[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to Reproduction (screening): Subacute / reproductive toxicity study oral (gavage), rat (Sprague-Dawley) m/f, 10/sex/dose, 0, 40, 130, 400 mg/kg bw/d (nominal) in corn oil (OECD TG 422, GLP):

NOAEL = 400 mg/kg bw/d (P, reproductive toxicity, no adverse effects observed in reproductive performance)

NOAEL = 130 mg/kg bw/d (P, systemic toxicity)

NOAEL = 130 mg/kg bw/d (F1, reproductive toxicity, based on live birth index, viability index, body weight)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-08 - 2017-10-27 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD 422 guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C) without protection from light.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

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) strain 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.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 10 to 11 weeks old.
Females 12 to 13 weeks old.
- Weight at study initiation:
Males 344 to 408 g.
Females 250 to 298 g
- Fasting period before study: no
- Housing:
Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Rodent facility was with limited access - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, 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.
Solid bottom cages contained softwood based bark-free fiber bedding that was sterilized by autoclaving, which was changed at appropriate intervals each week.
Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary and returned following removal of offspring.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations)
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, non-restricted. Bottles were changed at appropriate intervals
- Acclimation period:
Males: 8 days before commencement of treatment.
Females: 22 days before commencement of estrous cycle evaluation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES:
Males: 31 May 2017 - 13 July 2017
Females: 17 May 2017 - 28 July to 09 August 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
Method of preparation: For each formulation, the required amount of test item was stirred with approximately 50% of the required vehicle until uniformly mixed. The remaining volume of vehicle was added and the suspension magnetically stirred until homogenous.
Formulations were prepared in ascending group order.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C) for up to 15 days, at ambient temperature (15 to 25°C) for one day

- VEHICLE
- Concentration in vehicle: 0, 8, 26, 80 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 4 (males) of treatment and Day 12 of lactation (females) were analysed for achieved concentration of the test item.
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 refrigerated for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 7% of the initial time zero value and the coefficient of variation was less than 5%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.
The mean concentrations were ±6% which is within the applied limits +10/-15%, confirming the accuracy of formulation.
The percentage difference from the mean values were within ±3.5% confirming the accuracy of the analysis.
Procedural recovery samples were within the acceptable range of 93.1% to 108.1% (mean recovery value at validation of 100.6% ±7.5%). Procedural recovery values were not used to correct the final result.

Duration of treatment / exposure:

Two weeks before pairing to necropsy (males: Week 5 (36 days), females: Day 14 past partum (51-54 days))

Frequency of treatment:

daily

Details on study schedule:

not applicable

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

130 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 40, 130 and 400 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were selected based on the results of a 14 day preliminary toxicity study in the Sprague-Dawley rat. In that study, dose levels of 250 or 500 mg/kg/day were generally tolerated, however at 1000 mg/kg/day one animal died on Day 5 of treatment and adverse signs included decreased activity, piloerection, loose faeces and hunched posture. Macroscopically, thickening of the non-glandular mucosa of the stomach was observed in males given 500 or 1000 mg/kg/day and in females given 1000 mg/kg/day, and liver weights were high in males given 250 or 500 mg/kg/day and in males and females given 1000 mg/kg/day. Body weight loss was seen in males and females given 1000 mg/kg/day, and although body weight gain was slightly low for males and females given 500 mg/kg/day, weight gain improved during Week 2 of treatment and the overall gains (Day 1 to 14) were similar to the controls.
A high dose level of 400 mg/kg/day was selected for this study in order to minimise the risk of progression of the macroscopic findings observed at 500 mg/kg/day (thickened nonglandular mucosa of the stomach) over the longer treatment period. The low dose level of 40 mg/kg/day was selected as it is 10% of the high dose, and the intermediate dose level of 130 mg/kg/day was selected to assess dose response.
- Rationale for animal assignment (if not random): On arrival and non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights and estrous cycles were reviewed by Study Management before dosing commenced.
Body weight of animals did not exceed ±20% of the mean for each sex.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Daily during the first week of treatment, weekly thereafter and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
1 to 2 hours after completion of dosing
As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of animals was recorded as follows:
P0 males:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
P0 females:
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 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:
P0 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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food (females deprived of food on Days 13-14 of lactation) at termination
- Anaesthetic used for blood collection: Yes
Animals were held under light general anesthesia induced by isoflurane.
- Animals fasted: Yes
- How many animals: The five lowest numbered surviving males per group, the first five lactating females with a litter per group
- Parameters: 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 analyser:
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 analyser. 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 an ACL series analyser and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food (females deprived of food on Days 13-14 of lactation) at termination
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes
Animals were held under light general anesthesia induced by isoflurane.
- How many animals: The five lowest numbered surviving males per group, the first five lactating females with a litter per group
- Parameters: 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Bile acids (BiAc)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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, 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.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sensory reactivity and grip strength
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 experimental 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
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalisation without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4 Exaggerated response e.g. excessive vocalisation, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
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.
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.

