Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.02.-01.06.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
- Name of test material: Humic acid, sodium salts- Physical state: solid flakes- Composition of test material, percentage of components: main component: 68131-04-4 68% (w/w) impurities: clay minerals 10% (w/w)- Lot/batch No.: 10.01.2015- Expiration date of the lot/batch: unlimited- Stability under test conditions: stable- Storage condition of test material: at laboratory conditions
Specific details on test material used for the study:
- Name of test material: Humic acid, sodium salts
- Physical state: solid flakes
- Composition of test material, percentage of components:
main component: 68131-04-4 68% (w/w)
impurities: clay minerals 10% (w/w)
- Lot/batch No.: 10.01.2015
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: stable
- Storage condition of test material: at laboratory conditions

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 11760500
- Age at study initiation: 11 weeks
-Sex: sexually adult females (males-only for mating)
- Fasting period before study: no
- Housing: plastic cages, sterilised clean shaving of soft wood
Before mating 2 rats of the same sex in one cage
During mating period – one male and two females in one cage were housed. Pregnant females were placed individually
- Diet: ad libitum, complete peleted diet for rats, Diet was sterilised before using.
- Water: ad libitum, water was sterilised before using
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

STUDY TIME SCHEDULE
Test substance delivery:14. 01.2015
Date of animal arrival: 18.02.2015
Acclimatisation: at least 5 days
Mating: 24.02.2015-04.03.2015
Start of administration: 02.03.2015
End of administration: 23.03.2015
Clinical observation: 02.03.-23.03.2015
Necropsies: 17.03.-24.03. 2015
Microscopical examination: 20.05.-01.06.2015
Evaluation of results and final report elaboration: 01.06-22.10. 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua pro iniectione
Details on exposure:
PPREPARATION OF DOSING SOLUTIONS: 1 mL per 100 g of body weight, prepared daily just before administration, laboratory conditions,
DIET PREPARATION
- Type of food: peleted diet for rats and mice in SPF breeding
- Composition of food: Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex

VEHICLE
Aqua pro injectione
- Concentration in vehicle: Two concentrations of application form were prepared (10 mg/10 mL and 1000 mg/10 mL).
- Amount of vehicle (if gavage): The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
- Lot/batch no.:
Batch No.: 1406100325, Exp: 06/2016
Manufacturer: ARDEAPHARMA a.s., Ševětín, Czech Republic
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined in CETA analytical laboratories (Physical and Chemical Testing Group I).Test substance stability and homogeneity were determined on the basis of an absorbance of a water solution (the application form) of the test substance. A spectrophotometric method developed and validated at the test facility was used.The application form was prepared by mixing with aqua pro iniectione. Two concentrations of application form were prepared (10 mg/10 mL and 1000 mg/10 mL).
Details on mating procedure:
MATING
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/2 female
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear - referred to as day 0 of pregnancy
Duration of treatment / exposure:
5th-19th day after fertilisation
Frequency of treatment:
7 days per week at the same time
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study have been chosen with respect to the information given in the Study No. 27/07/18: Humic acids, sodium salts – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 09152, 2009).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
MORTALITY CONTROL: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
FOOD CONSUMPTION: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus
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 viable foteus: Yes
- Number of dead foteus: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes, all per litter - sex, body weights, symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla

- Soft tissue examinations: Yes, half per litter - placing and morphology of organs, spinal cord, nasopharyngeal tract, thyroid gland, thymus, trachea, esophagus, diaphragm, adrenal gland, great vessels, veins, arteries- Skeletal examinations: Yes, half per litter- scull, clavicle, scapula, sternebrae, sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb and fingers

- Skeletal examinations: yes, half per litter - scull, clavicle, scapula, sternebrae, sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb and fingers
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 are indicated in the summary tables.
Indices:
Preimplantation loss, postimplatation loss were calculated from number of implantations, corpora lutea and resorptions

