Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for reproductive toxicity was concluded to be greater than 1000 mg/kg bw/day (the highest dose tested; equivalent to 800 mg/kg bw/day active acid) based on no adverse effects (Harlan Laboratories, 2013a).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The in-life phase of the study was conducted between 13 June 2012 (first day of treatment) and 04 August 2012 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 306 to 351g, the females weighed 189 to 219g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C
- Humidity (%): The relative humidity control was set to achieve target values of 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water. Formulations were prepared fortnightly and stored at approximately 4ºC in the dark.

All formulation were adjusted for 10.95% water content. All dose levels of expressed in terms of Active Ingredient

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken and analysed for concentration of test item.

The concentration of test item in the test item formulations was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using an external standard technique.

The results indicate that the prepared formulations were within ± 3% of the nominal concentration.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation).

Frequency of treatment:

The test item was administered daily.

Details on study schedule:

Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

low dose group (active ingredient)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Remarks:

middle dose group (active ingredient)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose group (active ingredient)

No. of animals per sex per dose:

10 males and 10 females per dose (including control)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were chosen based on the results of a 7-day range-finding study.

Positive control:

No positive control.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
- Motor Activity
- Forelimb/Hindlimb Grip Strength
- Sensory Reactivity


BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

REPRODUCTIVE SCREENING:
Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

Oestrous cyclicity (parental animals):

During mating a vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

Sperm parameters (parental animals):

During histopathology, the male testes and epididymides were examined.

Litter observations:

Litter Data:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

All tissues were despatched to the histology processing Test Site for processing.
Microscopic examination was conducted by the Study Pathologist

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.

Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Reproductive indices:

Mating Performance and Fertility:

The following parameters were calculated from the individual data during the mating period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated ÷ Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females ÷ Number of animals mated) x 100

Gestation and Parturition Data :
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring ÷ Number of pregnant females) x 100

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were detected in any treated animal.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in body weight development.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any significant intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the haematological parameters examined.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences. There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related microscopic findings were detected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on mating performance. One female treated with 100 mg/kg bw/day. was non-pregnant. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of this missing pregnancy. There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 23½ days.
In total nine females from control and 100 mg/kg bw/day dose groups and ten females from 350 and 1000 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
No significant differences were detected for corpora lutea, implantation counts for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
Pre and post implantation losses were increased in all treated groups when compared to control females. Statistical analysis of the data did not reveal any significant intergroup differences and a true dose related response was not evident. The number of corpora lutea, implantation sites and number of offspring born were also comparable to control females therefore the intergroup differences were considered not to be of toxicological importance.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related effects detected in the reproductive parameters observed.

Remarks on result:

other: Equivalent to 800 mg/kg bw/day active acid.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There were no adverse systemic toxicity effects observed.

Remarks on result:

other: Equivalent to 800 mg/kg bw/day active acid.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant differences were detected for litter viability for treated animals when compared to controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected. No significant differences were detected for litter size for treated animals when compared to controls.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects on offspring observed.

Remarks on result:

other: Equivalent to 800 mg/kg bw/day active acid

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for reproductive toxicity was concluded to be greater than 1000 mg/kg bw/day (the highest dose tested) based on no adverse systemic effects

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test for HEBMP-xNa, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for systemic and reproductive toxicity was concluded to be greater than 1000 mg/kg bw/day (the highest dose tested) based on no adverse effects (Harlan Laboratories, 2013a).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 1000 mg/kg bw/day active ingredient (adjusting for 10.95% water content). A control group of ten males and ten females was dosed with vehicle alone (distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male:one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post-partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study period. No clinical signs of toxicity were detected in any treated animal. There were no treatment-related changes in the behavioural parameters measured, in the functional performance test or in the sensory reactivity assessment. There were no toxicologically significant effects detected in body weight development, food or water consumption. There were no toxicologically significant effects detected in the haematological or blood chemical parameters examined. No toxicologically significant macroscopic abnormalities were detected or any changes in organ weights. Histopathology did not reveal any treatment-related findings.

There were no treatment-related effects on mating or fertility of the treated animals. There were no differences in gestation lengths and the distribution for treated females was comparable to controls. Litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. Sex ratio was also comparable to controls. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. Surface righting was also comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Effects on developmental toxicity

Description of key information

In the key pre-natal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal toxicity and developmental effects was concluded to be at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects in maternal animals and foetuses at any dose level (VUOS, 2021b).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/08/2020 - 12/11/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25th June 201

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The dose levels of the test item were recalculated on 100 % of active ingredient HEBMP-H (Total Active Acid).

