N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: OECD Guideine 422. GLP study. The NOAEL for reproduction (P0) was determined to be 500 mg/kg bw/day since no test item related adverse effects were observed at the highest dose tested.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2017 - 09 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 14-15 weeks old at start of treatment and 16-17 weeks old at mating
- Weight at study initiation: Males: 441-564 g, females: 246-303 g
- Housing: Standard laboratory conditions
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6-24.1 °C
- Humidity (%): 34-70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared up to 6 days before use (formulation were kept at room temperature in that case), containers were closed immediately after preparation and were kept at room temperature until use. Stability of the test item in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 1-200 mg/mL concentration range (using olive oil as vehicle) were proven as being stable for at least 7 days when stored at room temperature.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): A short solubility test was performed.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): BCBT7822

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed using an HPLC-UV method. Top, middle and bottom duplicate samples were taken from test item formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse (which could be collected in replicates as practical), one set to analyse (which can be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Duration of treatment / exposure:

2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available acute oral toxicity data (LD50 between 300 and 2000 mg/kg bw in rats) and information from a Dose Range Finding study in the rat. In the DRF study, some test item related toxicity was seen at 300 mg/kg bw/day. In order to achieve sufficient effects to demonstrate an MTD was achieved, a High dose level of 500 mg/kg bw/day was selected.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before the first exposure, then at least weekly.
- Parameters: Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:
on Day -1, on Day 0, then afterwards at least weekly and at termination.
- Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partum Days PPD0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Animal food consumption was determined by re-weighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood:
Immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (
pentaorbital)
- Animals fasted: Yes
- How many animals:
5 males and 5 females/group
- Parameters: RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte) count, Hgb Haemoglobin concentration, Hct Haematocrit (relative volume of erythrocytes), MCV Mean Corpuscular (erythrocyte) Volume, MCH Mean Corpuscular (erythrocyte) Haemoglobin, MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, RDW Red Cell (erythrocyte) volume, Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume, RETIC % Reticulocyte count, NE % Neutrophil, LY % Lymphocyte, MO % Monocyte, BA % Basophil, EO % Eosinophil , LUC % Large Unstained Cells, APTT Activated Partial Thromboplastin Time, PT Prothrombin Time.


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
Immediately prior to scheduled necropsy
- Animals fasted: Yes
- How many animals:
5 males and 5 females/group
- Parameters:
Glucose Blood sugar concentration, T-BIL Total Bilirubin concentration, Urea Urea concentration, Chol. Cholesterol concentration, Creat. Creatinine concentration, Phos. Phosphorus concentration, Na+ Sodium concentration, K+ Potassium concentration, Ca++Calcium concentration, Cl- Chloride concentration, Tot. Prot. Total Protein concentration, Alb. Albumin concentration, A/G Alb/glob ration, AST/GOT Aspartate Aminotransferase activity, ALT/GPT Alanine Aminotransferase activity, GGT Gamma-Glutamyl transferase activity, ALKP Alkaline Phosphatase activity, Bile acids.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
Prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters:
LEU / Leukocyte, NIT / Nitrite, pH, PRO / Protein, GLU / Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY, Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Last exposure week (males on Day 23; females on PPD7-9)
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No

OTHER: THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: PND14 (females), at termination (males)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: All male and female adults.
- Parameters: T4, TSH

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight, spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), the presence of gross abnormalities, abnormal behaviour of the offspring, anogenital distance (AGD), presence of nipples/areolae in pups (at PND13). Body weights were measured within 24 hours of parturition (PND0) and on PND4 and PND13. All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4. All pups were culled on PND13. For thyroid hormone analysis, blood samples were taken from up to two pups per litter on PND4. Pup blood was pooled by litter and assessed for T4 levels.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals after 28 days treatment period (14 days pre-mating and 14 days mating/post-mating).
- Maternal animals: All F1 offspring were terminated on Day 13 post-partum. In order to allow for overnight fasting of dams with urine collection on PPD14, the dams were euthanized on PPD/PND14.

GROSS NECROPSY
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

At the time of termination, body weight and the weight of the following organs from all adult animals: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. Adrenal gland, Animal identification, Aorta, Brain, Epididymis, Eye with the optic nerve, Oesophagus, Femur with marrow, Heart, Kidney, Large intestine, Extraorbital lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph node, Ovary, Oviduct, Pancreas, Pituitary, Prostate, Salivary gland (including mandibular, sublingual and parotid glands), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, subcutis with mammary gland (inguinal), Skeletal muscle (quadriceps), Small intestine, Spinal cord, Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina.

A detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group). Moreover: Liver and kidneys of all male and female animals in all dose groups, thymus of all male animals in all dose groups.

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND13 days of age.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities.

ORGAN WEIGHTS
Thyroid glands from one male and one female PND13 pup.

Statistics:

The normality and heterogeneity of variance between groups is checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett’s (Multiple Range) test is used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests show significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis is required. A Kruskal-Wallis analysis of variance is used after Rank Transformation. If there is a positive result, the inter-group comparisons are performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

For non-continuous data, the Cochran-Armitage test for trend is applied and the Chi-squared test is used for statistical differences relative to control.

For most data the “n” number of independent variables is the number of animals/samples. For food intake, during the periods of group housing the independent variables are the cages, hence “n” is the number of cages.

For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend is applied, then if appropriate, the Chi-squared test homogeneity test. If significance is plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group is made. If the group size is <5 then Fisher’s Exact Test is used, if the group sizes are bigger then the Chi-squared test is used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Reproductive indices:

- Number of pairings
- Number of pregnant females †
- Number of sperm positive, but non-pregnant females †
- Number of non-mated females †
- * Female mating index †
- * Female fertility index †
- * Gestation index †
- Duration of pregnancy (days) †
- Number of corpora lutea / dams †
- Number of implantations / dams †
- Number of dams with live pups Day 0, 4 and 13 †
- * Pre-implantation mortality †
- * Intrauterine mortality †
- * Total mortality (intra and extra uterine mortality) †

Offspring viability indices:

- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13 †
- * Survival Index of pups on postnatal Days 0, 4 and 13 †

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Low and Mid dose groups during the study. In the High dose males, slight to moderate decreased activity and ataxia were observed mainly in the first week of treatment, and then diuresis during the second half of the observation period. In the High dose females, the same pattern was seen but with higher frequencies and severity: slight to moderate decreased activity and ataxia mainly during the first 8 days of treatment and then diuresis transiently from Day 9 until Day 23. Additionally, slight to extreme decreased activity, hunched back and piloerection were seen in the single preterminally euthanized animal before its euthanasia; not attributed to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the High dose group was preterminally euthanized on Day 38 / PPD2 (two days after littering), because of animal welfare reasons. The death was considered to be related to a difficult parturition, and was not ascribed to a direct test item effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, slightly lower bodyweight values were recorded in all dose groups compared to the control during the whole observation period, but without reaching statistical significance. The animals in all dose groups during the first week of treatment lost weight, but the extent of loss was significant only in the High dose group. The bodyweight gain values were also below the control values in all dose groups during almost the whole observation period, with occasional statistical significances in the Mid and High dose groups only. In females, lower bodyweight gain values were recorded in the first two weeks of treatment in the Mid and High dose groups, reaching statistical significance only in the High dose group. However, the animals fully compensated this difference later on and thus the overall bodyweight and bodyweight gain values were comparable with the Control group in all dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, occasionally slightly lower food consumption values were recorded in the Mid and High dose groups compared to the controls during the observation period, reaching statistical significance only at the Day 0-7 period. In females, the food consumption during the first two weeks of treatment was also significantly lower in the Mid and High dose groups compared to the controls, but after that, there were no test item related differences in the mean daily food consumption in any of the test item treated groups when compared to the control.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly lower (p<0.01) prothrombin time (PTT) in the male Mid dose and female Mid and High dose groups were recorded. There was no other supporting evidence of any changes in these animals (other clinical pathology parameters, histopathology, etc.) so the relationship between the PPT values and treatment was considered to be equivocal. Generally, slightly shorter clotting time parameters is not considered to be an adverse effect. While it is plausible that liver changes may have slightly affected these parameters, these differences are not considered to be an adverse effect of the test item.

