Trimethoxy(2,4,4-trimethylpentyl)silane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan - 4 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan - 4 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 2016

Deviations:

yes

Remarks:

food consumption was not measured in recovery males, clinical observation was not conducted on two days in females.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit- und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 13-15 weeks
- Weight at study initiation: males: 324 - 378 g, females: 203 - 250 g
- Fasting period before study: no
- Housing: 5 animals per sex per cage during pre-mating and post-mating period. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: maintenance diet for rats and mice (Nr. 1324; Altromin, Lage, Germany), ad libitum
- Water: tap water (sulphur acidified to a pH of approximately 2.8), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was prepared at least once every 10 days which is with the stability frame.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline
- Concentration in vehicle: 0, 12.5, 37.5, 75 mg/mL
- Amount of vehicle: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Remarks:

The determination of formulation concentrations of the test substance was based on a validated GC-FID method.

Details on analytical verification of doses or concentrations:

Before the beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study (No. 178283).
Study pre-start stability analysis was conducted on the samples from high dose and low dose group and the investigation was performed for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day at below -15 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item was shown to be homogenous (after at least 30 min without stirring). Consequently, samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
The mean recoveries observed for the low-dose dose group was between 93.2% and 99.1% of the nominal value, between 93.4% and 98.6% for the mid-dose dose group and between 94.3% and 98.6% of the nominal value for high-dose group. The mean recoveries observed in the low-, mid-, and high-dose groups were 96.6%, 95.3%, and 96.6% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Duration of treatment / exposure:

males: 28 days
satellite males: 28 days and 14 days post-exposure period
females: up to 61 days (during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 12)
satellite females: up to 63 days and 14 days post-exposure period

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (main study)
6 (satellite control and high dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level of 300 mg/kg bw/day was chosen on the basis of a dose range finding study, where mortality was observed after repeated oral administration at a dose of 1000 mg/kg bw/day (later reduced to 600 mg/kg bw/day). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects.
- Post-exposure recovery period in satellite groups: Male animals in the recovery groups were observed for a period of 15 days following the last administration and female animals of the recovery groups were treated up to 2 days after the first scheduled necropsy of dams and subjected to necropsy 15 days thereafter (end of recovery period). Satellite animals were not mated.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: health condition, morbidity, moratility

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and at least once a week thereafter
- Observations were made outside the home cage in a standard arena and included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, 9 and 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food weight of male recovery animals was not taken during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment for the main group; for the recovery group additionally in the last week of the recovery period
- Dose groups that were examined: main study: in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated); recovery study: all males and females

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: alanine aminotransferase (ALAT); aspartate-aminotransferase (ASAT); alkaline phosphatase (AP); creatinine (Crea); total protein (TP); albumin (Alb); urea; total bile acids (TBA); total cholesterol (Chol); glucose (Gluc); sodium (Na); potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: specific gravity; nitrite; pH-value (pH); protein; glucose; ketone bodies (Ket); urobilinogen (UBG); bilirubin (BIL); erythrocytes (Ery); leukocytes (Leuc); urine colour and appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week before the first treatment and during the last week of the treatment for main study group; animals of the recovery groups were examined once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. For the recovery groups all males and females were examined.
- Battery of functions tested: sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature

IMMUNOLOGY: No

OTHER: From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination, blood samples were collected. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary, based on the fact that no major histopathological finding was observed in thyroid/ parathyroid gland of selected male and female adult animals and no effect was observed on hormone levels of males and day 13 pups.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All adult animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGHTS: Yes
The wet weight of the following organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each main group was recorded: testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight), spleen, adrenal (paired weight), brain, pituitary gland, heart

HISTOPATHOLOGY: Yes: see table 1
A full histopathology was carried out on the preserved organs and tissues (Table 1) of 5 randomly selected males and females (only lactating females were evaluated) from the control and high dose main groups, which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and the adult animals were examined.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups as well as to animals subjected to necropsy at the end of the recovery period.
For the testes of control and high dose group animals sacrificed at the end of the treatment period, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides as well as the histopathology of interstitial testicular cell structure.