IMMUNOLOGY: Yes
- Time schedule for examinations: Blood samples were collected from animals at the following occasions:
At termination:All adults.
Day 4 of age F1 offspring, two females per litter (where possible)
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
No pups were allocated to these procedures if the resultant litter size fell below 10 pups, or the resultant number of female pups fell below 3 pups.
If only 4 female offspring were available within a litter but the overall litter size was >10, one female may be selected with priority given to the serum sample.
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample
- Parameters:
Sequence of blood sampling on each occasion : In order to minimise 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:
Offspring: No overnight deprivation of food.
Adults: Following overnight deprivation of food.
Anesthetic:
Offspring: None.
Adults: Isoflurane.
Blood sample site:
Offspring: Decapitation.
Adults: Sublingual vein.
Parameter:Thyroid stimulating hormone (TSH) , Thyroxine (T4)

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; animals that failed to exhibit 4-5 cycles were not allocated to the study.
After pairing until mating.
Females showing no evidence of mating – following completion of the pairing period females were separated from the male and vaginal smearing was continued for up to six days or until the first oestrus smear was seen.
For four days before scheduled termination (Days 11 to 14 of lactation).

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
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:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

Parturition Observations and Gestation Length
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.

Records Made During Littering Phase
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 size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex 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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Thyroid Hormone Analysis: Blood samples were collected as follows:
Day 4 of age F1 offspring, two females per litter (where possible ensuring that the number of female offspring did not fall below three).
No pups were allocated to these procedures if the resultant live litter size would fall below eight pups/litter.
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
Day 13 of age:F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Necropsy
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
P0 males: After Week 5 investigations completed.
P0 females failing to mate: Day 25 after last day of pairing.
If an oestrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible.
P0 females failing to produce a viable litter: Day 25 after mating.
P0 females whose litter died before Day 13: On or after day the last offspring died.
P0 females:Day 14 of lactation.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in the respective table in "Any other information on materials and methods"

HISTOPATHOLOGY: Yes
Histology
Processing: 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.
Full List: The five lowest surviving P0 males and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
All animals killed or dying prematurely.
Thyroids, liver and stomach: The five lowest surviving P0 males and the first five lactating females with a surviving litter in Groups 2 and 3 at scheduled termination.
Kidneys: The five lowest surviving P0 males in Groups 2 and 3 at scheduled termination.
Abnormalities only: All P0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Additional staining: Kidneys from the five lowest surviving P0 males in Groups 1, 2, 3 and 4 at scheduled termination were subjected to immunohistochemical staining for alpha 2-u globulin.
Light Microscopy: Tissues preserved for examination were examined as detailed in the respective table in "Any other information on materials and methods"

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Method of kill:
Intraperitoneal injection of sodium pentobarbitone.
For offspring selected for thyroid hormone sampling, decapitation.
F1 offspring:
Selected offspring for thyroid hormone analysis – Day 4 of age
Scheduled kill - 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 was performed.
Offspring at scheduled termination : Examined externally, with particular attention being paid to the external genitalia. If found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Statistics:

Due to limitations of this free-text field, see "Any other information on materials and methods"

Reproductive indices:

Estrous Cycle
The percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating / Animals paired) * 100
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) * 100
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) * 100

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. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born / Number pregnant) * 100

Offspring viability indices:

Litter Size
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.