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: 12 weeks old
- Weight at study initiation:
- Fasting period before study: not specified
- Housing: Before mating, 2 rats of the same sex were housed together in one cage; during mating period – one male and two females were housed together in one cage; pregnant females were housed individually during gestation.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice, ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 – 70 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua pro iniectione

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose levels of the test item HEBMP (1-3Na) were recalculated on 100 % of active ingredient HEBMP-H (Total Active Acid) for the main study. The application forms (the test item in Aqua pro iniectione) were prepared daily just before administration. The calculated volumes of the test item and vehicle were measured into a glass beaker. The application form was stirred by magnetic stirrer (250 rpm) for 30 minutes and then during the administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro iniectione. Due to it being an ionised substance, additional water was used as a vehicle.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 1mL per 100 g of body weight
- Lot/batch no. (if required): 2004010227

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the application form was checked by determination of a concentration P of the test item in three places of application form (at the bottom, in the middle and on the surface).
Stability of the application form was checked by analyses of the application form within 120 min (at the time 0, 30, 60, 90 and 120 min). Time interval 0 min represents for concentration 18.3 mL /100 mL the time after 30 minutes of mixing by magnetic stirrer (250 rpm) and for concentration 1.8 mL/100 mL the time after 30 minutes of mixing by magnetic stirrer (250 rpm).
the solution of the test item in vehicle at defined laboratory conditions is homogenous and stable at least for 120 minutes from a finalization of the application form preparation.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male : 2 females
- Length of cohabitation: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

from gestation day (GD) 5 to 19

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose (LD)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Remarks:

Middle Dose (MD)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose (HD)

No. of animals per sex per dose:

24 pregnant females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the oral (gavage) combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rat, conducted according to OECD Test Guideline 422 and in compliance with GLP (Harlan Laboratories, 2013a). The doses were also selected in order to align with the doses tested in the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP.
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were examined for vitality or mortality changes daily during the acclimatization, mating and pregnancy. The health condition was controlled daily during the acclimatization period, during the mating period and during pregnancy. During the administration period clinical observation was made in order to record possible clinical effects of the test item application and all changes in behaviour of females. Females were observed in natural conditions in their cages after application, once a day at the similar time each day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: First weighing was performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. Weight increments were computed as a mean per group (in grams).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was determined at three-day intervals; it coincided with the terms of body weight recording.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined. During macroscopic examination the thyroid glands were removed from all pregnant females and were preserved in fixation medium. The thyroid gland of control and high dose group females were examined at histopathology.

OTHER:
THYROID HORMONES: Yes
- Time schedule for examinations: The blood samples for this examination were taken from orbital plexus by glass micropipette under the light ether narcosis on the 20th day of pregnancy.
- Parameters: triiodothyronine (T3); thyroxine (T4); thyroid stimulating hormone (TSH).

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one half per litter]
- Skeletal examinations: Yes: [second half per litter]
- Head examinations: Yes (as part of skeletal examinations): [second half per litter]
- Anogenital distance: Yes
- Individual body weight: Yes
- Sex: Yes

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.

The parametric tests were used for statistical evaluation of:
• body weight of females
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses
• anogenital distance
• thyroid hormones
• biometry of thyroid gland (absolute and relative weight)
• biometry of uterus (absolute and relative weight)
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
• number of dead foetuses
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (skeletal foetal findings) were analyzed using the generalized linear mixed models with Poisson distribution.

Indices:

Preimplantation loss: Preimplantation loss = [(corpora lutea - implantations) / corpora lutea] x 100
Postimplantation loss: Postimplantation loss = (resorptions/implantations) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical changes indicating dysfunction of the organism were found in the control and treated groups during the whole study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No unscheduled death of maternal animals was recorded during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weights of treated maternal animals in all dose groups were comparable to the control group during whole study. No statistically significant changes were observed. The body weight increment was slightly decreased in the 350 mg/kg bw/day group and increased in the 100 mg/kg bw/day group in comparison with the control group. In the highest dose group body weight increment was comparable with the control group.
Corrected body weight of all treated females (the necropsy body weight of female minus weight of uterus) was not statistically significantly changed compared to the control females. Mean values of corrected body weight of treated females were similar to the control females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average food consumption of all treated groups was comparable with the control group during the whole study. Statistically significant differences were not detected.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus: The mean absolute and relative weights of the uterus were slightly increased in the 100 mg/kg bw/day group in comparison with control group. Statistically significant differences in uterus biometry were not detected in females of any group.
Thyroid gland: Absolute and relative weights of the thyroid glands were similar in the treated and control females. Statistically significant differences of thyroid weights were not detected in females of any dose level