Other examinations:

Oestrous cycles were monitored using vaginal smears for 14 days before start of treatment to select the study females with regular cyclicity. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating. A vaginal smear was also examined on the day of necropsy.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Dunn’s Test, Dunnett’s Test or Student’s T-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

On single study days or only occasionally, most of the animals animals treated with the test substance at 300 mg/kg bw/day were observed moving the bedding with the nose or/and salivating. Moving the bedding was also observed in female animals at 150 mg/kg bw/day. These signs were seen transiently in timely relation to dose administration and were considered as clinical signs elicited by local effects of the test item formulation and/or attributed to the discomfort of the animals due to the oral administration, but not systemic toxicity.
Transiently observed piloerection in few female animals of the high-dose group is considered to be a sign of discomfort most likely related to gavaging and not caused by the test item. Mild background findings, like scratch, hairless area, diarrhoea, transient dehydration. Regurgitation of the formulation was observed at single instances in a control and low-dose animal. Besides, no test item related clinical findings were observed in the treatment period.
Also, during the recovery period, no clinical signs were noted, with the exception of a hairless area observed in a single animal, which is not considered to be test item-related.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period or recovery period of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The test item had no effect on body weight development in this study. Body weights of male and female animals were in the normal range of variation throughout the treatment and recovery period. There were no considerable differences between the means of dose groups and the control group. In female animals a tendency towards lower body weight was observed at the high-dose level during the lactation period, when compared to controls (approx. 5% below controls).

The body weight of female recovery animals of the high-dose group was slightly and statistically significantly lower than body weight of controls on treatment day 14 but not thereafter, including the recovery period. Statistically significant transient variations in body weight gain of female recovery animals between day 7 and 14 and 35 and 49 are not considered toxicologically relevant. In male recovery animals of the high-dose group no significant difference was observed during treatment and at the end of the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The test item had no effect on food consumption in this study. The mean daily food intake of male and female animals was in the normal range of variation throughout the treatment period. A statistical significantly lower food consumption during the first lactation week in the mid- and high-dose group corresponds to the tendency towards lower body weight observed in female animals of this group.
No considerable difference in food consumption between the high-dose group and the control group was observed in female animals during the recovery period of this study. Food consumption of male recovery animals could not be calculated for the recovery period as food weight was not measured at the end of the first recovery week.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmoscopic findings in any of the animals of this study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period no considerable differences were observed in haematological parameters of male and female animals between control and dose groups. A statistically significantly lower rate of basophils in female animals of the mid dose, when compared to controls, is not considered to be biologically relevant.

Also at the end of the recovery period no test item related effect was observed in the high-dose group, when compared to controls. A slight but statistically significantly higher count of platelets in male animals of the high-dose group is not considered to be toxicologically relevant. A slightly but statistically significant longer prothrombin time in males and activated partial thromboplastin time in females of this group is considered to be incidental and without biological relevance. Values were within the range of historical control data. In female animals of the high-dose group mean corpuscular volume and mean corpuscular haemoglobin were minimally but statistically significantly lower than in controls. As values were within the normal range of historical control data, this is not considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on clinical biochemistry parameters of male and female animals analyzed at the end of the treatment period. There were no considerable differences between dose groups and the control group.

Also at the end of the recovery period there were no considerable differences in clinical biochemistry parameters of males and females between high-dose group and control group. Slightly but statistically significant lower alanine aminotransferase and aspartate-aminotransferase levels (28% and 15% below controls, respectively) were observed in male animals of the dose group. Serum aspartate-aminotransferase levels of female high-dsoe group animals were also slightly but statistically significantly lower (18% below controls) compared to controls.
All values were within the range of historical control data and are not considered toxicologically relevant. In treated female animals of the high-dose group a slight but statistically significantly higher cholesterin levels (20% above controls) and statistically significantly lower TBA levels were found. These slight differences are not considered to be toxicologically relevant, as values were within the range of historical data

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T4, coagulating gland histopathology, epididymis histopathology, epididymis weight, estrous cyclicity, liver weight, mammary gland histopathology, ovary histopathology, ovary weight, prostate histopathology (with seminal vesicles and coagulating gland), prostate weight, siminal vesicles histopathology, seminal vesicles weight, testis histopathology, testis weight, thyroid histopathology, thyroid weight, uterus histopathology (with cervix), uterus weight, vagina histopathology, adrenals histopathology, adrenals weight, brain weight, pituitary histopathology and pituitary weight. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on urinary parameters of male and female animals analyzed at the end of the treatment and recovery period.
The single finding of high protein level in one female animal of the mid-dose group is considered to be an incidental finding and not toxicologically relevant.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on functional behavioural parameters evaluated at the end of the treatment and recovery period. There were no considerable differences in body temperature between dose groups and control group. A slight isolated single statistically significant difference in supported rearings observed in females of the mid-dose group compared to controls before treatment is considered incidental and not biologically relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Moderately and statistically significantly higher absolute and relative liver weight was observed in male animals of the high-dose group (19 and 20% above controls, respectively). Slight increases were also seen in the low- and mid-dose groups (between 7% and 11% above controls), where relative liver weights of low-dose males were also statistically significantly higher than in controls. Only a tendency towards an increased liver weight was observed in female animals of these groups.