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) * 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) * 100
Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) * 100
Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) * 100
Group mean values were calculated from individual litter values.

Sex Ratio
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.
Percentage males = (Number of males in litter / Total number of offspring in litter) * 100
Group mean values were calculated from individual litter values.

Offspring Examinations
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. As no nipples were present, no data are included.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted 1-2 hours after dosing, and rarely at the end of day check, was seen on isolated occasions in some animals given 130 or 400 mg/kg/day. For females the frequency was more noticeable during gestation and lactation.
The distribution of other minor signs noted at the detailed physical examination and arena observations showed no test item related effects.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two decedents, neither of which were considered to be test item related.
Control group female 65 was killed for welfare reasons on Day 18 of gestation following signs of piloerection, hunched posture irregular breathing and brown stained muzzle. Gross macroscopic findings of a perforated oesophagus, pale thoracic adhesions, and enlarged adrenal glands were noted. Microscopic findings included thoracic lesions of neutrophilic inflammation and adhesions, moderate, mixed inflammation of lung pleura and sub-pleura, multifocal inflammation of the oesophagous, thymic atrophy, congestion in the adrenal glands, mixed inflammation in the epicardium and increased granulopoiesis in the bone marrow. Mis-dosing was considered to have caused the deterioration in clinical condition.
Female 56, given 40 mg/kg/day, was found dead immediately prior to terminal sacrifice on Day 14 of lactation, having been noted as prostate prior to anaesthetizing for the terminal bleed. Gross macroscopic findings included pale kidneys and heart, and stomach distension, with clear liquid present and dark areas on the glandular mucosa and caecum, and yellow viscous fluid in the jejunum. Microscopic findings included moderate cortical tubular dilatation with moderate mineralisation of the tubular epithelium, and moderate cortical tubular degeneration. This mineralisation was seen in the myocardium and vasculature of the heart as well as the glandular epithelium of the stomach. The cause of death was concluded to be due to the combination of lesions seen in the heart, kidneys and stomach. In the absence of any similar effects at higher doses this death is considered to be unrelated to the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related effects on group mean body weight gain of males over Weeks 0-5.
Group mean body weight gain of females given 130 or 400 mg/kg/day was higher than the controls in the 2 weeks prior to pairing (1.56X and 1.47X Control respectively), and whilst group mean body weight gain during gestation was similar in all groups, group mean body weight gain during Days 1-13 of lactation was higher than the concurrent control in females given 40, 130 or 400 mg/kg/day (1.48X, 1.55X or 2.12X Control respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption of males given 400 mg/kg/day was slightly higher than the control group (1.12X Control), with the difference being apparent from Week 2.
There were no test item related effects on group mean food consumption in males given 40 or 130 mg/kg/day.
Group mean food consumption of females given 400 mg/kg/day was slightly lower than the controls from Day 4 of lactation (0.90X Control). There were no test item related effects on group mean food consumption prior to pairing or during gestation in females given 400 mg/kg/day and no test item related effects on food consumption in females given 40 or 130 mg/kg/day.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Visual assessment of water consumption indicated that daily water consumption was higher for males and females given 400 mg/kg/day compared with the control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were considered to be no test item related effects on the haematological parameters investigated.
Occasion slight intergroup differences from controls, some of which attained statistical significance, were considered to be unrelated to the test item as the magnitude of response was low, there was no dose relationship and no correlation across the sexes and, for differential white cell parameters, no effect on total white blood cell counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were statistically significantly higher than the control for males given 130 or 400 mg/kg/day (1.29X and 1.66X Control for ALT and 1.26X and 1.27X Control for AST respectively).
Group mean bile acid levels for males given 400 mg/kg/day were statistically significantly lower than the controls (0.31X Control).
Group mean alkaline phosphatase (ALP) levels were statistically significantly higher than control for females given 400 mg/kg/day (2.05X Control).
Occasion slight intergroup differences from controls, some of which attained statistical significance, were considered to be unrelated to the test item as the magnitude of response was low, there was marked variability in individual results, there was no dose relationship and no correlation across the sexes.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were considered to be no test item related effects on sensory reactivity and grip strength.
Group mean forelimb grip strength values for all treated groups of males and females were slightly lower than the current control group. The group mean hindlimb value for females given 400 mg/kg/day was slightly higher than the control group and attained statistical significance. However, all values were within the historical control data range, therefore these minor differences were considered to be due to individual variation and were considered not to be test item related.

