles River Laboratories Hungary Kft. Study code: 18/288-316AN) in the Analytical Department of the Test Facility under GLP two times during the study.

Top, middle and bottom duplicate samples were taken from test item formulations for formulation analysis for homogeneity and concentration, one set (Set1G) to analyse (which was collected in replicates as practical) and two sets (Set2G and Set3G) as back-up, if required for any confirmatory analyses. Duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

This gravimetric formulation analysis was conducted under the control of the Contributing Scientist (at the Analytical Department of Charles River Laboratories
Hungary Kft.) in compliance with the relevant SOPs of the Test Facility. The results of the formulation analysis is included in Appendix 5.

Acceptance criteria for homogeneity was that the relative standard deviation (RSD) of the replicates must be less than 10%.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing, and then daily, up to the day of mating. On the first week of the mating, only half of the animals were examined daily for their oestrus cycles. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male:1-3 female) until 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 (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Randomisation

The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software (Provantis) was used to verify homogeneity/variation among/within groups.
Duration of treatment / exposure:
Start of treatment Gestation Day GD6
End of treatment Gestation Day GD19
Frequency of treatment:
daily by oral (gavage) administration
Duration of test:
07 January 2020 - 13 February 2020
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
100 female animals, 25 mated female animals/group, 4 groups (one control and 3 test item-treated groups);
22, 24, 20, 24 pregnant and evaluated female animals (with implantation sites at necropsy) per Control, Low, Mid and High dose groups, respectively;
56 male animals for mating;
no study-procedures were carried out on the male animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were set by the Sponsor in consultation with the Study Director, based on the available data and information from previous experimental work, including the results of a limit test in a dose range finding (DRF) study.
In the DRF study, no signs of systemic toxicity were observed at the limit dose of 1000 mg/kg bw/day. A statistically significant increase of the number of runts in the DRF study was considered to be incidental, and the top dose of 1000 mg/kg for the main study is considered appropriate. Based on this information, the doses of 1000, 300 and 100 mg/kg bw/day are deemed suitable for the purpose of the study. The oral route was selected as it is the most relevant route of human exposure.
- Rationale for animal assignment (if not random):
The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software (Provantis) was used to verify homogeneity/variation among/within groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones:
- Time of day for (rat) dam blood sampling: At termination

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made usually twice daily (at the beginning and end of each working day, or before treatment and when the peak of the clinical observations,
if any, is observed after treatment). Only one clinical observation was made in the afternoon on those days when detailed clinical observation is made in the morning.
When signs of toxicity were noted, animals might be observed more frequently.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD 6) 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. lachrymation,
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.

BODY WEIGHT: Yes
- Time schedule for examinations: The 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. Body weight gain were calculated for each interval, including GD0-6, GD6-20 and GD0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD6-10, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: dam's viscera, weight of the thyroid gland with parathyroid glands, ovaries, uterus, cervix, placentas, anogenital distance of each foetus, foetuse's thymus, great arteries and skeleton.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: Yes
- Serum: No
- Volume collected: volume target of at least 125 µL for the first aliquot and at least 75 µL
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Anogenital distance of all live rodent pups: The anogenital distance of each foetus was measured, and the sex of each assigned based on the distance. The sex of the foetuses was reconfirmed by examining the internal sex organs.
Statistics:
SPSS PC+4.0: The heterogeneity of variance between groups: Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test (for inter-group differences). Where significant heterogeneity was found, Kolmogorow-Smirnow test was used. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the were performed using Mann-Whitney U-test (inter-group comparisons). The Chi-squared test was used for non-continuous data.
SAS v9.2 (within Provantis): The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests. If no significant heterogeneity, an Anova/Ancova test was carried out. If the result was positive, Dunnett (Multiple Range) test was used for inter-group differences (<0.05 or <0.01). If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. In case of a positive result, the inter-group comparisons were performed using Dunn test (<0.05 or <0.01). For non-continuous data, the Cochran-Amitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. Non-pregnant females, females with no implantation or ≤ 5 implantation sites, and females showing signs of abnormal pathology and/or misdosing were excluded from selected statistical analysis; in-life individual data was reported as applicable.
The limit for growth-retarded foetuses (runt) was calculated from the average and standard deviation of the body weights of the vehicle control foetuses. A foetus was considered as growth retarded (runt) when their body weight is less than the control average – 2 standard deviations), by using the SAS v9.2.
Historical control data:
Historical control data is presented in the Appendix 9 HISTORICAL CONTROL DATA OF HANNOVER
WISTAR RATS of the attached study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Noisy respiration was observed in 1 out of 20 and 1 out of 24 animals in the Mid and High dose group. The finding appeared from Day 12 until Day 19; the symptom occurred for a maximum of eight days.