Markedly but not statistically significantly higher absolute and relative kidney weights were noted in male animals of the high-dose group (18% and 19% above controls, respectively). A tendency was also observed in the low- and mid-dose groups. This coincided with histopathological alterations in the kidneys. No considerable increases in kidney weight were seen in female animals of the dose groups.

Relative pituitary gland weight of female animals of the mid-dose group was slightly and statistically significantly higher than in controls (20% above controls). As this was not observed in the high-dose group, this is not assumed to be test item-related.

The relative adrenal gland weight was slightly but statistically significantly higher in females of the high-dose group, when compared to controls (19 % above controls). This is assumed to be related to slight stress-related hypertrophy.

A 10% increase in relative heart weight of males in the low-dose group, when compared to controls, is not assumed to be toxicologically relevant, as it was not statistically significant and no notable increase was seen in the mid- and high-dose groups. A slightly lower heart weight (10% below controls) was observed in female animals of this group at the end of the recovery period. Changes in heart weight were not associated with histopathological findings and are not assumed to be toxicologically relevant.

Slightly but not statistically significantly higher absolute and relative ovary weight was observed in the high-dose group, when compared to controls (18% and 23% above controls, respectively). This did not coincide with any histomorphological alteration and was therefore considered not toxicologically relevant.

Slightly but not statistically significantly lower absolute uterus weight seen in all test item groups (between 11 and 17% below controls) is not considered toxicologically relevant as it was not associated with any adverse histopathological finding.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only single macroscopic findings were noted at necropsy at the end of the treatment period: in the high-dose group a red ileum wall in male animal no. 32, reduced right seminal vesicles of animal no. 33, red axillary lymph nodes in animal no. 40, red and enlarged right mammary gland of female no. 88 were found. In the mid-dose group enlarged right mandibular lymph node of animal no. 28 and in the low-dose group a red-spotted thymus of female no. 68 were observed. All above-mentioned findings are not assumed to be toxicologically relevant.

At the end of the recovery period dilated uterus was observed in 2/6 control and 1/6 high-dose animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Peripheral Nerves
There was a chronic, peripheral polyneuropathy affecting several studied peripheral nerves (sciatic, brachial, tibial, peroneal, soleus, femoral and dorsal spinal root nerves) in animals (mostly females) treated at 300 mg/kg bw/day from the main and recovery groups. This finding was histologically characterized by peripheral nerve degeneration that includes the following findings: swelling or loss of myelin sheaths (demyelination), with formation of myelin-digestion chambers (Wallerian-type degeneration), and occasional axonal tumefaction.
The brain, spinal cord and dorsal root spinal ganglia remained intact. No lesions are detected in the optic cranial nerve or within other peripheral ganglia occasionally detected in screened organs such as the eye, adrenal gland or reproductive organs. No pathological changes associated with denervation were noticed in the studied muscles.

Urinary Bladder and Kidneys
Adverse histological findings were observed in the urinary system from males and females at ≥150 mg/kg bw/day from main group and at 300 mg/kg bw/day from recovery group.
Findings in the urinary bladder from males and females ≥150 mg/kg bw/day from main and recovery group were characterized by a chronic and diffuse urothelial cell hyperplasia (syn. transitional cell hyperplasia): the mucosa was thickened by simple or papilliform diffuse proliferation of the urothelial cells (more than 4 cell layers) without atypia, often associated with a submucosal mixed inflammatory infiltrate.
In one male from control group (0 mg/kg bw/day) there was a focal area of urothelial hyperplasia of the urinary bladder.
In the kidneys of some rats from both sexes exposed to 300 mg/kg bw/day, there was a urothelial hyperplasia within the renal pelvis, often associated with tubular and pelvic dilation and a mixed inflammatory infiltrate.
Hyaline droplets and tubular basophilia within the renal cortex of males at ≥ 50 mg/kg bw/day mildly exceeded the background incidence levels. The excessive presence of hyaline droplets in the renal cortex might represent intratubular accumulation of protein, and based on the presence only in males most likely an increase in alpha 2-microglobulin. Tubular basophilia is associated with renal tubular regeneration. This is not considered adverse.
Other findings were considered within the range of spontaneous or background lesions in the kidney of rats at this age.
Some cross sections of the ureters and urethra were observed at the level of the prostate or uterus/vagina in some animals and none showed any lesion in the mucosa