There were considered to be no test item related effects on motor activity.
Group mean low beam activity scores for all treated groups of females were low compared with the concurrent control group. Generally, a dose-related trend was apparent, specifically for total scores. Statistical significance was attained at the 30-minute interval for females given 400 mg/kg/day and at the 36-minute interval for females given 130 or 400 mg/kg/day. However, the majority of these values were within the historical control data range, including all the total scores. In the absence of a similar effect in high beam scores these differences were attributed to individual variation in results and were considered not to be test item related.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Incidental Findings
Mineralisation was seen in the cortical tubules within the kidneys of females from all groups. This was accompanied by tubular dilatation and tubular degeneration. This mineralisation was also seen in the myocardium and cardiac vasculature, and in the glandular portion of the stomach. In some of these animals, microscopic mineralisation could be correlated macroscopically to pale kidneys and multifocal pale areas on the heart. The distribution of mineralisation in the kidneys of control group females indicates alteration in calcium/phosphorus homeostasis.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or
stage-specific abnormalities were noted.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females assigned to the study had regular estrous cycles prior to the start of treatment.
There were no test item related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related effects on mating performance or fertility.
The pre-coital interval was generally 1-4 days for all pairings, indicating mating occurred at the first estrous opportunity. One pairing each at 130 or 400 mg/kg/day did not mate at the first estrous opportunity but the low incidence of this did not suggest any test item relationship.
With the exception of one control group pairing, all other pairings showed evidence of mating and conception occurred.

There was a slight, but statistically significant, shift in gestation length with more females given 130 or 400 mg/kg/day having a shorter duration of gestation (22 – 22.5 days) compared with the control group. However, all gestation length values were within the historical control data range, and the minimum and maximum values observed for females given 130 or 400 mg/kg/day were closer to the normal range than the current control group. Therefore, the differences in gestation length were attributed to individual variation in results and were considered not to be test item related.
The gestation index was 100% in all treated groups, and was 89% in the control group due to the accidental death of one female on Day 18 of gestation.
Prior to termination all females smeared were in diestrus. There were no test item related effects.

Key result

Dose descriptor:

NOAEL

Effect level:

130 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

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 test item related effects related to parental treatment in the few clinical signs noted in the offspring from birth to Day 13 of age.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There were no test item related effects on the group mean number of implantations or post implantation survival index such that there were no test item related effects on the total litter size on Day 1.
In litters from parental treatment at 400 mg/kg/day, there was a lower live birth index (0.76X Control) and viability index on Day 4 (0.94X Control), and following the litter size adjustment on Day 4 of age the survival index to Day 13 of age was also lower (0.85X Control). On Day 1 of age at 400 mg/kg/day, the live litter size was 0.83X Control and by Day 13 of age, the live litter size was 0.76X Control.
There were no test item related effects on litter size, sex ratio and survival indices in litters from parental treatment at 40 or 130 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight for offspring derived from parental treatment at 400 mg/kg/day was statistically significantly lower than the control on Day 1 of age, and the rate of body weight gain to Day 13 of age remained slightly lower than the controls such that group mean body weight on Day 13 of age was lower than the controls.
There were no test item related effects related to parental treatment at 40 or 130 mg/kg/day on offspring body weight.
Body weight gain of the offspring derived from parental treatment at 130 mg/kg/day was slightly yet statistically significantly lower (0.87X and 0.86X Control for males and females respectively) than the concurrent control group but review with historical control data (see Annex 5) shows the current control group values for absolute body weight at birth and body weight gain to Day 13 of age are close to or above the upper limit of historical data, and the same values for offspring derived from parental treatment at 130 mg/kg/day was within the 5 to 95 percentile historical range. As such the apparent intergroup difference in body weight gain is considered to reflect normal biological variation and is not considered attributable to the test item.

