Trimethoxy(2,4,4-trimethylpentyl)silane

Endpoint summary

Description of key information

Oral:

OECD 422, rat: NOAEL (systemic) = 50 mg/kg bw/day

OECD 408, rat: NOAEL (systemic) ≥150 mg/kg bw/day

Inhalation:

OECD 412, rat: NOAEC (systemic) = 2890 mg/m³ air

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jan - 20 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 133 – 177 g males; 100 – 142 g females
- Housing: Up to 5 animals of the same sex and same dosing group together in polycarbonate cages
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH provided ad libitum
- Water: Municipal tap water provided ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:
Periodic analysis of the water was performed. Results of analysis for nutritional components and environmental contaminants of the diet were provided by the supplier. There were no known contaminants in the water and feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 39 - 68
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations (w/w) were homogenized to visually acceptable levels. The formulations were prepared weekly and stored at 2-8°C.
An adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 5, 12.5 and 37.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Specific gravity: 0.92

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in Week 1, 6 and 12 for substance concentration verification of all dose groups and for homogeneity investigation in the low and high-dose group. For the homogeneity investigation the samples were collected from various levels (top, middle, bottom).

Accuracy
The concentrations analyzed in the formulations of 20, 50 and 150 mg/kg bw/day dose groups were in agreement with target concentrations (i.e. mean sample concentration results were within 90% and 110% of target concentration).
A small response at the retention time of the test item was observed in the chromatograms of the control group formulation prepared for use in Week 6. It was considered not to derive from the test item since the response was in the range of noise response. The maximum contribution to the Group 2 samples was 0.020%. In the control group formulations for use in Weeks 1 ad 12, no response at the retention time of the test item was detected.
Homogeneity
The formulations of 20 and 150 mg/kg bw/day dose groups were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

at least 90 days (control and test groups) and 28 days post-exposure observation period (recovery groups for control and high-dose)

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (main study)
5 (satellite control and high-dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on information provided by the Sponsor and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
In a previously conducted 50-day (female) and 30-day (male) repeated dose toxicity (OECD 422) study, rats were treated at 50, 150 and 300 mg/kg bw/day. Adverse polyneuropathy, hyperplasia of the bladder and hyperplasia of the renal pelvis were seen at 300 mg/kg bw/day. Hyperplasia of the bladder was also observed at 150 mg/kg bw/day. The dose levels of current study are based on the expectation that prolonged exposure (90 days) to the test item could lead to adverse polyneuropathy at 150 mg/kg bw/day. In addition, a 14-day dose range finder performed to select the dose levels for the 50- and 30-day repeated dose OECD 422, showed mortalitiy at 1000 and 600 mg/kg bw/day.

- Fasting period before blood sampling for clinical biochemistry: overnight with a maximum of 24 h
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily for cage side observations; at least twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-treatment and weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

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: Yes
- Time schedule for examinations: Regular basis throughout study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pretreatment period and during Week 13 of the main study. At the end of recovery period only if treatment-related effects are seen at the end of the treatment.
- Dose groups that were examined: control and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: White Blood Cell Count (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red Blood Cell Count, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets; Prothrombin time (PT); Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Triglycerides, HDL and LDL Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos), Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

SERUM HORMONES: Yes
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12-13 (main study); once during Week 17 (recovery period)
- Dose groups that were examined: all dose groups (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength; locomotor activity

IMMUNOLOGY: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes (see table 1)

Optional endpoint(s):

None

Other examinations:

Estrus stage for all females was determined by taking vaginal smears at the end of treatment or recovery period on the day of necropsy.

Statistics:

For body weights, body weight gains, haematology variables, coagulation variables, clinical chemistry variables, functional observation battery variables (quantitative), organ weights and organ weights relative to body weight, Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

For qualitative functional observation battery variables Fisher’s exact test were used to conduct pairwise group comparisons of interest.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were observed during the Dosing Period. During the Recovery Period, no clinical signs were observed.

