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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:
4.3.-20.5.2015
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, potassium salts- Physical state: solid flakes- Composition of test material, percentage of components: main component: 68514-28-3 63% (w/w) impurities: clay minerals 10% (w/w)- Lot/batch No.: 7.01.2015- 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, potassium salts
- Physical state: solid flakes
- Composition of test material, percentage of components
main component: 68514-28-3 63% (w/w)
impurities: clay minerals 10% (w/w)
- Lot/batch No.: 7.01.2015
- 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 - sterilized LIGNOCEL (raw material - spruce)
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 (polycarbonate cages TECNIPLAST, type 1291H for rats)

- 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: 04.03.2015
Acclimatisation: at least 5 days
Mating: 17.03.2015 – 30.03.2015
Start of administration: 23.03.2015
End of administration: 18.04.2015
Clinical observation: 23.03. – 18.04.2015
Necropsies: 07.04. – 19.04.2015
Microscopical examination: 27.04. – 20.05.2015
Evaluation of results and final report elaboration: 30.06. – 22. 10. 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua pro iniectione
Details on exposure:
PREPARATION 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 (ST1); sterilised before using
Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Storage temperature of food: laboratory conditions

VEHICLE
Aqua pro iniectione
- 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. (if required):
Batch No.: 1406100325, Exp: 06/2016
Batch No.: 1501210043, Exp: 01/2017
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. 26/07/18: Humic acids, potassium salts – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 09156, 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
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
Description (incidence and severity):
No test substance related changes or abnormalities in clinical signs occurred in any group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significantly decreased body weight of females at the middle dose from 14th day to 20th day of pregnancy (related to lower body increment) was detected. The body weight of the females at the low and at the highest doses was similar to control.
Food efficiency:
not examined
Description (incidence and severity):
Food consumption was examined only - decreased food consumption was recorded in females of the middle dose levels (correlation with decreased mean body increment).
Mean food consumption in other treated groups was similar compared to control group for the whole study.
Ophthalmological findings:
not examined
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
Description (incidence and severity):
The absolute and relative weight of uterus was decreased at the middle dose (related to lower body weight) but without statistically significant differences. Corrected body weight of females (the necropsy body weight of female minus weight of uterus) was statistically significantly decreased at the middle dose (related to lower mean body weight of females and mean uterus weight).
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):
Number of females with implantation sites was sufficient and similar in all groups.
The number of non-pregnant females after mating was 8-6-8-8. One female at the middle group aborted. Number of treated females with foetuses on the 20th day of pregnancy was similar to control (17-19-16-17).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of implantations and corpora lutea was statistical significant decreased at the middle group.
Preimplantation losses (IUDE) were increased at the middle dose level (18.02 % versus 13.84 % in control).
Postimplantation losses (IUDL) were increased at the middle dose (11.66 %) in comparison with control (8.38 %).
The influence of the treatment on maternal animals could be more likely spontaneous than caused by the test substance treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The mean number of resorptions was decreased at the all three doses.
The influence of the treatment on maternal animals could be more likely spontaneous than caused by the test substance treatment.
Early or late resorptions:
effects observed, non-treatment-related
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 changes in health conditions status, no pathological findings, no dose related changes in reproduction parameters

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in mean body weights of foetuses were observed.
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): No statistically significant changes in mean body weights of foetuses were observed.
Although the decrease in mean body weight of foetuses at the highest dose was observed it did not reach limit 0.15 – 0.25 times range (US EPA, 1991) against the control foetus to evaluation of teratogenic effect of the test substance.
Males were heavier than females in all group (include control).
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The total number and the mean number of live foetuses in litters was decreased at the middle group compared to control with statistically significant difference.
The variations in litter size are spontaneous and not correlated with treatment.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The total number and the mean number of live foetuses in litters was decreased at the middle group compared to control with statistically significant difference.
The variations in litter size are spontaneous and not correlated with treatment.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
no effects

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 and no serious structural abnormality