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:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment on the ano-genital distance on Day 1 of age, corrected for the body weight.
There were no nipples/areolae noted in any male offspring on Day 13

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related findings at macroscopic examination of offspring.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

130 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential reproductive toxicity (and repeated dose toxicity) of the test item.
Upon oral administration of the test item to parental Sprague Dawley rats at dose levels of 40, 130 or 400 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and also during gestation and lactation and up to termination of females on Day 14 of lactation, it was concluded that the exposure to 400 mg/kg/day was associated with signs of slight systemic toxicity.
Slight effects indicating a local mild irritation were observed in the stomachs of some adult rats given 400 mg/kg/day. While the observed pathological changes found in the kidneys are specific for male rats and not relevant for humans, pathological findings in the liver and thyroids in rats given 400 mg/kg/day, as well as the associated slight increase in transaminase levels in the blood were considered likely to be an adaptive response. However, due to the nature of the observed effects, it cannot be absolutely excluded that these effects may be slightly adverse, which could be associated with a general poorer condition not affecting parental animals to a high extent, but suffice to adversely affect the offspring as a secondary response. As an adverse nature of the effects associated with the oral administration of this highest dose of 400 mg/kg/day cannot be excluded, for precautionary reasons 130 mg/kg/day is considered as no observed adverse effect level for the test item.
Lower live litter size (fewer male offspring) and survival to Day 13 of age, and lower offspring body weight on Day 1 of age and growth to Day 13 were noted at 400 mg/kg/day. There were no other effects on reproductive performance, fertility, litter size or offspring survival and, in the context of this study, the test item showed no evidence of being an endocrine disruptor.
The no-observed-adverse-effect level (NOAEL) of the test item for systemic toxicity was considered to be 130 mg/kg/day and for reproductive/developmental toxicity the NOAEL was also considered to be 130 mg/kg/day. Hence, as reproductive/developmental toxicity only occurs when maternal toxicity is present, is must be considered as secondary effect, especially taking into account the non-specific nature of the effects in offspring, i.e. effects on body weight and viability.
According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.
Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
In consequence, the test item does not need to be regarded as reproductive toxicant.

Executive summary:

The purpose of this study according to OECD 422 under GLP was to assess the general systemic toxic potential of the test item, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, when administered to Sprague Dawley rats by oral gavage administration for at least 5 weeks.

Four groups of ten male and ten female rats received the test item at doses of 40, 130 or 400 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, 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, body weight, ano-genital distance, nipple counts (male only) and macropathology for all offspring were also assessed.

 

Results

P0 responses

There were two decedents, neither of which were considered test item related. A control female was sacrificed following mis-dosing, and a female given 40 mg/kg/day was found dead on Day 14 of lactation with cause of death being mineralisation in heart, stomach and kidneys which are occasionally also seen in control animals on these study types.

Post dose salivation was seen in animals given 130 or 400 mg/kg/day. Body weight gain was higher in females given 130 or 400 mg/kg/day prior to pairing and was higher during lactation in all treated female groups. Food consumption from Week 2 was slightly higher for males given 400 mg/kg/day, and was slightly lower in females given 400 mg/kg/day from Day 4 of lactation. Water consumption was higher in males and females given 400 mg/kg/day.

Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by treatment.

Alanine aminotransferase and aspartate aminotransferase levels were higher in males given 130 or 400 mg/kg/day, bile acid levels were lower in males given 400 mg/kg/day and alkaline phosphatase levels were higher for females given 400 mg/kg/day. There were no test item related effects on haematological parameters. There was no effect upon circulating levels of thyroxine (T4) in adult males.