Salivation was seen after dosing in 1 and 3 females dosed at 20 and 50 mg/kg bw/day, respectively, and in 6 females and 11 males dosed at 150 mg/kg bw/day. This was considered not to be toxicologically relevant, taking into account the nature and minor severity of the effects and its time of occurrence (i.e., after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Any other clinical signs (e.g., bent tail, broken teeth, skin lesion and skin scab) noted during the Dosing Period occurred within the range to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item.
Any statistically significant changes in body weight gain were considered to be unrelated to treatment with the test item, since no trend was apparent regarding dose and duration of the treatment and/or changes were seen at end of the Recovery Period only.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption were recorded during the Dosing and the Recovery Period.
Any differences seen during the Recovery Period were considered to be not toxicologically relevant as no test item-related changes in food consumption were observed during the Dosing Period.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted in Week 13 (i.e. last week of treatment) was limited to the control animals and/or observed in the pre-treatment period only.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters were considered not to have been affected by treatment with the test item.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.

At the end of the Recovery Period, a slightly shorter prothrombin time (PT) was observed in males at 150 mg/kg bw/day (0.96x of control). As no effect was observed in animals at the end of the Dosing Period, and the opposite effect (i.e. an increase) would be expected in case of target organ toxicity, this was considered a chance finding.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes were noted in clinical biochemistry parameters in males up to 150 mg/kg bw/day.

In females at 150 mg/kg bw/day, a slight increase in total protein (TPROT), albumin (ALB) and calcium (CA) concentration was observed at the end of the Dosing Period (1.06x, 1.06x and 1.07x of control, respectively) which normalized at the end of the Recovery Period.

Variable, not statistically significant, results were observed in the thyroid stimulating hormone (TSH) concentrations, without a clear pattern. Lower TSH values were observed in males dosed at 20 mg/kg bw/day (0.59x of control), while TSH in males dosed at 50 and 150 mg/kg bw/day were comparable to control values (0.9x and 0.83x of control, respectively). Lower TSH concentrations were observed in females dosed at 20, 50 and 150 mg/kg bw/day (0.75x, 0.72x and 0.75x of control, respectively). At the end of the Recovery Period, TSH concentration was slightly higher in males at 150 mg/kg bw/day (1.10x compared to control), while TSH concentration in females at 150 mg/kg bw/day was considerably lower (0.54x of control). These differences in TSH parameters were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Remaining differences in clinical chemistry parameters (including creatinine and inorganic phosphate) at end of Treatment or end of Recovery Period, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of the control values, were of a magnitude of change commonly observed in rats under similar study conditions and/or were likely the result of a relatively low control value.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4, TSH, coagulating gland histopathology, epididymis histopathology, epididymis weight, 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, vaginal smears, 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:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals during the Dosing and Recovery Period.

Males at 150 mg/kg bw/day showed a lower mean grip strength of the foreleg compared to the control at the end of the Dosing Period, which was not present at the end of the Recovery Period. In females, no effect on grip strength was observed during the Dosing Period. At the End of Recovery, females at 150 mg/kg bw/day showed a lower mean grip strength of the hindleg compared to the control. As these effects were not observed at the end of Dosing Period for females at 150 mg/kg bw/day and/or remained within the range of historical control data, these findings were considered unrelated to the treatment with the test item.

Motor activity was considered similar between control and high dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.

Some absolute organ weight differences reached statistical significance. These were noted in females at 150 mg/kg bw/day at the end of the Dosing Period in the kidney and liver, and in Recovery males at 150 mg/kg bw/day in the pituitary gland and were regarded to be related to the higher terminal body weights of these dose groups.

Any other organ weight differences (significant for mean relative kidney weight of males at 20 and 150 mg/kg bw/day and females at 150 mg/kg bw/day at end of the Dosing Period) were considered not test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the urinary bladder of the 50 and/or 150 mg/kg bw/day group males and females; see table 2.