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Although in prenatal developmental study the changed reproduction parameters at the middle dose were observed (decreased number of implantations, corpora lutea and related increased preimplantation and postimplantation losses, sporadic occurrence of late resorptions and one aborted female). These findings could be more likely spontaneous than caused by the test substance treatment.
Examination of foetal skeleton indicated delayed development of the skeleton at all doses including control group.
It appears that the delayed development of foetuses in this study was not induced by the test substance treatment and could be connected with low individual body weight of foetuses.
The structural changes (bipartite ossification of supraoccipital bone and misaligned sternebrae) were recorded also in control or sporadic (misshapen humerus in one foetus at the low dose).
The occurrence of structural skeletal variations 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 changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.
Executive summary:

Introduction

The test substance, Humic acids, potassium salts was tested for prenatal developmental toxicity using the Method 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 100 g of body weight. 

The dose levels for study – 250, 500 and 1000 mg/kg/day – have been chosen with respect tothe informationgiven in the Study No. 26/07/18:Humic acids, potassium salts – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No.09156, 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 animals and reproduction parameters:

No deaths occurred in any group and there were no test substance related changes or abnormalities in clinical signs.

The statistically significantly decreased body weight of females at the middle dose from 14thday to 20thday of pregnancy (related to lower body increment) was detected. The body weight of the females at the low and at the highest doses was similar to control. The decreased food consumption at the middle dose was related to lower body weight at the middle dose. No changed health condition of females was detected. No female with pathological findings was observed during necropsy.

Number of females with implantation sites was sufficient and similar in all groups.The absolute and relative weight of uterus was decreased at the middle dose(related to lower body weight) but withoutstatistically significant differences.Corrected body weight of females (the necropsy body weight of female minus weight of uterus) was statistically significantly decreased at the middle dose (related to lower mean body weight of females and mean uterus weight).

Number of females with implantation sites was sufficient and similar in all groups. One female at the middle dose aborted. The examination of reproductive parameters revealedthe statistically significant decreased number of implantations and corpora luteaat the middle dose in comparison with control group. Decreased number of resorptions at all dose levels was recorded. The preimplantation losses(Intra Uterine Death Early)and postimplantation losses(Intra Uterine Death Late) wereincreased at the middle dose level.

These 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 and the mean number of live foetuses in litters was decreased at the middle group compared to control with statistically significant difference. The sex ratio was well balanced. The variations in litter size is considered as not correlated with treatment.

Development of foetuses in uterus was assessed according to the results of weighing, detailed necropsy and skeletal examination of foetuses. No statistically significant changes in mean body weights of foetuses were observed. Males were heavier than females in all group (include control). 

No macroscopic changes of soft tissues and external alteration were found out.

One malformation (absent lumbar vertebrae) was found only in two foetuses from one litter in control group.

Structural skeletal variationswere detected without treatment relation. Themisaligned sternebrae were recorded only in litters in control and at the low dose. Supernumerary ribswere recorded in litters at all doses. The wavy ribs (undulation along the length of a rib) were observed in two foetus at the middle dose.The occurrence of structural changes mentioned above could be considered as accidental.

Incomplete ossification of cranial bones (total) was detected in all groups. The increased number of foetuses with decreased number of ossification sites of sternum was recorded also in litters of all groups.The incomplete ossification of sacral vertebrae was recorded in one litter at the middle and highest dose.Thesefindings were not related to the treatment and probably related with foetal body weight.

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

Examination of foetal skeleton indicated delayed development of the skeleton at all doses including control group.Delayed development of the skeleton can be associated with low individual body weights of some foetuses.

Conclusion

Although in prenatal developmental study the changed reproduction parameters at the middle dose were observed (decreased number of implantations, corpora lutea and related increased preimplantation and postimplantation losses, sporadic occurrence of late resorptions and one aborted female). These findings could be more likely spontaneous than caused by the test substance treatment. 

Examination of foetal skeleton indicated delayed development of the skeleton at all doses including control group.

It appears that the delayed development of foetuses in this study was not induced by the test substance treatment and could be connected with low individual body weight of foetuses. 

The structural changes (bipartite ossification of supraoccipital bone and misaligned sternebrae) were recorded also in control or sporadic (misshapen humerus in one foetus at the low dose).

The occurrence of structural skeletal variations 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 changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.