After five weeks of treatment for males and on Day 14 of lactation for females, liver and adrenal weights were higher in males and females given 400 mg/kg/day and females given 130 mg/kg/day, and heart and spleen weights were lower in males and females given 400 mg/kg/day.

Thickened fore stomach was noted macroscopically in males and females given 400 mg/kg/day. Histologically hyperkeratosis of the non glandular epithelium, with or without hyperplasia and erosion was seen in males and females given 400 mg/kg/day and in one female given 130 mg/kg/day.

Centrilobular hepatocellular hypertrophy was seen in the livers of males given 130 mg/kg/day and in males and females given 400 mg/kg/day. Kidney changes of cortical tubules with the presence of hyaline droplets were seen in some males given 40, 130 or 400 mg/kg/day, with cortical tubular basophilia being seen in one male given 40 mg/kg/day and some males given 400 mg/kg/day and tubular casts seen in some males given 400 mg/kg/day. There was a dose related increase in staining of kidneys for alpha2µ globulin in treated males. Follicular cell hypertrophy of the thyroids was seen in males given 400 mg/kg/day.

 

F1 responses

At 400 mg/kg/day there was a lower live birth index with a lower number of male offspring on Day 1 of age, and lower survival indices through to Day 13 of age, which was associated with lower litter size.

Body weight at birth and body weight gain to Day 13 of age was lower in male and female offspring from the 130 and 400 mg/kg/day groups were lower than Controls.

The clinical condition, litter size, sex ratio and survival indices of offspring were unaffected by parental treatment at 40 mg/kg/day.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to parental treatment were recorded.

 

Conclusion

Upon oral administration of the test item to parental Sprague Dawley rats at dose levels of 40, 130 or 400 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and also during gestation and lactation and up to termination of females on Day 14 of lactation, it was concluded that the exposure to 400 mg/kg/day was associated with signs of slight systemic toxicity. 

Slight effects indicating a local mild irritation were observed in the stomachs of some adult rats given 400 mg/kg/day. While the observed pathological changes found in the kidneys are specific for male rats and not relevant for humans, pathological findings in the liver and thyroids in rats given 400 mg/kg/day, as well as the associated slight increase in transaminase levels in the blood were considered likely to be an adaptive response.  However, due to the nature of the observed effects, it cannot be absolutely excluded that these effects may be slightly adverse, which could be associated with a general poorer condition not affecting parental animals to a high extent, but suffice to adversely affect the offspring as a secondary response. As an adverse nature of the effects associated with the oral administration of this highest dose of 400 mg/kg/day cannot be excluded, for precautionary reasons 130 mg/kg/day is considered as no observed adverse effect level for the test item.

Lower live litter size (fewer male offspring) and survival to Day 13 of age, and lower offspring body weight on Day 1 of age and growth to Day 13 were noted at 400 mg/kg/day. There were no other effects on reproductive performance, fertility, litter size or offspring survival and, in the context of this study, the test item showed no evidence of being an endocrine disruptor.   

The no-observed-adverse-effect level (NOAEL) of the test item for systemic toxicity was considered to be 130 mg/kg/day and for reproductive/developmental toxicity the NOAEL was also considered to be 130 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

130 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study was conducted on the registered substance itself acc. OECD TG 422 under GLP. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

See above, Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

A mode of action analysis in its classic sense cannot be performed for repeated dose toxicity as there is only one repeated dose study available in one species, inclusive reproductive screening. However, the rat is an established model for human risk assessment, interspecies differences are well studied and allow transfer conclusions to humans for most chemicals exhibiting certain effects in the rat. Nevertheless it is aimed to follow the WHO IPCS template as far as possible.

 

Problem formulation

The present MoAA aims to show that reproductive toxicity occurred only in the presence of maternal toxicity, and hence litter effects were a secondary non-specific consequence of the other toxic effects, the substance is as such no reproductive toxicant.