Small Intestines
Several males at ≥ 150 mg/kg bw/day from main and recovery groups and one female at 300 mg/kg bw/day (recovery group) showed multifocal lipid accumulation in the duodenum, jejunum and ileum. Lipid deposits appeared as well-defined, empty-like droplets, ranging in size from 15 µm up to 100 µm in the lamina propria, mainly at the tips of the villi inside the lymphatic vessels causing engorgement (lymphangiectasia) or within lipid-laden macrophages. Oil red O technique showed red positive staining of the vacuoles confirming the lipidic composition in the animal No. 32, 37 and 39.

Mesenteric Lymph Node, Peyer’s Patches and Thymus
In several female and male animals at 300 mg/kg bw/day, similar lipid accumulation to the ones describe in the intestine were also multifocally detected in the cortex of the mesenteric lymph nodes with a centripetal distribution. These droplets also presented a positive red stain with Oil Red O staining in animal Nos. 84 and 92 that confirmed the lipidic composition.
Brown pigment deposition (most likely haemosiderin) is mildly increased in treated animals (≥ 50 mg/kg bw/day) from main group compared to control group.
In the Peyer’s patches, the accumulation of lipids at 300 mg/kg bw/day mildly exceeded the background levels in control animals. The minimal and occasional lipid accumulation observed at 50 mg/kg bw/day and 150 mg/kg bw/day is considered in the normal range of background/spontaneous findings this organ in rats.
In the cortex of the thymus, there were multifocal small, well-defined, lipid-containing vacuoles in treated females rats (≥ 50 mg/kg bw/day).

Presence of lipid accumulation in the intestine, Peyer's patches, mensenteric lymph nodes, and thymus from high dose male rats suggests a test-item related impairment in the process of lipid transport through the lateals (small intestine --> Peyer's patches --> mesenteric lymph nodes). The pathogenesis to explain the presence of lipid vacuoles in the thymic cortex is not clear, and its significance is uncertain. Under the conditions of this study and in the absence of associated tissue damage, lipid accumulation in high dose males was considered non-adverse, and deemed to be an adaptative change to alteration in lipid transport from the intestine to the lymphatic vessels.

Liver
Hepatocellular hypertrophy was found to exceed findings in the control group in males and females from the main group treated at ≥ 50 mg/kg bw/day: the cytoplasm appeared hypertrophic with occasional karyomegaly, mainly with centrilobular distribution. This is a relatively common finding following enzymatic induction by xenobiotic and is considered an adaptive response to chemical stress and was not considered to be an adverse effect of the test item. The induced hepatocellular hypertrophy in females at 300 mg/kg bw/day explains the increase in this organ weight.

Lung
Minor histological findings were observed in animals treated at 300 mg/kg bw/day: there was an increase in multifocal perivascular inflammatory infiltrates of granulocytes (perivasculitis) and mixed cellular infiltrates in peribronchiolar areas. In addition, alveolar histiocytosis in female rats exceeded the background incidence levels.

Stress-related
Minor lymphoid depletion was observed in animals treated at 300 mg/kg bw/day at the mandibular lymph nodes, and in few males and females at ≥ 150 mg/kg bw/day in the mesenteric lymph nodes.

The adrenal gland from females and some treated males show also minimal to slight hypertrophy of the fasciculata cortical layer with cytoplasmic vacuolation.


Female Reproductive Organs
No test-item induced histological findings were observed.

Male Reproductive Organs
All the findings within the testes were within the range of normal background lesions that may be recorded in rats at these ages. During sperm staging of PAS stained testicular sections, there were no indicators for any induced lesions.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The test item had no considerable or statistically significant effect on serum T4 levels in male animals at the end of the treatment and recovery period.