Piloerection was present in 1 out of 24 animals in the High group. The finding appeared from Day 12 until Day 19; the symptom occurred maximum eight days.

Red discharge around the right eye was observed in 1 out of 24 animals in the High dose group.

The clinical signs were attributed to local respiratory effect of test item (aspiration of a small amount of formulation), there was no evidence for systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related body weight decrease was observed in any of the dose groups.

No significantly lower or higher corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in any of the dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes in the food consumption was observed in any of the dose groups during the treatment period.
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. There were no statistical differences in the organ weights between the control and the Dose groups.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. The result of the examination showed that there was a significant difference between the control and the low dose group in the number of multinucleated giant cell (parathyroid, unilateral, minimal), but this finding was considered to be a normal background finding and not related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

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: Parameters according to OECD Test Guideline 414

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was statistically significant higher number of foetuses with retarded body weight
in the High dose group (p<0.05). However, the number of affected litters was identical
to the concurrent controls (and it is in line with Historic Data).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external variations in the test item treated groups. There was an incidental external malformation only in the High dose group. Absent of lower jaw (mandible) was observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Most of the skeletal findings correspond to the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response. Based on the skeletal findings the number of malformed / intact foetuses were comparable with the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups. The number of affected foetuses with variation was statistically significantly higher in the Mid dose group compared to the control group (p<0.05). However, all data was considered to be in the normal range and there was no dose related trend.

In the case of most of the skeletal variations, the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

Most of the findings correspond to the study control data or have isolated occurrence that were considered incidental ascribed to individual variability.

Based on these results the test item did not affect adversely the intrauterine development by higher incidence of malformations, there were no adverse effects on external, visceral or skeletal evaluated data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The findings correspond with the current historical control or the study control data or have isolated occurrence that was considered incidental, ascribed to individual variability and not related to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parameters according to OECD Test Guideline 414

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, Mixed Ash, when administered daily by oral gavage to pregnant
Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day
induced no maternal systemic toxicity and no effect on body weight or growth. No
effect on the embryotoxicity and foetotoxicity was observed (number of foetuses
with retarded body weight and malformations) in the study based on overall
development.
The following no-observed-adverse-effect (NOAEL) levels were derived:
NOAELmaternal toxicity: 1000 mg/kg bw/day
Based on no maternal systemic effects at 1000 mg/kg bw/day.
NOAELembryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intra-uterine effect in any treatment group.
NOAELfoetotoxicity: 1000 mg/kg bw/day
Based on no effect on runts at 1000 mg/kg bw/day.
NOAELteratogenecity: 1000 mg/kg bw/day
Based on the lack of treatment related malformations in any dose group.
Executive summary:

Mixed Ash, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced no maternal systemic toxicity and no effect on body weight or growth in a GLP-compliant OECD 414 Prenatal Developmental Toxicity test. No effect on the embryotoxicity and foetotoxicity was observed (number of foetuses with retarded body weight and malformations) in the study based on overall development. No mortality occurred during the study.


All test item formulations were within the range of 98-108% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples.


In the higher dose group, some symptoms were present. Noisy respiration was observed in 1 out of 20 and 1 out of 24 animals in the Mid and High dose group. The finding appeared from Day 12 until Day 19, the symptom occurred for a maximum of eight days.


Piloerection was present in 1 out of 24 animals in the High group. The finding appeared from Day 12 until Day 19, the symptom occurred for a maximum of eight days. Red discharge around right eye was observed in 1 out of 24 animals.


No test item related body weight effects were observed in any of the dose groups. No changes in the food consumption in any of the dose groups was observed during the treatment period. No test item related macroscopic and microscopic findings were present at necropsy in the surviving animals.


There were no statistically significant changes in the concentration of the T3, T4 and TSH level between the Control and the Dose groups. There were no statistical differences in the organ weights between the control and the Dose groups. There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls. There was no toxicologically significant difference in the foetal weights of either sex of foetuses, sex distribution or on anogenital distance between the control and treatment groups. There were no adverse test item related effects on external, visceral or skeletal development of foetuses in the study.


Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as a test item related effect.


The following no-observed-adverse-effect (NOAEL) levels were derived:


NOAELmaternal toxicity: 1000 mg/kg bw/day


Based on no maternal systemic effects at 1000 mg/kg bw/day.


NOAELembryotoxicity: 1000 mg/kg bw/day


Based on the lack of any test-item related intra-uterine effect in any treatment group.


NOAELfoetotoxicity: 1000 mg/kg bw/day


Based on no effect on runts at 1000 mg/kg bw/day.


NOAELteratogenecity: 1000 mg/kg bw/day


Based on the lack of treatment related malformations in any dose group.

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