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The uterus dilatation (the change probably related with oestrous cycle) was detected sporadically at the dose levels of 100 and 350 mg/kg bw/day. No finding related with treatment was noted at necropsy in treated females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid gland: Histological examination of thyroid glands revealed a spontaneous finding; a squamous cell cyst in one female in the 1000 mg/kg bw/day group. Because treatment-related changes were not recorded for the high dose group, histopathology of thyroid gland at doses 100 and 350 mg/kg bw/day was not performed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant differences were recorded in serum levels of thyroid hormones T3, T4 and TSH in females from treated groups against control females.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of implantations in treated groups were comparable with the control group. Preimplantation losses (IUDE) in all treated groups were similar or lower in comparison with control group. Postimplantation losses (IUDL) were increased in the 350 mg/kg bw/day group in comparison with control. Statistically significant differences were not detected in these all parameters.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of resorptions in treated groups were comparable with the control group.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One dead foetus was recorded in the control group and two dead fetuses in the 100 mg/kg bw/day group. No dead foetuses were recorded for the mid and high dose groups. The lowest number of foetuses was recorded for the 350 mg/kg bw/day group, because the lowest number of pregnant females was in this group. However, the average total number of foetuses per litter was comparable across the treated groups and control group.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of non-pregnant females was 4, 4, 8 and 4, respectively for the four groups, 0, 100, 350 and 1000 mg/kg bw/day. The highest number of non-pregnant females was recorded for the dose of 350 mg/kg bw/day. Sperms were present in the vaginal smears of the non-pregnant females; however, no implantations or resorptions were recorded after necropsy. The females were concluded not to have been fertilized/pregnant. The administration of the test item started from the 5th day of pregnancy. Therefore, the non-pregnant status of the females was not related to the test item treatment.

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea in treated groups were comparable with the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

active acid

Basis for effect level:

other: No adverse effects observed in maternal animals.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of foetuses was slightly decreased in the 350 mg/kg bw/day group compared to the control group, without statistical and toxicological significance. Male foetuses were heavier than females in all groups.

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):

One dead foetus was found in the control group and two in the the 100 mg/kg bw/day goup. The dead foetuses could not be examined due to autolysis. Foetuses without a tail were recorded sporadically for doses of 100 and 1000 mg/kg bw/day. These findings were not treatment related and were of spontaneous origin. No other external changes were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels as well as in the control group.
Statistical evaluation was performed for 'number of foetuses with skeletal findings‘ and also for 'number of litters with skeletal findings‘.
Incomplete ossification of foetal cranium was observed during examination in all groups including the control group. The delayed development in parietal bone, interparietal bone, supraoccipital bone was not related to the treatment with because the occurence of these findings in litters was high in all groups, including the control group. There was a statistically significantly increased number of foetuses with incomplete ossification of supraoccipital bone in the mid dose group, but in the case of conversion to litter no statistical significance was detected. The proportions of litters with incomplete ossification of the squamosal part of temporal bone were similar or lower in treated groups compared to the control group. The proportions of litters with incomplete ossification of nasal and frontal bones were statistically significantly increased in the mid dose in comparison with control litters. This delayed development could be associated with the lower foetal body weight that was recorded for the mid dose group. The proportions of litters with incomplete ossification of arcus zygomaticus (15.00 % – 25.00 % – 33.33 % – 11.11 %) was increased in the mid dose group, but without statistical significance and dose dependence. Litters with holes in the supraoccipital bone were also observed in high percentage in all treated groups as well as in the control group.

Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as the control group. It is normal biological variability for ossification of sternebra to be incomplete on the 20th day of pregnancy. These findings were not related to the treatment, because the occurence of these findings was also high in the control litters and dose dependence for these findings was not recorded. Bipartite ossification of ossification sites of sternebra was detected sporadically.

Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including the control group. The proportion of litters with bipartite ossification of vertebrae thoracic centrum was increased (close to statistical significance: Pvalue = 0.056) in the 1000 mg/kg bw/day group in comparison with control group (10.00 % – 25.00 % – 20.00 % – 50.00 %). This finding was also quantitatively compared with findings in the control group of other developmental study performed simultaneously at our facility. In this simultaneous study the incidence of bipartite ossification of vertebrae thoracic centrum was 21.05 % in control litters, which is also lower than the incidence in the high dose group (50.00 %) for the current study. This delayed vertebrae development is not related to the lower body weight of foetuses. The foetal body weight at 1000 mg/kg bw/day was comparable with foetal body weight in the control group. The bipartite ossification of vertebrae thoracic centrum is included in the Transitional Findings (grey zone). These may be upgraded to malformation or downgraded to variations status, depending on severity and/or frequency of occurrence. Ossification delay is a transient finding that may indicate a delayed schedule of events but does not indicate disrupted development. Changes in the size, shape, or symmetry of sternebrae or vertebral centra are transient and have no implications for the health or survival of the offspring .