The test item had no biologically significant effect on the estrous cycle in treatment groups when compared to the controls prior to mating. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: nervous, urinary

Organ:

bladder

kidney

other: peripheral nerves

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

Under the conditions of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 and in compliance with GLP the systemic NOAEL was determined to be 50 mg/kg bw/day for male and female rats based on adverse polyneuropathy (at 300 mg/kg bw/day) and urinary damage (at ≥150 mg/kg kg/bw).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit- und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxy(2,4,4-trimethylpentyl)silane

EC Number:

251-995-5

EC Name:

Trimethoxy(2,4,4-trimethylpentyl)silane

Cas Number:

34396-03-7

Molecular formula:

C11H26O3Si

IUPAC Name:

trimethoxy(2,4,4-trimethylpentyl)silane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 13-15 weeks
- Weight at study initiation: males: 324 - 378 g, females: 203 - 250 g
- Fasting period before study: no
- Housing: 5 animals per sex per cage during pre-mating and post-mating period. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: maintenance diet for rats and mice (Nr. 1324; Altromin, Lage, Germany), ad libitum
- Water: tap water (sulphur acidified to a pH of approximately 2.8), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at least once every 10 days which is with the stability frame.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 0, 12.5, 37.5, 75 mg/mL
- Amount of vehicle: 4 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug and/or sperm referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages

Analytical verification of doses or concentrations:

yes

Remarks:

The determination of formulation concentrations of the test substance was based on a validated GC-FID method.

Details on analytical verification of doses or concentrations:

Before the beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study (No. 178283).
Study pre-start stability analysis was conducted on the samples from high dose and low dose group and the investigation was performed for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day at below -15 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item was shown to be homogenous (after at least 30 min without stirring). Consequently, samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
The mean recoveries observed for the low-dose dose group was between 93.2% and 99.1% of the nominal value, between 93.4% and 98.6% for the mid-dose dose group and between 94.3% and 98.6% of the nominal value for high-dose group. The mean recoveries observed in the low-, mid-, and high-dose groups were 96.6%, 95.3%, and 96.6% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Duration of treatment / exposure:

males: 28 days
satellite males: 28 days and 14 days post-exposure period
females: up to 61 days (during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 12)
satellite females: up to 63 days and 14 days post-exposure period

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 (main study)
6 (satellite control and high dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level of 300 mg/kg bw/day was chosen on the basis of a dose range finding study, where mortality was observed after repeated oral administration at a dose of 1000 mg/kg bw/day (later reduced to 600 mg/kg bw/day). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects.
- Post-exposure recovery period in satellite groups: Male animals in the recovery groups were observed for a period of 15 days following the last administration and female animals of the recovery groups were treated up to 2 days after the first scheduled necropsy of dams and subjected to necropsy 15 days thereafter (end of recovery period). Satellite animals were not mated.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: health condition, morbidity, moratility

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and at least once a week thereafter
- Observations were made outside the home cage in a standard arena and included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, 9 and 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food weight of male recovery animals was not taken during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination, blood samples were collected. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary, based on the fact that no major histopathological finding was observed in thyroid/ parathyroid gland of selected male and female adult animals and no effect was observed on hormone levels of males and day 13 pups.

Oestrous cyclicity (parental animals):

Oestrous cycles were monitored using vaginal smears for 14 days before start of treatment to select the study females with regular cyclicity. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating. A vaginal smear was also examined on the day of necropsy.

Sperm parameters (parental animals):

For the testes of control and high dose group animals sacrificed at the end of the treatment period, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides as well as the histopathology of interstitial testicular cell structure.

Litter observations:

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

GROSS EXAMINATION OF DEAD PUPS: Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals of the main group were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days).
- Maternal animals: All surviving animals were sacrificed on their respective PND 13.

GROSS NECROPSY
- All adult animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY
- A full histopathology was carried out on the preserved organs and tissues (Table 1) of 5 randomly selected males and females (only lactating females were evaluated) from the control and high dose main groups, which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and the adult animals were examined.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups as well as to animals subjected to necropsy at the end of the recovery period.

