Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There were no effects of treatment on mating performance, fertility or the post natal survival, development or growth of the offspring.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16005P040.
- Expiration date of the lot/batch: 4 December 2017
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST (Han Wistar) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Strain/Species RccHan™:WIST rat.
Supplier Envigo RMS Limited.
Number of animals ordered 44 males and 48 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: six days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment.
Age of the animals at the start of treatment: Males: 85 to 92 days old. Females: 99 to 106 days old.
Weight range of the animals at the start of treatment: Males: 301 to 345 g. Females: 195 to 230 g.
Diet: ad libitum
Water: ad libitum

Allocation and Identification
Allocation: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and
body weights were reviewed before dosing commenced by Study Management to ensure variations in body weight of animals did not exceed +- 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.


DETAILS OF FOOD AND WATER QUALITY:
Diet Supply
Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. A sample of each batch of diet used was retained until finalization of the report.
Availability: Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).
Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
PREPARATION OF DOSING SOLUTIONS:
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE
- Justification for use and choice of vehicle: corn oil was a suitable vehicle for the test material (non water-soluble)
- Volume dose: 4 mL/kg body weight.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
The homogeneity and stability of formulations between 5 and 200 mg/mL during storage were determined as part of another study, Envigo Study Number: JN37SD.

Achieved concentration:
Samples of each formulation prepared for administration in Week 1 of treatment (all groups) and on Day 12 of lactation (females only) were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.
Frequency of treatment:
once daily
Details on study schedule:
Males Two weeks before pairing up to necropsy after minimum of five weeks. Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation (termination on Day 14 of lactation).
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels are based on the results of a 2-week preliminary toxicity study in the Han Wistar rat (Envigo Study No. KQ28DL). On that study, 600 mg/kg/day led to body weight loss, and the three males and three females on study were killed for that reason on Day 9 of treatment, since the dose would have been too high for longer administration.
Macroscopic examination of animals that received 600 mg/kg/day revealed dark areas on the stomach/non glandular mucosa of the stomach, thickening of the forestomach and various abdominal adhesions between organs/tissues, and clear liquid within the abdomen. There was also a low incidence of depression(s) on the antrum/corpus of the stomach. All females had pale jejunum and one female had multiple abdominal masses. A dose of 200 mg/kg/day was tolerated, however macroscopic examination following 14 days of treatment at 200 mg/kg/day revealed thickened fore stomach for 3/4 males and 4/4 females. No other treatment related findings were observed at macroscopic examination. The significance of the forestomach thickening in the 200 mg/kg/day group together with the more severe changes at 600 mg/kg/day were discussed with the Director of Pathology. It was concluded that it was unlikely that the longer duration of treatment of both sexes at 200 mg/kg/day required on this study would result in the more severe macroscopic changes observed at 600 mg/kg/day after nine days of treatment. This conclusion was based on the absence of marked clinical signs and stomach depressions/dark areas following 14 days of treatment at 200 mg/kg/day with the only change seen being forestomach thickening.
200 mg/kg/day were therefore selected as the high dose for this study and, with a low dose of 20 mg/kg/day and an intermediate dose of 60 mg/kg/day.
- Rationale for animal assignment: random
- Section schedule rationale: random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 - daily, Week 2 to 4 - twice weekly (middle and end of each week), Week 5 - once each week
F0 females: Week 1 - daily, Week 2 - twice weekly (middle and end of the week), Gestation phase - Days 0, 7, 14 and 20,Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, One to two hours after completion of dosing all groups, As late as possible in the working day

Detailed physical examination and arena observations:
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation (See Section 4), detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”. After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- The weight of animals was recorded as follows:
F0 males: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0)
and weekly thereafter. On the day of necropsy.
F0 females: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0)
and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7, 13 and 14 of lactation. On the day of necropsy. Animals were deprived of food, body
weight was recorded on the day prior to necropsy (before food withdrawal).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows: F0 animals Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording: Days 0-6, 7-13 and 14-19 after mating Days 1-3 , 4-6 and 7-12 of lactation. From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OTHER:

HAEMATOLOGY
Blood samples were collected after overnight withdrawal of food at the following occasion: At termination. The five lowest numbered surviving males per group and the first five lactating females per group. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
* Derived values calculated in ClinAxys
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of Prothrombin time (PT) - using IL PT Fibrinogen reagent - and activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY
Blood samples were collected after overnight withdrawal of food at the following occasion: At termination. The five lowest numbered surviving males per group and the first five lactating females per group. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyltranspeptidase (gGT)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

NEUROBEHAVIOURAL EXAMINATION
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”. After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on theseindividual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced
across the groups as far as possible on each day of testing. The following measurements, reflexes and responses were recorded. Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, Grip strength.
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
- Dry smears: For 15 days before pairing, using cotton swabs.
- Wet smears: Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 11 to 14 of lactation).
Sperm parameters (parental animals):
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age. A check was performed to assess for the presence or absence of nipple/areolae for the male offspring. There were no counts and consequently no data are presented in this report.
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring
on Days 1, 4 (before and after blood sampling) and 13 of age.
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.
The following were calculated for each litter:
Post-implantation survival index, Live birth index, Viability index, Lactation index

Premature deaths: Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
F1 offspring on Day 13 of age Blood sampling required. All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Thyroid glands were preserved from one male and one female in each litter.
Postmortem examinations (parental animals):
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ
and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy:
- F0 males: During Week 5 investigations completed.
- F0 females failing to produce a viable litter: Day 25 after mating.
- F0 females whose litter died before Day 13: On or after day the last offspring died.
- F0 females: Day 14 of lactation.
F1 offspring: Day 13 of age.

Pathology procedures for first five males surviving to scheduled termination and first five lactating females with a surviving litter per group at scheduled termination:
- The following organs were weighed: Adrenals, Brain (including cerebrum, cerebellum and pons), Epididymides, Heart (including auricular and ventricular regions), Kidneys, Liver (section from two lobes), Ovaries, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid + parathyroid (Weighed after partial fixation), Uterus with cervix.
- The following tissue samples were fixed: Abnormalities, Adrenals, Aorta, Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femur (with knee joint, and marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Lacrimal glands, Larynx, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (left axillary and mesenteric), Nose (nasal cavity), Pancreas, Pharynx, Preputial/clitoral gland, Optic nerve, Ovaries, Oviducts, Peyer’s Patch, Pituitary, Prostate, Rectum, Salivary glands (sublingual and mandibular), Sciatic nerve, Seminal vesicles with coagulating glands, Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina
- Histology was conducted on the following tissue samples: Abnormalities, Adrenals, Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Eyes, Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (left axillary and mesenteric), Preputial/clitoral gland, Optic nerve, Ovaries, Oviducts, Peyer’s Patch, Prostate, Rectum, Sciatic nerve (Only one examined), Seminal vesicles with coagulating glands, Skeletal muscle (Only one examined), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina.
- Light microscopy was conducted on the following tissue samples: Abnormalities, Adrenals, Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Eyes, Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (left axillary and mesenteric), Preputial/clitoral gland, Optic nerve, Ovaries, Oviducts, Peyer’s Patch, Prostate, Rectum, Sciatic nerve (Only one examined), Seminal vesicles with coagulating glands, Skeletal muscle (Only one examined), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina.