Asymmetric ossification of vertebrae thoracic centrum was recorded sporadically in foetuses. The proportions of litters with dumbbell ossification of vertebrae thoracic centrum were similar or lower in treated groups than in the control group. Test item treatment relation was not evident for these findings associated with vertebrae.

Changes such as wavy ribs and ribs-supernumerary site were detected also in all groups. The proportion of litters with wavy ribs was similar or lower in treated groups than in the control group. These findings did not relate to treatment with the test item. The proportion of litters with ribs-supernumerary site was increased in the high dose group in comparison with controls (35.00 % – 35.00 % – 26.67 % – 50.00 %), this increase is not dose-dependent and moreover statistical evaluation revealed that it is non-significant. This finding was also quantitatively comparable with findings in the control group of other developmental study performed simultaneously at the test facility. In this simultaneous study the incidence of ribs-supernumerary site was 57.89 % in control litters, which is comparable with the incidence in the high dose group (50.00 %) recorded in the current study. This change is not considered to be adverse.

Incomplete ossification of scapula was recorded only in treated groups. The proportions of litters with scapula was 0.00 % – 15.00 % – 20.00 % – 11.11 %, respectively for the four groups. However, this finding was recorded in a relatively low number of foetuses in the treated groups (0 – 3 – 3 – 2). The occurence of this finding was sporadic. Test item treatment relation was not evident for this sporadic variation.

Visceral malformations:

no effects observed

Description (incidence and severity):

No adverse findings were observed in any of the test groups or the control group.

Other effects:

no effects observed

Description (incidence and severity):

The AGD and corrected AGD of male and female foetuses at all doses were comparable with control group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: scapule

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

- cranial bones: delayed development at all dose groups and the control, non-treatment related
- sternebra: delayed development at all dose groups and the control, non-treatment related
- vertebrae: dumbbell and bipartite ossification of vertebrae thoracic centrum all dose groups and the control, non-treatment related
- ribs: wavy ribs and ribs-supernumerary site in all groups, non-statistically significant compared to control, non-treatment related
- scapula: incomplete ossification in all dose groups with evidence for treatment relevance

Key result

Developmental effects observed:

no

TNotes for tables 20 - 23:          

i.o.      – incomplete ossification

u.        – unossified

d.o.     – dumbbell ossification

b.o.     – bipartite ossification

as.os.  – asymmetric ossification

able 1: Pregnancy results

Group code	Number of pregnant females	Number of pregnant females	Number of Non-pregnant females**
	Number of females with foetuses	Number of females without live foetuses but with implantations and resorptions**
0	20	0	4 (102,108,116, 122)
100	20	0	4 (127,128,130,134)
350	15	1 (164)	8 (152,154,155,156,157,158,163,169)
1000	20	0	4 (180,183,184,197)

Note:  **numbers in parentheses = individual labels of single animals.

Only the data from females with live foetuses were used for calculations of means. Data from females with uterus implantations were used for calculation of preimplantation (IUDE) and postimplantation (IUDL) losses.

Table 2: Body weight in grams (mean ± standard deviation) 

Day of pregnancy	Group	Group	Group	Group
	0	100	350	1000
1^stday	277.7± 14.9	277.8± 14.1	276.4 ± 17.1	273.8 ± 11.9
5^thday	296.9 ± 18.5	294.4± 16.4	291.6 ± 17.0	294.4 ± 14.4
8^thday	305.5 ± 16.8	304.9 ± 16.9	300.8 ± 18.8	305.6 ± 14.1
11^thday	322.8 ± 20.1	324.1 ± 19.6	316.6 ± 21.5	322.1 ± 16.3
14^thday	339.2 ± 19.1	342.0 ± 18.0	332.2 ± 23.1	340.6 ± 18.6
17^thday	376.5 ± 29.3	380.2 ± 21.6	365.4 ± 33.7	375.4 ± 25.9
20^thday	423.1 ± 40.1	433.8 ± 28.2	414.5 ± 41.8	419.3 ± 31.5
Mean increment	145.4	156.1	138.0	145.5

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 3: Food consumption /animal/day (grams)

Day of pregnancy	Group	Group	Group	Group
	0	100	350	1000
5^thday	24.01	23.22	21.87	22.48
8^thday	26.73	26.67	24.94	25.02
11^thday	26.75	28.28	26.63	27.00
14^thday	29.19	29.63	28.45	28.93
17^thday	29.22	30.38	28.54	28.69
20^thday	32.00	34.26	31.95	31.72

