Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Remarks:

source study on the analogous substance

Adequacy of study:

key study

Study period:

From May 04 May to November 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

Test animals

Species:

rat

Strain:

Sprague-Dawley

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 Crl:CD(SD) 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
- Source: Charles River (UK) Ltd.
- Age at study initiation: approx. 71 days old males; approx. 85 days old females.
- Weight at study initiation: 317 to 371 g males; 239 to 281 g females. Variations in body weight of animals did not exceed ± 20% of the mean for each sex.
- Housing: during pre-pairing up to five animals of one sex; during pairing one male and one female; after mating, males up to five animals; during gestation one female; during lactation one female + litter. Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum.
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, ad libitum.
- Acclimation period: males six days prior to the commencement of treatment; females 20 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber be dding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 ºC
- Humidity: 40 - 70 %
- Air changes: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ADMINISTRATION
- Volume dose: 10 ml/kg body weight.
- Individual dose volume: calculated from the most recently recorded scheduled body weight. A correction factor (including purity) of 1.165 was used when calculating quantities of test item to be used during dose preparation.
- Control (Group 1): vehicle at the same volume dose as treated groups.

PREPARATION OF DOSING SOLUTIONS
Method of preparation: the required amount of test item was weighed and approximately 50 to 60 % of the final volume of vehicle was added. It was magnetically stirred for a minimum of one hour to ensure accurate mixing. It was made up to the required volume with vehicle and mixed using a high shear homogenizer until homogenous. The suspension was transferred to the final containers, via syringe, whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

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 investigations demonstrated that formulations in the 1 to 100 mg/ml concentration range are stable following ambient storage (15-25 °C) for one day and following refrigerated storage (2-8 °C) for 15 days.
The homogeneity and stability of test item in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/ml and 100 mg/ml.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 4 % of the initial time zero value and the coefficient of variation was less than 2 %.
Recovery results during the trial remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method.

ACHIEVED CONCENTRATION
Samples of each formulation prepared for administration in Week1 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.
The mean concentrations of test item in test formulations analyzed during the study were within 5 %, confirming the accuracy of formulation, with the exception of Week 1, Group 4 (+12.0 %), this result was confirmed by the analysis of contingency samples and reported as the mean of four values (two original and two contingency). The difference from mean was within 4 %, confirming precise analysis.

Duration of treatment / exposure:

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.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 300 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories.
In the preliminary study dose levels of 250, 500 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.
The high dose level for the current OECD 422 study was set at 1000 mg/kg/day. The intermediate and low dose levels of 300 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS
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 severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule: F0 males daily during week 1, onwards, once each week, during week 2; F0 females daily during week 1 and once during week 2; during gestation phase, on days 0, 7, 14 and 20; during lactation phase on 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; as late as possible in the working day.

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.

BODY WEIGHT
The weight of animals was recorded as follows:
- F0 males weekly during acclimatization; before dosing on the day that treatment commenced (Day 1) and weekly thereafter; on the day of necropsy.
- F0 females: weekly during acclimatization; before dosing on the day that treatment commenced (Day 1) and weekly before pairing; days 0, 7, 14 and 20 after mating; day 1, 4, 7 and 13 of lactation; on the day of necropsy.

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows: F0 animals: weekly before pairing, from the day that treatment commenced; food consumption was not recorded for males and females during the period when paired for mating (Day 15-22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: 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 daily consumption per animal (g/animal/day) was calculated for each phase.

HAEMATOLOGY
Blood samples were collected after overnight withdrawal of food at the following occasion: at termination, the five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose 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.
- Parameters: 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).
- 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) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY
Blood samples were collected after overnight withdrawal of food at the following occasion: at termination, the five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose 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.
- Parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), 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), Globulin (Glob).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

THYROID HORMONES
Blood samples were collected as follows: at termination, in all adults; two females per litter (where possible).
Parameters: T4 (serum) and SH (plasma)
Sequence of blood sampling on each occasion: in order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: no overnight deprivation of food.
Anesthetic: F0 animals Isoflurane.

NEUROBEHAVIOURAL EXAMINATION
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 these individual 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.

PARTURITION OBSERVATIONS AND GESTATION LENGHT
Duration of gestation: time elapsing between the detection of mating and commencement of parturition.
Parturition observations: from Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

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.

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:

CLINICAL OBSERVATIONS
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

LITTER OBSERVATIONS
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

THYROID HORMONES
Blood samples were collected as follows: at day 13 of age in F1 offspring, two males and two females per litter (where possible).
No offspring were allocated to these procedures if the resultant live litter size would fall below ten offspring or if it would leave less than three females.
Parameters: T4 (serum) and SH (plasma)
Sequence of blood sampling on each occasion: in order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: no overnight deprivation of food.
Anesthetic: F1 offspring none.

Postmortem examinations (parental animals):

SACRIFICE
Carbon dioxide asphyxiation with subsequent exsanguination.
Time of Necropsy: F0 males after at least five weeks of treatment; F0 females failing to produce aviable litter on day 25 after mating; F0 females - on day 14 of lactation (following terminal blood sampling).

GROSS PATHOLOGY
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. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.
Organ weight: for bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled termination.
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below: testes, initially in modified Davidson’s fluid; eyes, in Davidson’s fluid.
Tissues: adrenals, brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum, jejunum, kdneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes - left axillary mmesenteric, ovaries, peyer’s Patch, prostate, rectum, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus with cervix (weighed with oviducts), vagina.

