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Effects on fertility

Description of key information

There are no reproductive toxicity data for HEBMP-H. Therefore, data are read-across from HEBMP-xNa.

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 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
800 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

There are no reproductive toxicity data for HEBMP-H. Therefore, data are read-across from HEBMP-xNa.

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test for HMDTMP-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; equivalent to 800 mg/kg bw/day active acid) 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

There are no prenatal developmental toxicity data for HEBMP-H. Therefore, data are read-across from HEBMP-xNa.

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 developmental 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).

In addition, in line with ECHA Final Decision No CCH-D-2114493149-38-01/F, there is an prenatal developmental toxicity study with HEBMP-xNa, conducted according to OECD Test Guideline 414 and in compliance with GLP. The study result will be submitted in December 2021.

Link to relevant study records
Reference
Endpoint:
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:
other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
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.
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.
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.
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.
Duration of test:
Up to eight weeks.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
350 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:
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.
Maternal 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)

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.

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





















Ovaries and uterine content:
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).
Fetal examinations:
Not examined.
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
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
Historical control data:
Normal range data for the different parameters examined were used in comparison against the test data.
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.

FUNCTIONAL PERFORMACE TEST:
There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related microscopic findings were detected.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
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.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
No significant differences were detected for litter viability for treated animals when compared to controls.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
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.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
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. 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.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on mating performance.
There were no treatment-related effects on fertility.
Dose descriptor:
NOAEL
Remarks:
maternal reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: other:
Remarks on result:
other: Equivalent to 800 mg/kg bw/day active acid.
Dose descriptor:
NOAEL
Remarks:
maternal systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
act. ingr.
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.
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant differences were detected for litter size for treated animals when compared to controls.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No significant differences were detected for litter viability for treated animals when compared to controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
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.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
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.
Changes in postnatal survival:
no effects observed
External malformations:
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.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
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 observed.
Remarks on result:
other: Equivalent to 800 mg/kg bw/day active acid.
Abnormalities:
no effects observed
Developmental 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 developmental toxicity was concluded to be 1000 mg/kg bw/day (the highest dose tested) based on no adverse effects.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 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

There are no prenatal developmental toxicity data for HEBMP-H. Therefore, data are read-across from HEBMP-xNa.

In the combined repeat dose toxicity study with reproduction/developmental toxicity screening test for HMDTMP-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; equivalent to 800 mg/kg bw/day active acid) 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 required for reproductive or developmental toxicity for HEBMP-H according to Regulation (EC) No 1272/2008.

Additional information