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 4: Clinical observation

Week of pregnancy	Group	Group	Group	Group
	0	100	350	1000
1^stweek	1	1	1	1
2^ndweek	1	1	1	1
3^rdweek	1	1	1	1

Note:1–no clinical signs of intoxication

Table 5: Biometry of uterus (mean ± standard deviation)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Mean necropsy body weight of females (g)	423.1 ± 40.1	433.8 ± 28.2	414.5 ± 41.8	419.3 ± 31.5
Mean absolute weight of uterus (g)	79.94 ± 27.21	90.54 ± 15.27	79.45 ± 26.61	78.09± 24.13
Mean relative weight of uterus (%)	18.56 ± 5.39	20.89 ± 3.38	18.81 ± 5.28	18.35 ± 5.08

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 6: Body weight - corrected* (mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Body weight(g)	343.2 ± 23.5	343.3 ± 27.9	335.0 ± 23.5	341.2 ± 17.4

Note:*body weight correction = necropsy body weight of female – weight of uterus

Statistically significant differences on probability level 0.05 were not detected.

Table 7: Macroscopic findings (number of females with pathological findings)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Number of examined females	24	24	24	24
Number of died females	0	0	0	0
Without pathological findings	24	24	24	24
Uterus: dilatation	0	1	1	0

Note:Uterus dilatation = probablynon-pathological finding.

Table 8: Parameters of reproduction (number per female, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
implantations	14.55± 4.22	16.65± 1.76	14.19± 5.87	14.70± 4.68
resorptions	0.70± 1.03	1.00± 2.10	0.75± 1.24	0.60± 0.88
corpora lutea	16.35± 2.32	17.45± 1.39	15.19± 5.00	15.75± 3.77

Table 9: IUDE and IUDL (% per female, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
IUDE	12.33± 20.42	4.61± 6.23	9.46± 16.96	10.01± 18.00
IUDL	5.71± 8.93	5.48± 10.86	9.23± 24.75	4.34± 7.05

Note: Statistically significant differences on probability level 0.05 were not detected.

The mean of preimplantation and postimplantation losses were calculated from individual data of females.

IUDE = Preimplantation losses

IUDL = Postimplantation losses

Table 10: Weight of thyroid gland (group mean ± SD)

Thyroid gland	Group	Group	Group	Group
	0	100	350	1000
Absolute weight (g)	0.0311 ± 0.0017	0.0305 ± 0.0012	0.0306 ± 0.0011	0.0308 ± 0.0013
Relative weight (%)	0.0074 ± 0.0008	0.0071 ± 0.0005	0.0075 ± 0.0008	0.0074 ± 0.0006

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 11: Pregnant females - Thyroid hormones (mean concentration ± SD)

Hormone	Group	Group	Group	Group
	0	100	350	1000
T3(ng/mL)	0.648 ± 0.056	0.652 ± 0.068	0.674 ± 0.085	0.623 ± 0.062
T4(µg%)	3.368 ± 0.525	3.271 ± 0.558	3.230 ± 0.428	3.086 ± 0.434
TSH(pg/mL)	809.5 ± 216.0	869.6 ± 223.5	776.2 ± 180.9	812.0 ± 225.0

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 12: Number of foetuses (total in group)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Total number of live foetuses	277	313	215	282
Number of live foetuses – males	142	162	112	141
Number of live foetuses – females	135	151	103	141
Number of dead foetuses	1	2	0	0

Table 13: Number of foetuses (average per litter; mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Total number of live foetuses	13.85 ± 4.56	15.65 ± 1.95	14.33 ± 5.25	14.10 ± 4.73
Number of live foetuses – males	7.10 ± 2.71	8.10 ± 1.97	7.47 ± 3.29	7.05 ± 2.61
Number of live foetuses – females	6.75 ± 3.34	7.55 ± 1.32	6.87 ± 2.88	7.05 ± 2.95
Number of dead foetuses	0.05 ± 0.22	0.10 ± 0.31	0.00 ± 0.00	0.00 ± 0.00

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 14: Body weight of foetuses (grams, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
weight of all foetues	3.80± 0.73	3.80± 0.74	3.57± 0.56	3.77± 1.12
weight of male foetus	3.89± 0.72	3.91± 0.76	3.68 ± 0.64	3.86± 1.16
weight of female foetus	3.70± 0.73	3.68± 0.74	3.46 ± 0.54	3.56± 0.99

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 15: Mean anogenital distance of foetuses (mm)

Group code	0	0	100	100	350	350	1000
Sex	Male	Female	Male	Female	Male	Female	Male	Female
Mean AGD	3.57	2.18	3.60	2.18	3.56	2.18	3.39	2.17
Corrected AGD	2.28	1.42	2.30	1.42	2.31	1.44	2.18	1.45

Note: Statistically significant differences on probability level 0.05 were not detected.