Diffuse hyperplasia of the urothelium of the urinary bladder was noted in 2/10 females of the 50 mg/kg bw/day group (minimal) and in 10/10 males and 9/10 females of the 150 mg/kg bw/day group (up to mild). In general, the hyperplastic cells formed a uniform linear thickening (simple growth pattern) and was seen without cellular atypia.

In a single male at 150 mg/kg day/day, the hyperplasia was accompanied by minimal submucosal infiltrate. In 2 and 1 females at 150 mg/kg bw/day the hyperplasia was accompanied by minimal mucosal lymphocytic infiltrate and mild submucosal (follicular) lymphocytic infiltrate, respectively.

After a 28-day treatment free period, the urothelial hyperplasia was present at a minimal degree in 3/4 males and 3/5 females of the 150 mg/kg bw/day group. There was no evidence for lymphocytic (sub)mucosal infiltrates. This was interpreted as partial recovery.

Based on earlier study results, where test item-related polyneuropathy had occurred at 300 mg/kg bw/day (performed at another test facility), an extensive examination of skeletal muscles and peripheral nerves was performed in the current study. There was no evidence of any test item-related finding in these organs. This was also the case for the other target organs (i.e. kidneys of males, liver, small intestines, mesenteric lymph node and thymus) identified in the previous study.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Table 2: Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

	Main Animals				Recovery Animals
Dose level (mg/kg bw/day):	0	20	50	150	0	150
URINARY BLADDER-MALES^a	10	8	10	10	5	4
Hyperplasia, urothelial, diffuse
Minimal	0	0	0	4	0	3
Mild	0	0	0	6	0	0
Infiltrate, lymphocytic, (sub)mucosal
Minimal	0	0	0	1	0	0
Mild	0	0	0	0	0	0
URINARY BLADDER-FEMALES^a	10	10	10	10	5	5
Hyperplasia, urothelial, diffuse
Minimal	0	0	2	4	0	3
Mild	0	0	0	5	0	0
Infiltrate, lymphocytic, (sub)mucosal
Minimal	0	0	0	2	0	0
Mild	0	0	0	1	0	0

^a = Number of tissues examined from each group.

Conclusions:

The test item was tested for subchronic oral toxicity according to OECD TG 408 and in compliance with GLP. The systemic NOAEL was determined to be ≥150 mg/kg bw/day for male and female rats. Non-adverse morphologic changes in the urinary bladder of females at 50 mg/kg bw/day and in both sexes at 150 mg/kg bw/day were noted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

other: urinary and peripheral nervous system

Organ:

bladder

other: peripheral nerves

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar (Hoe: WISKf(SPF71))
- Source: Hoechst AG, Pharma-Forschung-Toxikologie, Kastengrund (breeding under SPF conditions)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males: 137-150 g (mean = 143.3± 3.74), females: 137-146 g (mean = 141.0±1.29)
- Fasting period before study: no
- Housing: individually during exposure, 5 per Makrolon cage (type 4) after exposure
- Diet: Altromin 1324-pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum except during exposure
- Water: tap water ad libitum except during exposure


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 50±20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 (7.00-19.00)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: air

Remarks on MMAD:

MMAD / GSD: Particle size (mean): 99.98% of the particle in 0.3 mg/L group, 99.98% of the particle in 1.5 mg/L group and 99.87% of the particle in 3.0 mg/L group are below 6 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Animals were exposed to the test atmosphere in nose-only inhalation units
- Exposure apparatus: The inhalation chambers used in the study were stainless steel/glass cylindrical columns placed in a hood with a volume of approximately 4 m³. Each column had a volume of 80 L and consisted of a top assembly with the inlet of the test atmosphere, one rodent tube section and the bottom, the base assembly with the exhaust port.
- Method of holding animals in test chamber: Nose only; cylindrical tubes.
- Method of conditioning air: Compressed air (4 bar), oil separation filter/absolute filter; to achieve requested air humidity moistened air was feeded directly in the chamber
- System of generating particulates/aerosols: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing test material through special nozzles. The operating pressure was 4 bar.
- Temperature, humidity: 20.0-23.5°C, 31.5-60.6%
- Continuous measurement of CO-, CO2- and O2-concentration during exposure in exposure chamber; CO: 0 ppm; CO2: 3800-7900 ppm; O2: 19.8-20.6 Vol %
- Air flow rate: Air inlet 800 L/h; 1100 L/h exhausted
- Method of particle size determination: The particle size distribution was measured using an APS 33 Aerodynamik Particle Sizer from TSI Inc., St Paul. The aerodynamic diameter range measured with this analyser covered 0.486 to > 15.4 micrometer. Measurements were performed daily 30 minutes, 2 and 4 hours respectively after starting the exposure. After the end of the exposure period, the arithmetic means together with the standard deviations based on the 3 points in time were calculated using a statistics program. The statistics program was used further to calculate an overall arithmetic mean for each dose group based on the arithmetic means for each single day of exposure.
- Treatment of exhaust air: Escaped particles were exhausted by a gas purifying plant and neutralised. Additionally, there was an exhaust device at the bottom of each inhalation chamber to remove the aerosol by filtering over a Buehler-Filter and a gas-washing bottle.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric verification of concentrations using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (Sartorius Membranfilter GmbH, Goettingen). Samples were taken daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.
Chemical verification of concentrations was performed using a gas chromatograph. Sampling was performed on days 1, 8, 15, 22, and 27 of the treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric verification of concentrations
Gravimetric measurements were performed using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (sartorius Membranfilter GmbH, Goettingen). The air flow rate was 3 L/min, which is equivalent to an intake velocity of 1.25 m/sec. Gravimetric measurements were performed daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.

Chemical verification of concentrations
Within 60 minutes 31 liter aerosol from the exposure chambers was pumped through 3 gas-washing bottles filled with acetone (PESTANAL, RIEDEL DE HAEN) connected in series and standing in a cool trap. Thereof aliquots were taken and analysed using a gas chromatograph. The concentration of isooctyltrimethoxysilane in the exposure chambers was calculated based on the results of the GC and considering the aerosol- and acetone volumina. Sampling was performed on days 1, 8, 15, 22 and 27 of the treatment.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.32 mg/L air (analytical)

Dose / conc.:

1.54 mg/L air (analytical)

Dose / conc.:

2.89 mg/L air (analytical)

Dose / conc.:

0.3 mg/L air (nominal)

Dose / conc.:

1.5 mg/L air (nominal)

Dose / conc.:

3 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Five animals of each sex and group were used to assess recovery from treatment-related effects
- Post-exposure recovery period in satellite groups: 15 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Mortality, behaviour and general state of health (before and after exposure and during exposure; one time per day during weekend); Neurological disorders, opacity of eyes, damage of oral mucosa, disorder of tooth growth (weekly)

BODY WEIGHT: Yes
- Time schedule for examinations: Before exposure began and then twice per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Twice per week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All dose groups and control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex (Nembutal narcosis)
- Parameter checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations (neurological disorders): Once per week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

None

Statistics:

The following parameters have been checked inter-collective concerning statistical significance (p = 0.05) in accordance with internal SOP: body weight, body weight gain; haematology (excluding differential blood count, heinz bodies, reticulocytes); clinical chemistry (excluding bilirubin direct, methaemoglobin, g-glutamyltranspeptitase); serum electrophoresis, relative organ weights, pH-value urine.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)

Mortality:

mortality observed, treatment-related

Description (incidence):

staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

increased for high dose males on day 7, high dose females on days 7-21, mid dose males on days 14 until recovery, and mid dose females on days 14-29

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

3 males and 1 female of the high dose group had signs of minimal intense irritation in the alveoli.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. One animal additionally showed ruffled fur and retracted belly flanks on day 10.
In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. The low dose and control group animals did not have any clinical signs.
No clinical signs were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN:
On days 1 and 3 males of the high and mid dose group had a slightly increased mean body weight (10 animals each dose group, experimental period) in comparison to the control animals. Females of the high dose group had a slightly reduced mean body weight on day 14 and days 21-29 (10 animals, experimental period) and days 29-31 (5 animals, recovery period) of the recovery period.