Besides this, the following other significant values were also recorded: Significantly higher (p<0.01) platelet count in the male Mid dose group, significantly higher haematocrit percentage (p<0.05) in the female High dose group and significantly higher (p<0.05) Large Unstained Cell (LUC) percentage in the female Low dose group. These differences were considered to be incidental, there was no relationship with dose and all recorded values were within the historical control ranges. These differences were considered to not reflect an effect of the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant differences were recorded in the males: Significantly higher (p<0.05 or p<0.01) cholesterol, phosphorus, total protein, albumin and bile acid concentrations in the High dose group. The concentrations of total protein and albumin were also significantly higher (p<0.01) in the Mid dose group. In females, significantly higher total protein, albumin, ALT/GPT and ALKP concentrations were measured in the High dose group.

Most statistical differences observed in clinical chemistry of the High dose animals were well within the historic control range and/or showed no clear dose response. None of the changes indicated a severe toxic effect. Moderate increases in cholesterol, total protein/albumin in both sexes, and increased ALT/GPT in females at the High dose may have been related to the hypertrophy and liver changes observed at histopathology. Increased total protein/albumin in Mid dose males may also be related to the liver changes, although all data were well within the historic control range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant values recorded in any of the dose groups.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

During the first week of the study (Days 1-8), a transient ataxia was observed in the male and female High dose groups. From Day 9 until the end of the observation period, the animals did not show any neurological symptoms. At the functional observation battery (FOB) performed during the last week of exposure, there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity. All dose groups of males and females had a normal locomotor activity.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In summary, the hepatic hypertrophy was regarded as adaptive response corresponding with the organ weight increase, and was non-adverse; the hepatic vacuolation was also considered to be a non-adverse finding, without any degeneration; the single case of centrilobular necrosis (with hepatocellular vacuolation) in one High dose female was considered to be an adverse finding. The renal eosinophilic tubular droplets in the males, and tubular vacuolation in the females, with some kidney weight increases, were considered to be non-adverse. It was considered that there was a degree of individual stress related with treatment in the thymus of some High Dose males, with a slightly decreased organ weight and evidence of decreased cortical size/cellularity by light microscopy, occasionally visualized as small thymus at necropsy. The thymus change was not considered to be a primary effect of the test item on the immune system.

Besides these, based on the low incidence and/or severity and/or distribution across control and dosed animals, the following observations were considered incidental or a common background: Single unilateral cyst in the piriform cortex of the brain, minimal multifocal inflammatory cell infiltration in the left harderian gland, minimal to moderate unilateral focal/multifocal degeneration of the tubule and/or cortex of the kidney in 3/12 High dose males, minimal tubular basophilia in the kidney in 1/12 Control male, 2/12 Mid dose males, 3/12 High dose males, 1/12 Mid dose female, 1/11 High dose female, minimal to slight focal or multifocal cast in the cortex/tubule/medulla of the kidneys in 1/12 Control male, 3/12 Low dose males, 2/12 Mid dose males, 4/12 High dose males, 3/12 Control females, 1/11 High dose female, minimal multifocal mineralisation in the tubule/outer stripe/papilla of the kidney in 2/12 Control females, severe dilatation of the left renal pelvis in one High dose male (#4009), slight multifocal bilateral haemorrhage in the mandibular lymph node of one High dose male (#4009), minimal to slight inflammation of inflammatory cell infiltration in the interstitium and dorsolateral lobe of the prostate in 2/12 Control males and 2/12 High dose males, minimal focal degeneration or atrophy in the tubular section of the testis in 1/12 High dose male, minimal to slight multifocal multinucleated giant cell in the testis in 2/12 High dose males, minimal extramedullary haematopoiesis in the spleen in 3/5 Control males, 4/5 Control females and in 3/5 High dose females.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In the adult male dose groups the T4 hormone concentration levels were statistically significantly higher than in the control group. The concentrations in the Low and Mid male dose groups were well within the historic control range and showed no dose response, therefore slight statistical differences were regarded as incidental and of no toxicological significance at these dose levels.

The T4 hormone is generally considered as a pro-hormone, that is converted into T3, the biologically active form, mainly in the liver and kidney. It is well known that the conversion of T4 to T3 may change as a consequence of liver changes if the metabolising enzyme levels are affected, resulting in high free T4 level in blood. Based on the other findings in the study (changes in clinical chemistry, necropsy findings in the liver) it is considered that the elevated T4 level in the High dose males was probably caused by the liver effects of the test item (hepatic hypertrophy, with a 36 liver weight increase relative to bodyweight).