Table 16: External alterations - foetuses

Alteration	Group	Group	Group	Group
	0	100	350	1000
Total number of examined foetuses	277	313	215	282
Total number of examined litters	20	20	15	20
Dead foetus	1	2	0	0
Foetus without tail	0	1	0	1
Number of examined foetuses in litter (mean ± SD)	13.85 ±4.56	15.65 ±1.95	14.33 ±5.25	14.10 ±4.73
Total number of foetuses with alteration	1	3	0	1
Number of foetuses with alteration in litter(mean ± SD)	0.05 ±0.22	0.15 ±0.49	0.00 ±0.00	0.05 ±0.22
Proportion of foetuses with alteration in litter (% mean ± SD)	0.36 ±1.60	0.88 ±2.88	0.00 ±0.00	0.31 ±1.40

Table 17: Macroscopic changes of soft tissues – individual foetuses

Alteration	Group	Group	Group	Group
	0	100	350	1000
Total number of examined foetuses	129	147	100	130
Total number of examined litters	20	20	15	20
With pathological findings - total(number of affected foetuses)	0	0	0	0
Number of examined foetuses in litter (mean ± SD)	6.45 ±2.31	7.35 ±0.99	6.67 ±2.55	6.50 ±2.35
Total number of foetuses with alteration	0	0	0	0
Number of foetuses with alteration in litter (mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00
Proportion of foetuses with alteration in litter (% mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00

Table 18: Skeletal alterations (number of affected foetuses)

Group code	0	100	350	1000
Number of examined foetuses	148	165	112	128
CRANIUM
Nasal bone – i.o.	4	0	24	7
Frontal bone – i.o.	7	6	17	7
Parietal bone – i.o.	45	59	37	29
Interparietal bone–i.o.	64	92	69	56
Supraoccipital bone – i.o.	97	115	93	80
Supraoccipital bone – hole	29	28	18	38
Arcus zygomaticus–i.o.	4	7	8	4
Squamosal part of temporal bone – i.o.	12	18	15	8
Basisphenoid–i.o.	1	1	0	0
STERNEBRA
Sternebra – ossification sites – i.o.	137	144	106	120
Sternebra – ossification sites – u.	73	65	63	53
Sternebra – ossification sites – b.o.	0	0	0	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	2	11	3	14
Vertebrae thoracic centrum – b.o., as.os.	0	0	2	2
Vertebrae thoracic centrum – d.o.	36	58	33	59
RIBS
Ribs – supernumerary site	23	15	11	16
Ribs – wavy	8	9	2	3
Scapula – i.o.	0	3	3	2

Note:The resultsstatistically significantly changedon probability level 0.05 were shaded in the summary table

Table 19: Skeletal alterations (number of affected litters)

Group code	0	100	350	1000
Number of examined litters	20	20	15	18
CRANIUM
Nasal bone – i.o.	3	0	9	5
Frontal bone – i.o.	5	4	9	6
Parietal bone – i.o.	17	16	12	14
Interparietal bone–i.o.	17	19	14	16
Supraoccipital bone – i.o.	19	19	15	17
Supraoccipital bone – hole	15	16	10	13
Arcus zygomaticus–i.o.	3	5	5	2
Squamosal part of temporal bone – i.o.	7	6	6	5
Basisphenoid–i.o.	1	1	0	0
STERNEBRA
Sternebra – ossification sites – i.o.	20	20	15	18
Sternebra – ossification sites – u.	15	14	12	13
Sternebra – ossification sites – b.o.	0	0	0	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	2	5	3	9
Vertebrae thoracic centrum – b.o., as.os.	0	0	2	2
Vertebrae thoracic centrum – d.o.	15	17	10	16
RIBS
Ribs – supernumerary site	7	7	4	9
Ribs – wavy	6	7	1	3
Scapula – i.o.	0	3	3	2

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 20: Skeletal alterations (% proportion of litters with affected foetuses)