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
The number of non pregnant females after mating was 6-6-5-5. Treated females from all doses aborted (0-1-3-2). Number of females with foetuses on the 20th day of pregnancy (19-18-17-18) was well-balanced to control.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The changed reproduction parameters (decreased number of implantations and corpora lutea at the highest dose level) were recorded but without statistical significance and the test substance did not influence early intrauterine death (even preimplantation loss was decreased at all dose levels).
Increased postimplantation losses at all dose levels (mainly at the middle dose) and the occurrence of aborted females (0-1-3-2) in treated doses is questionable and despite not changed most of other indicator of maternal toxicity this changes are more likely spontaneous than caused by the test substance treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Females with resorptions were recorded in all treated group as well as in control group.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No death of foetuses was recorded in any litter.
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:
no effects

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no mortality, no statistically significant decreased body weight, no changes in health condition status, no pathological findings, no changes in reproduction parameters

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean number of foetuses was well balanced with control and at administered dams the mean body weight of foetuses was not influenced by the test substance treatment. Males were heavier than females in all group (include control).

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean number of foetuses was well balanced with control and at administered dams the mean body weight of foetuses was not influenced by the test substance treatment. Males were heavier than females in all group (include control).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of foetuses was decreased at the highest group compared to control group (without statistically significant difference). Mean number of foetuses in litters of treated mothers was well-balanced.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The number of female foetuses was lower than of males. Sex ratio was well-balanced in all groups.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of foetuses was decreased at the highest group compared to control group (without statistically significant difference). Mean number of foetuses in litters of treated mothers was well-balanced.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic changes of soft tissues were found out. But one external alteration (after birth tissue and skin adherent to trunk in limb region) were recorded in four foetuses at the lowest level and in four foetuses at the highest dose level. This alteration was probably accidental than treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal variations (misaligned sternebrae, supernumerary ribs) were detected at all doses including control group. The occurrence of wavy ribs was observed at four foetuses at the low and middle dose level.
Examination of foetal skeleton indicated delayed development of the skeleton at all doses including control group. These findings were not related to the treatment.
Delayed development of the skeleton can be associated with low individual body weights of some foetuses.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
For further detail see below attached document with tables of results.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no altered growth, no serious structural abnormality of treated foetuses

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Although the increased number of aborted females, increased number of resorptions and related postimplantation losses were recorded in treated females, most of other indicator of maternal toxicity (mortality of females, statistically significant decreased body weight of females, changes in health condition status, pathological findings in the dams, increased early intrauterine death) were not observed and this changes are more likely spontaneous than caused by the test substance treatment. The sporadic incidence of malformation and occurrence of variations also in control group could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus. Causes of their occurrence are questionable and they can depend on many factors (one of them can have genetic background). The NOAEL (No Observed Adverse Effect Level) for prenatal development is higher than 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality of treated foetuses.The NOAEL (No Observed Adverse Effect Level) for toxicity in pregnant females is higher than 1000 mg/kg/day. This NOAEL value is based on no mortality of females, no statistically significant decreased body weight of females, no changes in health condition status, no pathological findings in the dams, no changes in reproduction parameters.
Executive summary:

Introduction

The test substance, Humic acids, sodium salts was tested for prenatal developmental toxicity using theMethod B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008.

Study performance

Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then administered to pregnant females - daily from the 5thto the 19thday of pregnancy. The study included four groups of females – 3 treated groups and 1 control group (vehicle only). The test substance was administered suspended in water for injections by stomach tube and the concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per of body weight. 

The dose levels for study – 250, 500 and 1000 mg/kg/day, have been chosen with respect to the information given in the Study No. 27/07/18: Humic acids, sodium salts – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No.09152, 2009).

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the 20thday of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes.

Results

Maternal toxicity:

No mortality and no signs of toxicity were observed in the dams.

No changed health condition of females was detected. The statistically significantly changes in body weight were not found out. The biometry of uterus did not show negative influence of the test substance treatment on absolute and relative weight of uterus. Corrected body weight of females (the necropsy body weight of female minus weight of uterus) at all dose levels was not statistically significantly changed.