 

Hypothesised Mode of action Statement

The substance is as such no reproductive toxicant, litter effects were a secondary non-specific consequence of maternal toxicity.

 

Summary of data for use in Mode of Action Analysis

For more details, see the respective endpoints. In brief, upon oral administration of the test item to parental Sprague Dawley rats at dose levels of 40, 130 or 400 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and also during gestation and lactation and up to termination of females on Day 14 of lactation, it was concluded that the exposure to 400 mg/kg/day was associated with signs of slight systemic toxicity. 

Slight effects indicating a local mild irritation were observed in the stomachs of some adult rats given 400 mg/kg/day. While the observed pathological changes found in the kidneys are specific for male rats and not relevant for humans, pathological findings in the liver and thyroids in rats given 400 mg/kg/day, as well as the associated slight increase in transaminase levels in the blood were considered likely to be an adaptive response.  However, due to the nature of the observed effects, it cannot be absolutely excluded that these effects may be slightly adverse, which could be associated with a general poorer condition not affecting parental animals to a high extent, but suffice to adversely affect the offspring as a secondary response. As an adverse nature of the effects associated with the oral administration of this highest dose of 400 mg/kg/day cannot be excluded, for precautionary reasons 130 mg/kg/day is considered as no observed adverse effect level for the test item.

Lower live litter size (fewer male offspring) and survival to Day 13 of age, and lower offspring body weight on Day 1 of age and growth to Day 13 were noted at 400 mg/kg/day. There were no other effects on reproductive performance, fertility, litter size or offspring survival and, in the context of this study, the test item showed no evidence of being an endocrine disruptor.

 

 

Listing of key events identified for a specific Mode of Action

 

Key Event 1	Maternal toxicity: - changes in foodconsumption, body weight gain - increasedalkaline phosphatase levels (ALP) - Higher liver and adrenal weights -Thickened forestomach,Epithelial Hyperplasia, Hyperkeratosis, Erosion - Centrilobular hepatocellular hypertrophy
Key Event 2	Offspring toxicity - lower live birth index, viability index, live litter size - lower offspring body weight

 

All other examined parameters were not considered relevant or different from control.

 

Bradford Hill Considerations for Weight of Evidence Analysis of available data/information for Mode of Action Analysis in experimental species

 

Dose Response Relationships and Temporal Association

Conclusions on temporal association cannot be drawn, as there is only one OECD 422 study available with a study duration of approx. 6-7 weeks. In the following table, only effects in the dams and offspring are denoted:

 

Dose (mg/kg bw/d)	Key event 1:Maternal toxicity					Key event 2: Offspring toxicity
	foodconsumption, body weight gain (fold over control)	ALP levels	liver and adrenal weights	Irritation, stomach	Centrilobular hepatocellular hypertrophy	live birth index, viability index, live litter size	body weight
40	- No treatment-related effects on bw 2 weeks prior to pairing -1.48X mean body weight gain during Days 1-13 of lactation	No effects	No effects on liver and adrenal weights	- Thickened forestomach (1F), no further effects	No effects	No test item related effects on litter size, sex ratio and survival indices	No effects on offspring body weight
130	-1.56X mean body weight gain2 weeks prior to pairing -1.55X mean body weight gain during Days 1-13 of lactation	No effects	- Liver weight: 1.12X (F) - Adrenal weight: 1.37X (F)	- No thickened forestomach - Hyperkeratosis, Non-glandular Region (slight (1F))	No effects	No test item related effects on litter size, sex ratio and survival indices	No effects on offspring body weight
400	-1.47X mean body weight gain2 weeks prior to pairing -2.12X mean body weight gain during Days 1-13 of lactation - Group mean food consumption slightly lower from Day 4 of lactation (0.90X Control).	2.05X ALP, females	- Liver weight:1.10X (F) - Adrenal weight: 1.36X (F)	- Thickened forestomach (1F) -Epithelial Hyperplasia, Non-glandular Region (minimal (3F)) - Hyperkeratosis, Non-glandular Region (minimal (2F), slight (1F)) -Erosion, Non-glandular Region (minimal (1F))	2 minimal, 2 slight (F)	lower live birth index (0.76X Control), viability index on Day 4 (0.94X Control), - on Day 4 of age the survival index to Day 13 of age was also lower (0.85X Control) - Day 1 of age live litter size was 0.83X Control and by Day 13 of age, live litter size was 0.76X Control	Statistically significantly lower than the control on Day 1 of age, and the rate of body weight gain to Day 13 of age remained slightly lower

 

A dose-response was observed, effects on the offspring occur only concurrent with effects on the dams.