Group code	0	100	350	1000
Number of examined litters	20	20	15	18
Alteration
CRANIUM
Nasal bone – i.o.	15.00	0.00	60.00	27.78
Frontal bone – i.o.	25.00	20.00	60.00	33.33
Parietal bone – i.o.	85.00	80.00	80.00	77.78
Interparietal bone–i.o.	85.00	95.00	93.33	88.89
Supraoccipital bone – i.o.	95.00	95.00	100.00	94.44
Supraoccipital bone – hole	75.00	80.00	66.67	72.22
Arcus zygomaticus–i.o.	15.00	25.00	33.33	11.11
Squamosal part of temporal bone – i.o.	35.00	30.00	40.00	27.78
Basisphenoid–i.o.	5.00	5.00	0.00	0.00
STERNEBRA
Sternebra – ossification sites – i.o.	100.00	100.00	100.00	100.00
Sternebra – ossification sites – u.	75.00	70.00	80.00	72.22
Sternebra – ossification sites – b.o.	0.00	0.00	0.00	11.11
VERTEBRAE
Vertebrae thoracic centrum – b.o.	10.00	25.00	20.00	50.00
Vertebrae thoracic centrum – b.o., as.os.	0.00	0.00	13.33	11.11
Vertebrae thoracic centrum – d.o.	75.00	85.00	66.67	88.89
RIBS
Ribs – supernumerary site	35.00	35.00	26.67	50.00
Ribs – wavy	30.00	35.00	6.67	16.67
Scapula – i.o.	0.00	15.00	20.00	11.11

Table 21: Skeletal alterations (% proportion of affected foetuses in litter (mean ± SD)

Group code	0	100	350	1000
Number of examined foetuses	148	165	112	128
Number of examined litters	20	20	15	18
Alteration
CRANIUM
Nasal bone – i.o.	2.22 ± 5.81	0.00 ± 0.00	20.42 ± 26.05	5.38 ± 10.68
Frontal bone – i.o.	3.96  ± 7.53	3.58 ± 7.84	14.58 ± 16.56	5.62 ± 10.28
Parietal bone – i.o.	30.54 ± 23.12	34.63 ± 28.39	29.03 ± 23.15	23.52 ± 22.55
Interparietal bone–i.o.	40.74  ± 29.42	55.08 ± 30.42	63.17 ± 32.26	44.91 ± 32.78
Supraoccipital bone – i.o.	63.44  ± 27.67	69.44 ± 27.24	81.02 ± 20.60	59.56 ± 30.89
Supraoccipital bone – hole	21.74 ± 20.49	17.39 ± 14.06	14.88 ± 15.67	30.39 ± 26.96
Arcus zygomaticus–i.o.	2.29  ± 5.93	3.85 ± 7.26	6.74 ± 11.35	2.64 ± 9.01
Squamosal part of temporal bone – i.o.	7.08 ± 11.70	10.13 ± 18.18	12.22 ± 17.39	5.93 ± 12.66
Basisphenoid–i.o.	0.56 ± 2.48	0.56 ± 2.48	0.00 ± 0.00	0.00 ± 0.00
STERNEBRA
Sternebra – ossification sites – i.o.	93.42  ± 7.93	86.50 ± 20.63	95.49 ± 5.79	94.24 ± 12.56
Sternebra – ossification sites – u.	49.54 ± 36.67	40.32 ± 32.27	48.06 ± 34.69	37.48 ± 29.86
Sternebra – ossification sites – b.o.	0.00  ± 00.00	0.00  ± 00.00	0.00 ± 0.00	1.17 ± 3.42
VERTEBRAE
Vertebrae thoracic centrum – b.o.	1.11  ± 3.42	6.74 ± 15.38	2.36 ± 4.96	8.70 ± 10.04
Vertebrae thoracic centrum – b.o., as.os.	0.00 ± 0.00	0.00 ± 0.00	1.62 ± 4.35	1.41 ± 4.14
Vertebrae thoracic centrum – d.o.	25.07  ± 21.52	34.66 ± 25.34	29.43 ± 28.30	41.83 ± 28.89
RIBS
Ribs – supernumerary site	13.61 ± 23.68	8.79 ± 15.00	10.14 ± 19.36	10.58 ± 13.17
Ribs – wavy	5.10 ± 9.59	5.26 ± 7.74	1.67 ± 6.45	3.10 ± 8.38
Scapula – i.o.	0.00 ± 0.00	1.89  ± 4.66	3.12 ± 6.62	1.35 ± 3.99

Notes for tables 18 - 21:          

i.o. – incomplete ossification

u. – unossified

d.o. dumbbell ossification

b.o. - bipartite ossification

as.os. – asymmetric ossification

See attachment for individual animal data.