There were no test item-related effects seen in the adult male TSH levels. The differences between the control and the dose groups did not show dose response and there were no statistical differences recorded in any of the dose groups. The higher means were considered to be incidental and not test item related.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

During the treatment (pre-mating and mating periods), the Control and the Low dose animals had regular oestrus cycles. In the Mid dose, 3/12 females showed irregular cycles. The smears from these 3 animals contained typical cell types of normal cytology. This Mid dose incidence is considered to be in the normal range of findings seen in previous studies. In the High dose, 11/12 animals had transient irregular oestrus cycles. From these, 3/12 were pseudo-pregnancy (diestrus ≥10 days) and 7/12 were prolonged diestrus. In one case a rat had irregular cycles but without prolonged diestrus and in one case the cycles were regular. In the smears of prolonged diestrus and pseudo-pregnancy animals had an atypical cellularity. These differences were observed in the second week of treatment only, on Day 14 pairs were put together and all High dose females had completely normal vaginal smears and mated normally. It is noted that the High dose animals displayed ataxia at the same time period as the prolonged diestrus. The female reproductive hormones are also regulated via signals from the central nervous system, therefore it is plausible that the transient ataxia was related to transient oestrus cycle observations. It is concluded that the irregular cycles were a test item-related secondary effect, caused by the clinical signs and bodyweight losses. As the affected animals were able to get pregnant and deliver healthy litters, this change is considered to be non-adverse.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE ABILITY:
There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant. The mating indices were 100% in all groups, except the Mid dose group where one mating was unsuccessful. The female fertility indices were 75% in the Control group (3 non-pregnant females out of the 12 animals), 100% in the Low dose group, 82% in the Mid dose group (one unmated and one non-pregnant female) and 100% in the High dose group. The gestation index was 100% in all dose groups, meaning that all impregnated females delivered living pups in the study. Test item administration was considered to have no impact on the duration of the mating period.

PRE-IMPLANTATION AND GESTATION PERIODS:
There was no effect of treatment noted during the pre-implantation and gestation periods. The mean duration of pregnancy was comparable in the control and test item treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related adverse effects at the highest dose tested.

Key result

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in each dose group. Overall, there were no treatment-related effects on pup mortality and on the viability of pups on PND0, 4 and 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The pups of the High dose dams were born with significantly lower bodyweights than the control pups. The High dose group’s bodyweight gain was similar to the Control group from a slightly lower starting value. Thus the initial percentage difference in the bodyweights remained until the end of the observation period although it was only statistically significant on Day0 (p<0.01) and Day4 (p<0.05). This effect was considered as probably secondary to maternal toxicity. In the Low and Mid dose groups, there were no test item related differences in the offspring body weights or weight gains when compared to the controls. As there were no other changes in any of the F1 parameters, the lower birth weights seen in the High dose were considered to be non-adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid gland weights of the PND13 pups were also statistically not different from the Control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the anogenital distance between the test item treated groups (males/females) and the control on PND0. There was no nipples/areolae presence seen in any of the male pups on PND13.

No test item-related macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.

During the lactation period 3 Control, 3 Low dose, 2 Mid dose and 6 High dose pups were found dead that remained intact (not cannibalized or autolysed). Those were subjected to necropsy with macroscopic examination. No test item-related macroscopic findings were seen. No other pups that died during the study could be examined for test item-related macroscopic findings.

Other effects:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant T4 thyroid hormone concentration levels recorded in any of the PND13 pup dose groups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related adverse effects at the highest dose tested.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The 28-days NOAEL (oral) for parental systemic toxicity (P0) was determined to be 150 mg/kg bw/day due to the clinical signs (ataxia, diuresis), lower body weight gain and food intake and the liver necrosis observed at the highest dose tested (500 mg/kg bw/day). The NOAELs for reproduction (P0) and developmental toxicity (F1) were determined to be 500 mg/kg bw/day since no test item related adverse effects were observed at the highest dose tested.