Reproduction parameters

The numbers of females which did not become pregnant were well-balanced to control group.

Number of females with implantation sites was sufficient and similar in all groups.

The examination of reproductive parameters revealed decreased number of implantations and corpora lutea at the highest dose level in comparison with control group, but the test substance did not influence early intrauterine death.

Increased number of resorptions, postimplantation loss (at all dose levels but mainly at the middle dose level) and highest number of aborted females at the middle dose level were observed compared to control group. 

The influence of the test substance on treated animals was not evident, so these mentioned findings could be more likely spontaneous than caused by the test substance treatment.

Foetuses:

No death of foetuses was recorded in any litter. The total number of foetuses was decreased at the highest group compared to control group (without statistically significant difference). Mean number of foetuses in litters of treated mothers was well-balanced. Sex ratio was well-balanced in all groups. The mean body weights of foetuses from treated females were well-balanced with control foetuses. Males were heavier than females in all groups (include control). No macroscopic changes of soft tissues were found out.

One external alteration (after birth tissue and skin adherent to trunk in limb region) was recorded in four foetuses at the lowest level and in four foetuses at the highest dose level. The only one malformation (absence lumbar vertebrae) was recorded in one foetus at the middle dose. This malformation was probably not treatment related due to extremely rare occurrence and absence dose-response curve.

Skeletal variations (misaligned sternebrae, supernumerary ribs) were detected at all doses including control group.The occurrence of wavy ribs was observed at four foetuses at the low and middle dose level.

Examination of foetal skeleton indicated delayed development of the skeleton at all doses including control group. These findings were not related to the treatment.

Delayed development of the skeleton can be associated with low individual body weights of some foetuses.

The cranium incomplete ossification of parietal bone, interparietal bone and their combination, interparietal bone with supraoccipital bone, interparietal bone in combination with parietal and supraoccipital bone was found out in all groups (including control). On the contrary some incomplete ossifications in cranium (supraoccipital bone and combination of incomplete ossification of parietal, interparietal, supraoccipital bone and arcus zygomaticus) were observed at all doses but not in control (slight increased occurrence in litters at the middle dose).

Total portion of litters with incomplete ossification of cranium (total) in comparison with control (78.95 %) was higher at the middle dose (88.24 %) and the lowest dose (94.44 %) and decreased at the highest dose (66.67 %).

The number of foetuses with decreased number of ossification sites of sternum was recorded also in all groups (percentual portion of litters was increased at the middle (76.47 %) and at the highest dose level (66.67 %) versus control (57.89 %).

The incomplete ossification of sacral vertebrae was recorded mostly in control.The incomplete ossification of lumbar vertebrae was recorded only in one foetus of middle dose level. The vertebrae variation (supernumerary lumbar vertebrae) was observed only in one foetus at the highest dose. Skeletal variations wavy ribs were observed only in low (16.67 % of litters) and middle dose (17.65 %). The supernumerary floating ribs were observed in all doses including control group. These findings are probably spontaneous not related to the treatment.

No sex-related difference was found in the effects at any of skeletal area.

Conclusion

Although the increased number of aborted females, increased number of resorptions and related postimplantation losses were recorded in treated females, most of other indicator of maternal toxicity (mortality of females, statistically significant decreased body weight of females, changes in health condition status, pathological findings in the dams, increased early intrauterine death) were not observed and this changes are more likely spontaneous than caused by the test substance treatment.

 

The sporadic incidence of malformation and occurrence of variations also in control group could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus. Causes of their occurrence are questionable and they can depend on many factors (one of them can have genetic background).

 

 

The NOAEL (No Observed Adverse Effect Level) for is higher than 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality of treated foetuses.

 

The NOAEL (No Observed Adverse Effect Level) fortoxicity in PREGNANT FEMALES is higher than 1000mg/kg/day.This NOAEL value is based on no mortality of females, no statistically significant decreased body weight of females, no changes in health condition status, no pathological findings in the dams, no changes in reproduction parameters.