Conclusions:

In the pre-natal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal toxicity and developmental effects was concluded to be at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects in maternal animals and foetuses at any dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key pre-natal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, pregnant female Wistar rats were exposed daily by oral gavage to 0, 100, 350 or 1000 mg HEBMP active acid/kg bw/day in aqua pro iniectione from gestation day 5 to 19 (VUOS, 2021b). The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during the study. On gestation day (GD) 20 blood was collected from pregnant animals to determine thyroid hormone levels (T3, T4, TSH). Then the animals were euthanised and the uterine contents were examined. Thyroid glands were also removed from the females and a histological examination was performed. Following removal from the dams, the foetuses were weighed, examined for external malformations and the anogenital distances (AGD) were measured. Then a thorough examination of foetal soft tissues and skeletons was conducted.

There were no unscheduled deaths of maternal animals during the study at any dose level.

No adverse changes in health condition and no clinical symptoms of intoxication were observed in maternal animals following administration of the test item at any dose. Nor were there any toxicologically significant treatment-related effects on body weight or food consumption in maternal animals.

Evaluation of uterine weights (absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. The mean absolute and relative weights of uterus were slightly increased at 100 mg/kg bw/day in comparison with the control group, without statistical and toxicological significance.

Macroscopical structure of examined organs of pregnant females and values relating to parameters (number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test item. Post-implantation losses (IUDL) were increased in the 350 mg/kg bw/day group in comparison with control, but without statistical significance and without dose dependence. The increase was due to one female in this group, which became pregnant and then all implanted conceptuses in the uterus were totally resorbed (female No. 164 without foetuses but with implantations).

Examination of the thyroid glands (absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones) did not reveal any changes associated with the application of the test item. Histological examination of thyroid glands revealed squamous cell cyst in one female at 1000 mg/kg bw/day. This sporadic finding was probably of spontaneous origin.

Test item-related foetal mortality was not evident at any dose level. Detailed necropsy of foetuses did not reveal an increase of external and visceral variations and malformations at any dose level. During the pathological examination of external alterations, one dead foetus in the control group and two in the low dose group were recorded. One foetus without a tail was recorded in each of the low and high dose groups. These sporadic findings were not treatment related and were probably of spontaneous origin.

Based on statistical evaluation of mean values of foetal body weight, no significant growth retardation was detected in treated groups. The foetal body weight was slightly decreased in the mid dose group, but without statistical or toxicological significance.

The mean AGD and corrected AGD of male and female foetuses in treated groups were not statistically significantly different from the control group.

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all doses and in the control group. The delayed development of cranial bones and sternebra was not related to the treatment, because the occurrence of these findings was observed commonly also at a high percentage in control foetuses or these findings were recorded without dose dependence and toxicological significance.

Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including the control group. The proportion of litters with bipartite ossification of vertebrae thoracic centrum were increased at 1000 mg/kg bw/day in comparison with the control group (10.00 % – 25.00 % – 20.00 % – 50.00 %) on the edge of the statistical significance. The proportion of litters with dumbbell ossification of vertebrae thoracic centrum was similar or lower at the dose levels in comparison with the control group. Wavy ribs and ribs-supernumerary site were also detected in all groups. The proportion of litters with wavy ribs was similar or lower in all dose groups compared to the control group. The proportion of litters with ribs-supernumerary site was increased in the highest dose group in comparison with control (35.00 % – 35.00 % – 26.67 % – 50.00 %), without statistical significance. The proportions of litters with incomplete ossification of scapula were recorded only in treated groups.  However, this finding was recorded in a relatively low number of foetuses in the treated groups (0 – 3 – 3 – 2). Test item treatment relation was not evident for this sporadic variation.

The NOAEL for maternal toxicity and developmental effects was concluded to be at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects in maternal animals and foetuses at any dose level.

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test for HEBMP-xNa, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for systemic, reproductive and developmental toxicity was concluded to be greater than 1000 mg/kg bw/day (the highest dose tested) based on no adverse effects (Harlan Laboratories, 2013a).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 1000 mg/kg bw/day active ingredient (adjusting for 10.95% water content). A control group of ten males and ten females was dosed with vehicle alone (distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male:one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post-partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study period. No clinical signs of toxicity were detected in any treated animal. There were no treatment-related changes in the behavioural parameters measured, in the functional performance test or in the sensory reactivity assessment. There were no toxicologically significant effects detected in body weight development, food or water consumption. There were no toxicologically significant effects detected in the haematological or blood chemical parameters examined. No toxicologically significant macroscopic abnormalities were detected or any changes in organ weights. Histopathology did not reveal any treatment-related findings.

There were no treatment-related effects on mating or fertility of the treated animals. There were no differences in gestation lengths and the distribution for treated females was comparable to controls. Litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. Sex ratio was also comparable to controls. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. Surface righting was also comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Justification for classification or non-classification

Based on the available information, no classification is proposed for reproductive or developmental toxicity in accordance with Regulation (EC) No. 1272/2008.