Pathology procedures for remaining males surviving to scheduled termination and remaining lactating females with a surviving litter per group at scheduled termination, decedent females plus any males that failed to sire a litter or which died before mating:
- The following organs were weighed: Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid + parathyroid (Weighed after partial fixation), Uterus with cervix.
- The following tissue samples were fixed: Abnormalities, Adrenals, Aorta, Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femur (with knee joint, and marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Lacrimal glands, Larynx, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (left axillary and mesenteric), Nose (nasal cavity), Pancreas, Pharynx, Preputial/clitoral gland, Optic nerve, Ovaries, Oviducts, Peyer’s Patch, Pituitary, Prostate, Rectum, Salivary glands (sublingual and mandibular), Sciatic nerve, Seminal vesicles with coagulating glands, Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina
- Histology was conducted on the following tissue samples: Abnormalities, Preputial/clitoral gland, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Uterus with cervix, Vagina.
- Light microscopy was conducted on the following tissue samples: Abnormalities, Preputial/clitoral gland, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Uterus with cervix, Vagina.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre-coital interval and stage of estrous cycle at termination the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
Pre-Coital Interval: Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating.
Mating Performance and Fertility: Percentage mating, Conception rate, Fertility index.
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
Offspring viability indices:
Post-implantation survival index, Live birth index, Viability index, Lactation index.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Signs seen in association with dosing were limited to only one single male (4M 7) receiving Laromer BDDA at 200 mg/kg/day showing salivation on Day 13 of treatment and are therefore most probably a randomly occurring non-substance related effect.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, bodyweight gain of males during the first week of treatment was statistically significantly lower than that of the controls. Afterwards, overall bodyweight gain of these males was similar to that of the controls.This effect was not considered adverse. Due to the corrosive effect of the test substance, it is assumed by the registrant that the rats had to get accustomed to the respective substance in the beginning of the study before they started to gain weight normally. Since the body weight of the males is higher than that of the females, they received a more concentrated solution, which would also explain the lack of effects in females. Furthermore p-values were not adjusted and therefore there is a small probability, that significant differences were simple coincidence.
At 20 or 60 mg/kg/day, overall bodyweight gain of males was similar to that of the controls.
The body weight and body weight change of females receiving Laromer BDDA at 20, 60 or 200 mg/kg/day was similar to that of the control group prior to pairing, and throughout gestation and lactation.
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:
effects observed, treatment-related
Description (incidence and severity):
Haematocrit and haemoglobin concentrations, reticulocyte counts, mean cell volume, and monocyte counts were significantly increased for males receiving Laromer BDDA at 200 mg/kg/day, when compared with the control animals. Monocyte counts were also significantly higher in mid and low dose animals.
Large unstained cell counts were significantly lower (p<0.05) for females receiving Laromer BDDA at 200 mg/kg/day when compared with the control.
Taken together these effects might be related to the also observed elevated potassium, calcium and phosphorus blood levels and are potentially caused by an imbalanced water intake.
Prothrombin time was significantly shorter (p<0.05) for males receiving Laromer BDDA at 200 mg/kg/day.
These effects were not considered adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations at scheduled termination revealed the following:
Bile acid, urea, potassium, calcium, phophorus, and albumin concentrations were significantly increased for males receiving Laromer BDDA at 200 mg/kg/day.
Glucose concentrations were significantly lower for mid and high dose males, although a dose related trend was not apparent.