 

Consistency & Specificity – Biological Plausibility

Treatment-related findings were seen in the liver and stomach of the dams, and also some rather general effects.

In animals given 400 mg/kg/day, hepatocellular hypertrophy and thyroid follicular cell hypertrophy were observed in males given 400 mg/kg/day. Hepatocellular hypertrophy and associated thyroid follicular cell hypertrophy is usually a result of hepatic enzymatic induction. Hepatic enzyme induction is generally an adaptive response associated with increases in liver weight, induction of gene expression, and morphological changes in hepatocytes. No hepatocyte necrosis, abnormal clinical chemistry parameters or obvious functional liver impairment was observed and, therefore, the liver and thyroid findings may be considered non-adverse. However, the increase in alanine aminotransferase and aspartame aminotransferase levels for males given 130 or 400 mg/kg/day (1.29X and 1.66X Control for ALT and 1.26X and 1.27X Control for AST respectively) may be nevertheless indicative for some treatment-related cell damage, especially the increased alanine aminotransferase in the 400 mg/kg group. In addition, the hepatobiliary enzyme alkaline phosphatase was increased 2.05-fold over background in females given 400 mg/kg/day, which is further indicative for liver involvement. No clear conclusion can be drawn to which extent those effects still can be regarded as adaptive, or whether they should be considered indicative for an already adverse effect. Also, even if adaptive, these changes may have influence on the general physiological state of the animals, which can further lead to secondary effects on the offspring, although adult animals may tolerate it.

In animals given 400 mg/kg/day, hyperplasia, hyperkeratosis and erosion of the non-glandular stomach were observed. These changes were considered to represent a mild irritant effect of the test item to the non-glandular epithelium, i.e a local response with an evident dose response. These effects should have contributed to the modified food uptake and body weight gain, which is further indicative for some slight systemic toxicity in the dams.

At 130 mg/kg/day there was an apparent lower offspring body weight at birth and through to Day 13 of age but as there was a shorter gestation length than the controls, this may be associated with this apparent lower offspring body weight at birth and subsequent weight gain. The control group was at or above the upper end of the historical background range for gestation length and offspring body weight and gain, whereas the values at 130 mg/kg/day were generally within the historical background range and within 10% variation from historical mean body weight. It was considered that the control group was atypical for gestation length and offspring body weight and as such the apparent differences from control seen at 130 mg/kg/day was due to general biological variations and not the test item. 

In consequence, as stated above, there are no specific effects in offspring noted, only general effects such as diminished viability or body weight were observed, indicating no specific MoA of the test item. A dose-response was observed, effects on the offspring occur only concurrent with effects on the dams.

 

Qualitative and Quantitative human concordance

There is no indication given that the obtained results may not be relevant for humans, no species-specificity was obvious.

 

Other potential Modes of Action

None identified

 

Uncertainties/Inconsistencies and Identification of Data Gaps

None identified

 

Conclusions in relation to problem formulation

It was shown that reproductive toxicity occurred only in the presence of maternal toxicity, and hence litter effects were a secondary non-specific consequence of the other toxic effects, the substance is as such no reproductive toxicant. There is no concrete evidence given that the obtained results may not be relevant for humans, no species-specifity was obvious.

Justification for classification or non-classification

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.

Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”

It was shown that reproductive toxicity occurred only in the presence of maternal toxicity, and hence litter effects were a secondary non-specific consequence of the other toxic effects. In consequence, the test item does not need to be regarded as reproductive toxicant.