Executive summary:

A combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rats was performed according to the OECD Guideline 422 (GLP study). Twelve male and female Wistar rats per group were treated at 50, 150 and 500 mg/kg bw/day by gavage for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13. Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups, dams and adult males were assessed. The thyroid-stimulating hormone (TSH) levels were also assessed in the dams and adult males. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups. Additionally, the liver and kidneys of all animals and the thymus of all male animals were also microscopically examined. The treatment did not result in test item related mortality, or any persistent clinical signs. However, at 500 mg/kg bw/day transient ataxia (mainly week 1) and diuresis (mainly week 3) were seen. Significant adverse effects on body weight gain were seen transiently in the male High dose group (500 mg/kg bw/day), particularly in the first two weeks of treatment, with similar changes in food intake. There were no clearly adverse effects on haematology, coagulation, clinical chemistry or urinalysis parameters. At the end of the study, there were no changes recorded during the neurological assessment of any of the dose groups. The T4 hormone level was apparently increased in the male High dose group, without any changes on organ weight or histopathology. Based on the other observed effects, it is considered that this change was probably caused by a decreased clearance of the hormone as a consequence of the liver effects. At necropsy, increased liver and kidney organ weights were recorded in the male and female Mid and High dose groups (150, 500 mg/kg bw/day) and thymus weights were decreased in the male High dose group (500 mg/kg bw/day). Liver enlargement and small thymus were seen macroscopically in the High dose male group (500 mg/kg bw/day), correlating with the organ weights and histopathology data. At histopathology, hepatocellular hypertrophy was seen in the Mid and High dose groups (150, 500 mg/kg bw/day) and hepatocellular microvesicular vacuolation at the High dose (500 mg/kg bw/day); eosinophil droplets and tubular vacuolation were seen in the kidneys only at the High dose. Decreased size/cellularity of the thymus was seen in 2/12 High dose males only, which was attributed to a stress effect rather than a direct effect on this organ. Besides these findings, in one High dose female slight necrosis of the liver was also seen; this single case of this liver degeneration was considered as adverse effect, while the other necropsy findings were attributed to treatment related, adaptive/non-adverse changes. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. However, before the mating period, transient irregular cycles (prolonged diestrus) were seen in the High dose females (500 mg/kg bw/day), although the High dose was unaffected from day 14 when mating started. It is concluded that the irregular cycles were a test item-related secondary effect, caused by the clinical signs and bodyweight losses. As the affected animals were able to get pregnant and deliver healthy litters, this change is considered to be non-adverse. The pups of the High dose group were born with significantly slightly lower bodyweights; this effect was considered as probably secondary to maternal toxicity. The weight gains of High dose pups to day 13 was comparable to controls. Besides this, there were no effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, nipple retention, general development, thyroid gland weights, thyroid hormone level, etc.). As there were no other changes in any of the F1 parameters, the lower birth weights seen in the High dose were considered to be non-adverse. In conclusion, the 28-days NOAEL (oral) for parental systemic toxicity (P0) was determined to be 150 mg/kg bw/day due to the clinical signs (ataxia, diuresis), lower body weight gain and food intake and the liver necrosis observed at the highest dose tested (500 mg/kg bw/day). The NOAELs for reproduction (P0) and developmental toxicity (F1) were determined to be 500 mg/kg bw/day since no test item related adverse effects were observed at the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available with Klimish score of 1.

Additional information

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. However, before the mating period, transient irregular cycles (prolonged diestrus) were seen in the High dose females (500 mg/kg bw/day), although the High dose was unaffected from day 14 when mating started. It is considered that this observation is probably related to the transient ataxia observed during the first week of exposure at the High dose only. As the affected animals were able to get pregnant and deliver healthy litters, this change was considered to be non-adverse.

Effects on developmental toxicity

Description of key information

Key study: OECD Guideline 422. GLP study. The NOAEL for developmental toxicity (F1) was determined to be 500 mg/kg bw/day since no test item related adverse effects were observed at the highest dose tesded.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available with Klimish score of 1.

Additional information

The pups of the High dose group were born with significantly slightly lower bodyweights; this effect was considered as probably secondary to maternal toxicity. The weight gains of High dose pups to day 13 was comparable to controls. Besides this, there were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, nipple retention, general development, thyroid gland weights, thyroid hormone level, etc.). In conclusion, as there were no other changes in any of the F1 parameters, the lower birth weights seen in the High dose were considered to be non-adverse.

Justification for classification or non-classification

Based on available information (no test item related adverse effects observed at the highest dose tested), the substance is not classified for toxicity to reproduction according to the CLP Regulation (EC) no. 1272/2008.