ORGAN WEIGHTS
- The wet weight of the following organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each main group was recorded: testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight), spleen, adrenal (paired weight), brain, pituitary gland, heart

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations as follows: From 1 pup/sex/litter/group sacrificed on PND 13 thyroid/parathyroid glands were preserved for potential histopathological examination and weighed after fixation.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Dunn’s Test, Dunnett’s Test or Student’s T-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring on day 4 / No. of live offspring at birth) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

On single study days or only occasionally, most of the animals animals treated with the test substance at 300 mg/kg bw/day were observed moving the bedding with the nose or/and salivating. Moving the bedding was also observed in female animals at 150 mg/kg bw/day. These signs were seen transiently in timely relation to dose administration and were considered as clinical signs elicited by local effects of the test item formulation and/or attributed to the discomfort of the animals due to the oral administration, but not systemic toxicity.
Transiently observed piloerection in few female animals of the high-dose group is considered to be a sign of discomfort most likely related to gavaging and not caused by the test item. Mild background findings, like scratch, hairless area, diarrhoea, transient dehydration. Regurgitation of the formulation was observed at single instances in a control and low-dose animal. Besides, no test item related clinical findings were observed in the treatment period.
Also, during the recovery period, no clinical signs were noted, with the exception of a hairless area observed in a single animal, which is not considered to be test item-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period or recovery period of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The test item had no effect on body weight development in this study. Body weights of male and female animals were in the normal range of variation throughout the treatment and recovery period of this study. There were no considerable differences between the means of dose groups and the control group. In female animals a tendency towards lower body weight was observed at the high-dose level during the lactation period, when compared to controls (approx. 5% below controls).

The body weight of female recovery animals of the high-dose group was slightly and statistically significantly lower than body weight of controls on treatment day 14 but not thereafter, including the recovery period. Statistically significant transient variations in body weight gain of female recovery animals between day 7 and 14 and 35 and 49 are not considered toxicologically relevant. In male recovery animals of the high-dose group no significant difference was observed during treatment and at the end of the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The test item had no effect on food consumption in this study. The mean daily food intake of male and female animals was in the normal range of variation throughout the treatment period. A statistical significantly lower food consumption during the first lactation week in the mid- and high-dose group corresponds to the tendency towards lower body weight observed in female animals of this group.
No considerable difference in food consumption between the high-dose group and the control group was observed in female animals during the recovery period of this study. Food consumption of male recovery animals could not be calculated for the recovery period as food weight was not measured at the end of the first recovery week.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmoscopic findings in any of the animals of this study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period no considerable differences were observed in haematological parameters of male and female animals between control and dose groups. A statistically significantly lower rate of basophils in female animals of the mid dose, when compared to controls, is not considered biologically relevant.

Also at the end of the recovery period no test item related effect was observed in the high-dose group, when compared to controls. A slight but statistically significantly higher count of platelets in male animals of the high-dose group is not considered toxicologically relevant. A slightly but statistically significant longer prothrombin time in males and activated partial thromboplastin time in females of this group is considered incidental and without biological relevance. Values were within the range of historical control data. In female animals of the high-dose group mean corpuscular volume and mean corpuscular haemoglobin were minimally but statistically significantly lower than in controls. As values were within the normal range of historical control data, this is not considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on clinical biochemistry parameters of male and female animals analyzed at the end of the treatment period. There were no considerable differences between dose groups and the control group.

Also at the end of the recovery period there were no considerable differences in clinical biochemistry parameters of males and females between high-dose group and control group. Slightly but statistically significant lower alanine aminotransferase and aspartate-aminotransferase levels (28 % and 15 % below controls, respectively) were observed in male animals of the dose group. Serum aspartate-aminotransferase levels of female high-dsoe group animals were also slightly but statistically significantly lower (18 % below controls) compared to controls.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T4, coagulating gland histopathology, epididymis histopathology, epididymis weight, estrous cyclicity, liver weight, mammary gland histopathology, ovary histopathology, ovary weight, prostate histopathology (with seminal vesicles and coagulating gland), prostate weight, siminal vesicles histopathology, seminal vesicles weight, testis histopathology, testis weight, thyroid histopathology, thyroid weight, uterus histopathology (with cervix), uterus weight, vagina histopathology, adrenals histopathology, adrenals weight, brain weight, pituitary histopathology and pituitary weight. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on urinary parameters of male and female animals analyzed at the end of the treatment and recovery period.
The single finding of high protein level in one female animal of the mid-dose group is considered to be an incidental finding and not toxicologically relevant.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on functional behavioural parameters evaluated at the end of the treatment and recovery period. There were no considerable differences in body temperature between dose groups and control group. A slight isolated single statistically significant difference in supported rearings observed in females of the mid-dose group compared to controls before treatment is considered incidental and not biologically relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Peripheral Nerves
There was a chronic, peripheral polyneuropathy affecting several studied peripheral nerves (sciatic, brachial, tibial, peroneal, soleus, femoral and dorsal spinal root nerves) in animals (mostly females) treated at 300 mg/kg bw/day from the main and recovery groups. This finding was histologically characterized by peripheral nerve degeneration that includes the following findings: swelling or loss of myelin sheaths (demyelination), with formation of myelin-digestion chambers (Wallerian-type degeneration), and occasional axonal tumefaction.
The brain, spinal cord and dorsal root spinal ganglia remained intact. No lesions are detected in the optic cranial nerve or within other peripheral ganglia occasionally detected in screened organs such as the eye, adrenal gland or reproductive organs. No pathological changes associated with denervation were noticed in the studied muscles.