Liver weights of test group receiving 200 mg test substance/kg bw/day were significantly higher compared to the control group. Bile acids, urea and albumin are predominantly formed in the liver. Also the liver is a driving factor in the regulation of glucose blood levels. Hence significant changes in blood levels are probably a secondary effect of an increased liver metabolism and not considered adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs seen at detailed physical examination and arena observations considered associated with dosing Laromer BDDA at 20, 60 or 200 mg/kg/day.
Sensory reactivity and group mean forelimb and hindlimb grip strength scores for males and females receiving Laromer BDDA at 20, 60 or 200 mg/kg/day were similar to the scores for males and females in the control group.
For males receiving Laromer BDDA at 200 mg/kg/day, several of the group mean low beam activity scores were low compared with the control animals, including the total score. Statistical significance was attained at the 12-minute time interval and this score is below the Historical Control Data (HCD) minimum. However, the control score at the same time interval is above the HCD maximum, so this difference from controls has contributed to the statistical significance that was attained. Some of the other 6-minute interval low beam scores for males in this group are also below the HCD range, including the total score, however, none of these scores have attained statistical significance. It is considered that these low scores are not a genuine treatment related effect. Compared with the HCD, many of the 6-minute interval scores in the other groups, including the controls, are below the HCD minimum. A similar pattern is seen in the high beam scores with the total score for group 4 males below the HCD range, but no statistical significance has been attained. There are also some scores that are below the HCD range throughout the rest of the groups, but again, this includes the controls. Activity scores for females were similar to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Squamous cell hyperplasia and hyperkeratosis were present in the nonglandular region of the stomach of all animals given 200 mg/kg/day and most animals given 60 mg/kg/day. Squamous cell hyperplasia appeared as a diffuse thickening of the epithelium with papillary projections and exaggerated rete peg formation. Hyperkeratosis presented as an increase in the thickness of the keratin layer lining the luminal surface of the epithelium and was orthokeratotic in nature. Focal regions of mucosal ulceration were present in the nonglandular region of the stomach in three females given 200 mg/kg/day and two males given 60 mg/kg/day. In these animals, there was typically an inflammatory reaction in the submucosa, while in others, submucosal inflammation was present without ulceration. In one male, submucosal inflammation was associated with minimal submucosal haemorrhage.
The test substance is corrosive. Hence hyperplasia and keratosis observed are most probably secondary effects of the corrosive nature of the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no clear effect of treatment with Laromer BDDA at 20, 60 or 200 mg/kg/day on the thyroid hormone levels of adult males at termination or F1 offspring on Day 13 of age. The 200 mg/kg/day values for males was heavily influenced by one grossly atypical value. In addition, the control group mean was lower than the mean in the Historical Control Data as was the mean for the control female offspring.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females showed normal estrous cycles before treatment. Estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment with Laromer BDDA. All females were not cycling before termination (Days 11-14 of lactation), and were in dioestrous at termination.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in the offspring considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter size, offspring survival and sex ratio were unaffected by treatment at 20, 60 or 200 mg/kg/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Offspring body weight and body weight change for males and females receiving Laromer BDDA at 20, 60 or 200 mg/kg/day was similar to concurrent Control group animals from Days 1-13 of age.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of parental treatment with Laromer BDDA on the ano-genital distance of male or female offspring on Day 1 of age.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Administration of BDDA was associated with test item-related changes in the nonglandular region of the stomach of all animals given 200 mg/kg/day. These changes consisted predominantly of hyperkeratosis and squamous cell hyperplasia, with additional mucosal ulceration, submucosal inflammation and haemorrhage in some animals. Similar changes were present in selected animals given 60 mg/kg/day where microscopic examination was performed. The changes were generally of lower severity. Examination of further animals from intermediate dose treatment groups could be undertaken to determine a No Observed Effect Level. The changes observed in the stomach were considered to be adaptive owing to an irritant effect of BDDA. Despite the changes in the stomach (which were considered adaptive), it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity was 200 mg/kg/day. There were no effects of treatment on mating performance, fertility or the post natal survival, development or growth of the offspring. The NOAEL for reproductive/developmental toxicity was also concluded to be 200 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

Toxicity to reproduction: other studies

Additional information

In an OECD 422 study, three groups of ten male and ten female rats received Laromer BDDA at doses of 20, 60 or 200 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. 

There were no adverse systemic effects associated with the administration of Laromer BDDA up to the highest dose in the parental or F1 generation, but there were treatment related macroscopic and microscopic pathology changes in the stomach in the parents due to local irritation at 60 and 200mg/kg. Consequently, the NOAEL for systemic toxicity, mating performance, fertility, and development of the offspring was 200 mg/kg/day. The NOAEL for local effects was 20 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. There were no effects on reproductive performance and fertility. As a result the substance is not classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.

Additional information