HISTOPATHOLOGY
Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 4 at scheduled termination were asssessed.
Abnormalities were checked in all F0 animals.
Tissues preserved for examination were examined as follows: scheduled kill, the five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 4; all F0 animals for abnormalities.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
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.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

SACRIFICE
Offspring selected for thyroid hormone sampling on Day 4 and 13 of age were decapitated; all other offsprings by intraperitoneal injection of sodium pentobarbitone.
Time of Necropsy: F1 offspring selected offspring for thyroid hormone analysis on day 4 of age; other offspring on day 13 of age.

GROSS NECROPSY
Premature deaths: where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.
F1 offspring on Day 4 of age: externally normal offspring discarded without examination. Externally abnormal offspring identified on dispatch to necropsy, an external macroscopic examination was performed and offspring retained pending possible future examination.
F1 offspring on Day 13 of age: all animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

Reproductive indices:

Mating Performance and Fertility: percentage mating; conception rate; fertility index
Gestation Length and 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Administration with test item at doses of 100, 300 and 1000 mg/kg/day during the 5-week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated. There were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment and no premature deaths occurred.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of animals receiving test item at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment throughout the 5 week dosing period for males, and throughout the 2-week pre-pairing period, gestation period and lactation period for females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption for all groups of animals receiving test item was similar to that of controls and no effect of treatment was inferred.
During Days 0-7 and Days 14-20 of gestation, the mean food intake of females given 300 or 1000 mg/kg/day was marginally higher than Control, with differences attaining statistical significance. There was no dose response apparent and the magnitude of the differences from Control was minor, therefore they were considered to be fortuitous.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of haematological parameters at the end of the treatment period did not reveal any adverse effects of treatment with test item.
All groups of treated males showed a slight but statistically significantly higher than Control mean cell haemoglobin concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day mean cell haemoglobin was slightly higher than Control, with statistical significance attained; this difference was attributable to a single female (No. 66) with an atypically high mean cell haemoglobin value and no effect of treatment was inferred. All other haematological differences from controls observed were minor, lacked dose relationship or occurred in one sex only and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biochemical changes in the plasma at scheduled termination which were attributable to treatment with test item.
Triglyceride concentrations in males receiving 300 mg/kg/day were higher than Control and sodium concentrations in males given 300 or 1000 mg/kg/day were slightly lower than Control; these differences attained statistical significance, however in the absence of a dose relationship were considered fortuitous and unrelated to test item administration. All other biochemical differences from controls were minor, lacked dose relationship or occurred in one sex only and were therefore attributed to normal biological variation.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity observations conducted during Week 5 of treatment for males and Days 7-9 of lactation for females revealed no findings which were considered treatment related.
Females in the 100 mg/kg/day group showed slightly increased grip strength compared to Controls, with statistical significance attained for hindlimb grip strength. In the absence of similar differences in the 300 or 1000 mg/kg/day groups, these differences were considered incidental and unrelated to treatment.
Motor activity scores for males and females given test item were considered to be unaffected by treatment.
Some minor differences from Control were evident in the high and low beam scores, in terms of the individual 6-minute recording periods, however there was no consistent pattern of change and therefore these minor differences were considered to be attributable to natural variation and were unrelated to treatment with test item.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination performed after 5 weeks of treatment for males and on Day 14 of lactation for females revealed no test item related lesions.
All findings were considered to be incidental and unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONES
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatisation period.
Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and index were unaffected by treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

With the exception of Group 2F No. 59 all females were pregnant, successfully gave birth to live young and reared their offspring to Day 13 of age.
There was no effect of parental treatment with test item on the mean numbers of implantation sites or litter size.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with test item.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment with test item on offspring survival to Day 13 of age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean offspring body weights on Day 1 of age and subsequent body weight gain up to Day 13 of age was similar in all groups and no effect of parental treatment with test item was inferred.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The mean ano-genital distance of male and female offspring was essentially similar to control in all treated groups and no effect of parental treatment with test item was inferred.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The single incidence of nipples observed among male offspring on Day 13 of age occurred in the control group, therefore was unrelated to parental treatment with test item.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no findings in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONES
There was no effect of treatment on the circulating levels of thyroxine (T4) in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age and none of the TSH (thyroid stimulating hormone) samples required analysis.

SEX RATIO
There was no effect of parental treatment with test item on the sex ratio of offspring.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (P0) (rat males/females) ≥ 1000 mg/kg bw day, reproductive toxicity
NOAEL (F1) (rat) ≥ 1000 mg/kg bw day

Executive summary:

The purpose of the study was to assess the general systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, following administration of test item by oral gavage administration for at least five weeks. The study was conducted according to OECD TG 422 guideline: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Three groups of ten male and ten female rats received test item at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a volume dose of 10 ml/kg/day. 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, purified water, at the same volume dose as treated groups.

Oral administration of test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 300 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after 5 weeks of treatment and females on Day 14 of lactation was well tolerated. There were no premature deaths, no test article-related signs observed during the detailed physical examination and arena observations, no post-dosing observations, no effects on sensory reactivity, grip strength or motor activity and no effects on body weight performance or food consumption. Organ weights at scheduled termination were essentially similar in all groups, and macroscopic and microscopic examination conducted after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any test item-related lesions.

Estrous cyclicity, pre-coital interval, mating performance, gestation length and index were unaffected by treatment with test item.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

The clinical condition, litter size, sex ratio, body weight, survival and ano-genital distances of the F1 offspring was unaffected by parental treatment and at scheduled termination there were no findings associated with treatment.

 

Conclusion

NOAEL (P0) (rat males/females) ≥ 1000 mg/kg bw day, systemic toxicity

NOAEL (P0) (rat males/females) ≥ 1000 mg/kg bw day, reproductive toxicity

NOAEL (F1) (rat) ≥ 1000 mg/kg bw day