Urinary Bladder and Kidneys
Adverse histological findings were observed in the urinary system from males and females at ≥150 mg/kg bw/day from main group and at 300 mg/kg bw/day from recovery group.
Findings in the urinary bladder from males and females ≥150 mg/kg bw/day from main and recovery group were characterized by a chronic and diffuse urothelial cell hyperplasia (syn. transitional cell hyperplasia): the mucosa was thickened by simple or papilliform diffuse proliferation of the urothelial cells (more than 4 cell layers) without atypia, often associated with a submucosal mixed inflammatory infiltrate.
In one male from control group (0 mg/kg bw/day) there was a focal area of urothelial hyperplasia of the urinary bladder.
In the kidneys of some rats from both sexes exposed to 300 mg/kg bw/day, there was a urothelial hyperplasia within the renal pelvis, often associated with tubular and pelvic dilation and a mixed inflammatory infiltrate.
Hyaline droplets and tubular basophilia within the renal cortex of males at ≥ 50 mg/kg bw/day mildly exceeded the background incidence levels. The excessive presence of hyaline droplets in the renal cortex might represent intratubular accumulation of protein, and based on the presence only in males most likely an increase in alpha 2-microglobulin. Tubular basophilia is associated with renal tubular regeneration. This is not considered adverse.
Other findings were considered within the range of spontaneous or background lesions in the kidney of rats at this age.
Some cross sections of the ureters and urethra were observed at the level of the prostate or uterus/vagina in some animals and none showed any lesion in the mucosa

Small Intestines
Several males at ≥ 150 mg/kg bw/day from main and recovery groups and one female at 300 mg/kg bw/day (recovery group) showed multifocal lipid accumulation in the duodenum, jejunum and ileum. Lipid deposits appeared as well-defined, empty-like droplets, ranging in size from 15 µm up to 100 µm in the lamina propria, mainly at the tips of the villi inside the lymphatic vessels causing engorgement (lymphangiectasia) or within lipid-laden macrophages. Oil red O technique showed red positive staining of the vacuoles confirming the lipidic composition in the animal No. 32, 37 and 39.

Mesenteric Lymph Node, Peyer’s Patches and Thymus
In several female and male animals at 300 mg/kg bw/day, similar lipid accumulation to the ones describe in the intestine were also multifocally detected in the cortex of the mesenteric lymph nodes with a centripetal distribution. These droplets also presented a positive red stain with Oil Red O staining in animal Nos. 84 and 92 that confirmed the lipidic composition.
Brown pigment deposition (most likely haemosiderin) is mildly increased in treated animals (≥ 50 mg/kg bw/day) from main group compared to control group.
In the Peyer’s patches, the accumulation of lipids at 300 mg/kg bw/day mildly exceeded the background levels in control animals. The minimal and occasional lipid accumulation observed at 50 mg/kg bw/day and 150 mg/kg bw/day is considered in the normal range of background/spontaneous findings this organ in rats.
In the cortex of the thymus, there were multifocal small, well-defined, lipid-containing vacuoles in treated females rats (≥ 50 mg/kg bw/day).

Presence of lipid accumulation in the intestine, Peyer's patches, mensenteric lymph nodes, and thymus from high dose male rats suggests a test-item related impairment in the process of lipid transport through the lateals (small intestine --> Peyer's patches --> mesenteric lymph nodes). The pathogenesis to explain the presence of lipid vacuoles in the thymic cortex is not clear, and its significance is uncertain. Under the conditions of this study and in the absence of associated tissue damage, lipid accumulation in high dose males was considered non-adverse, and deemed to be an adaptative change to alteration in lipid transport from the intestine to the lymphatic vessels.

Liver
Hepatocellular hypertrophy was found to exceed findings in the control group in males and females from the main group treated at ≥ 50 mg/kg bw/day: the cytoplasm appeared hypertrophic with occasional karyomegaly, mainly with centrilobular distribution. This is a relatively common finding following enzymatic induction by xenobiotic and is considered an adaptive response to chemical stress and was not considered to be an adverse effect of the test item. The induced hepatocellular hypertrophy in females at 300 mg/kg bw/day explains the increase in this organ weight.

Lung
Minor histological findings were observed in animals treated at 300 mg/kg bw/day: there was an increase in multifocal perivascular inflammatory infiltrates of granulocytes (perivasculitis) and mixed cellular infiltrates in peribronchiolar areas. In addition, alveolar histiocytosis in female rats exceeded the background incidence levels.

Stress-related
Minor lymphoid depletion was observed in animals treated at 300 mg/kg bw/day at the mandibular lymph nodes, and in few males and females at ≥ 150 mg/kg bw/day in the mesenteric lymph nodes.

The adrenal gland from females and some treated males show also minimal to slight hypertrophy of the fasciculata cortical layer with cytoplasmic vacuolation.


Female Reproductive Organs
No test-item induced histological findings were observed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The test item had no considerable or statistically significant effect on serum T4 levels in male animals at the end of the treatment and recovery period.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The test item had no biologically significant effect on the estrous cycle in treatment groups when compared to the controls prior to mating. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

All the findings within the testes were within the range of normal background lesions that may be recorded in rats at these ages. During sperm staging of PAS stained testicular sections, there were no indicators for any induced lesions.

Reproductive performance:

no effects observed

Description (incidence and severity):

In each groups, 10/10 animals were pregnant. There was no test item-related or statistically significant effect on the duration of the precoital interval and duration of gestation in the dose groups when compared to the control group. Maternal behavior was normal in all groups.
There were no test item-related effects on number of corpora lutea or implantation sites, live pups on PND 0, 4 and 13, percent pre-implantation loss and post-implantation loss in low-, mid- and high-dose groups when compared with the control group.
There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse reproductive effects observed.

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: nervous, urinary

Organ:

bladder

kidney

other: peripheral nerves

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

All 3 pup recorded at first litter check were found dead at PND 0 for one high-dose female (No. 86). Furthermore, one female (No. 61) of the control group was recorded sperm positive but did not litter. In the secluded final necropsy on post mating date 26 one implantation site and 13 corpora lutea were recorded. Values are well in range of historical control data are therefore not considered to be test item related.
No test item-related effect was observed on mortality of pups between PND 0 and PND 4 and between PND 4-13 in low-, mid- and high-dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related or statistically significant effect on male and female litter weight on PND 0 and PND 4 in test item treated animals, when compared with the controls.
A slightly but statistically significantly lower pup mean weight in the high-dose group on PND13 and a tendency towards a slightly lower total and male litter weight coincided with a slightly lower body weight of dams during the lactation period, but is not considered as test item related developmental toxicity.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

In male pups of the high-dose group slightly but statistically significantly smaller absolute (approx. 9% below controls) and relative (approx. 6% below controls) anogenital distance (AGD) was found. Contrary to this, the anogenital distance of female pups of the mid- and high-dose groups was slightly but statistically significantly larger than in controls (absolute AGD: 12% and 18% above controls, respectively and relative AGD: 13% above controls, respectively).
These slight differences in male and female animals are not assumed to be toxicologically relevant as values were in the range of historical control data.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

The retention of nipples in male animals on PND 13 was statistically significantly more frequent in the low-dose group when compared to controls. Due to a smaller mean number in the mid-dose group and a high variability in the high-dose group no dose-dependency was observed. Moreover, the low-dose mean was within the range of historical control data. Thus, this is not considered to be a test item related relevant effect.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effect on thyroid gland weight (post-fixation) was noted for male and female pups on PND13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pup external findings:
One pup of the control group was small.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels of male and female PND 13 pups were also not affected by the test item. The values of the high-dose group pups did not considerably or statistically significantly differ from the values of the respective control pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter data:
There were no test item-related or statistically significant effects observed in low-, mid- and high-dose groups on litter data parameters like total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runts on PND 0 as well as number of live pups, number of male and female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 and in compliance with GLP the systemic NOAEL was determined to be 50 mg/kg bw/day for male and female rats based on adverse polyneuropathy (at 300 mg/kg bw/day) and urinary damage (at ≥150 mg/kg bw/day). Reproductive and developmental parameters were not affected. Therefore, the NOAELs for reproductive toxicity and developmental toxicity were considered to be ≥300 mg/kg bw/day.