FOOD CONSUMPTION:
Males from high and mid dose groups on days 3-29 (experimental period) and females on days 3-29 (experimental period) and days 29-31 (recovery period) had slight reductions in feed intake.

WATER CONSUMPTION:
In high dose males there was an increased water consumption on day 7 until the end of the experimental period. Females of this group had elevated water consumption on days 7-29 (experimental period). In the mid dose group males, water consumption was increased from day 14 until start of recovery period, and in females of this group from day 14-29 (experimental period).

OPHTHALMOSCOPIC EXAMINATION:
No effects on the eyes.

HAEMATOLOGY:
Statistically significant effects one day after the end of exposure were as follows: increased albumin in mid dose females, increased alpha2 globulin in high dose males, decreased alpha3 globulin in mid and high dose females, increased albumin to globulin ratio in mid dose females.
Statistically significant effects 15 days after the end of exposure (recovery group) were as follows: increased albumin in low dose males and high dose males and females, decreased beta1 globulin in low and high dose males, decreased g1 globulin in low dose male and females and high dose females, and increased albumin to globulin ratio in low and high dose males. However, all values were in the normal range and were not considered to be toxicologically relevant.

URINALYSIS:
No treatment-related effects.

NEUROBEHAVIOUR:
Not examined but there were no neurological disorders observed in the clinical observations.

ORGAN WEIGHTS:
There were statistically significant increases in lung weight in mid dose males one day after exposure, and lung weight in high dose females sacrificed 15 days after exposure. However, all values were within the normal range, and there were no corresponding histopathological findings. Therefore, the effect was considered non-adverse.

GROSS PATHOLOGY:
There were no treatment-related macroscopic changes identified.

HISTOPATHOLOGY:
There were no treatment related findings in the control, low and mid dose groups. In the high dose group three males and one female, which were sacrificed one day after the last exposure, had signs of minimal intense irritation in the alveoli (isolated and small clusters of foam cells). There were no other treatment-related effects.

Dose descriptor:

NOAEC

Effect level:

ca. 3 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.

Dose descriptor:

NOAEC

Effect level:

2 890 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.

Critical effects observed:

no

Conclusions:

The test item was tested for subacute inhalation toxicity according to OECD TG 412 and in compliance with GLP. The NOAEC was determined to be 3000 mg/m³ (nominal). No specific target organ toxicity was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 890 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was according an apropriate OECD test guideline, and in compliance with GLP.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar (Hoe: WISKf(SPF71))
- Source: Hoechst AG, Pharma-Forschung-Toxikologie, Kastengrund (breeding under SPF conditions)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males: 137-150 g (mean = 143.3± 3.74), females: 137-146 g (mean = 141.0±1.29)
- Fasting period before study: no
- Housing: individually during exposure, 5 per Makrolon cage (type 4) after exposure
- Diet: Altromin 1324-pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum except during exposure
- Water: tap water ad libitum except during exposure


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 50±20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 (7.00-19.00)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: air

Remarks on MMAD:

MMAD / GSD: Particle size (mean): 99.98% of the particle in 0.3 mg/L group, 99.98% of the particle in 1.5 mg/L group and 99.87% of the particle in 3.0 mg/L group are below 6 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Animals were exposed to the test atmosphere in nose-only inhalation units
- Exposure apparatus: The inhalation chambers used in the study were stainless steel/glass cylindrical columns placed in a hood with a volume of approximately 4 m³. Each column had a volume of 80 L and consisted of a top assembly with the inlet of the test atmosphere, one rodent tube section and the bottom, the base assembly with the exhaust port.
- Method of holding animals in test chamber: Nose only; cylindrical tubes.
- Method of conditioning air: Compressed air (4 bar), oil separation filter/absolute filter; to achieve requested air humidity moistened air was feeded directly in the chamber
- System of generating particulates/aerosols: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing test material through special nozzles. The operating pressure was 4 bar.
- Temperature, humidity: 20.0-23.5°C, 31.5-60.6%
- Continuous measurement of CO-, CO2- and O2-concentration during exposure in exposure chamber; CO: 0 ppm; CO2: 3800-7900 ppm; O2: 19.8-20.6 Vol %
- Air flow rate: Air inlet 800 L/h; 1100 L/h exhausted
- Method of particle size determination: The particle size distribution was measured using an APS 33 Aerodynamik Particle Sizer from TSI Inc., St Paul. The aerodynamic diameter range measured with this analyser covered 0.486 to > 15.4 micrometer. Measurements were performed daily 30 minutes, 2 and 4 hours respectively after starting the exposure. After the end of the exposure period, the arithmetic means together with the standard deviations based on the 3 points in time were calculated using a statistics program. The statistics program was used further to calculate an overall arithmetic mean for each dose group based on the arithmetic means for each single day of exposure.
- Treatment of exhaust air: Escaped particles were exhausted by a gas purifying plant and neutralised. Additionally, there was an exhaust device at the bottom of each inhalation chamber to remove the aerosol by filtering over a Buehler-Filter and a gas-washing bottle.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric verification of concentrations using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (Sartorius Membranfilter GmbH, Goettingen). Samples were taken daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.
Chemical verification of concentrations was performed using a gas chromatograph. Sampling was performed on days 1, 8, 15, 22, and 27 of the treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric verification of concentrations
Gravimetric measurements were performed using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (sartorius Membranfilter GmbH, Goettingen). The air flow rate was 3 L/min, which is equivalent to an intake velocity of 1.25 m/sec. Gravimetric measurements were performed daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.

Chemical verification of concentrations
Within 60 minutes 31 liter aerosol from the exposure chambers was pumped through 3 gas-washing bottles filled with acetone (PESTANAL, RIEDEL DE HAEN) connected in series and standing in a cool trap. Thereof aliquots were taken and analysed using a gas chromatograph. The concentration of isooctyltrimethoxysilane in the exposure chambers was calculated based on the results of the GC and considering the aerosol- and acetone volumina. Sampling was performed on days 1, 8, 15, 22 and 27 of the treatment.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.32 mg/L air (analytical)

Dose / conc.:

1.54 mg/L air (analytical)

Dose / conc.:

2.89 mg/L air (analytical)

Dose / conc.:

0.3 mg/L air (nominal)

Dose / conc.:

1.5 mg/L air (nominal)

Dose / conc.:

3 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Five animals of each sex and group were used to assess recovery from treatment-related effects
- Post-exposure recovery period in satellite groups: 15 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Mortality, behaviour and general state of health (before and after exposure and during exposure; one time per day during weekend); Neurological disorders, opacity of eyes, damage of oral mucosa, disorder of tooth growth (weekly)

BODY WEIGHT: Yes
- Time schedule for examinations: Before exposure began and then twice per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Twice per week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All dose groups and control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex (Nembutal narcosis)
- Parameter checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations (neurological disorders): Once per week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

None

Statistics:

The following parameters have been checked inter-collective concerning statistical significance (p = 0.05) in accordance with internal SOP: body weight, body weight gain; haematology (excluding differential blood count, heinz bodies, reticulocytes); clinical chemistry (excluding bilirubin direct, methaemoglobin, g-glutamyltranspeptitase); serum electrophoresis, relative organ weights, pH-value urine.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)

Mortality:

mortality observed, treatment-related

Description (incidence):

staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

increased for high dose males on day 7, high dose females on days 7-21, mid dose males on days 14 until recovery, and mid dose females on days 14-29

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

3 males and 1 female of the high dose group had signs of minimal intense irritation in the alveoli.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. One animal additionally showed ruffled fur and retracted belly flanks on day 10.
In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. The low dose and control group animals did not have any clinical signs.
No clinical signs were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN:
On days 1 and 3 males of the high and mid dose group had a slightly increased mean body weight (10 animals each dose group, experimental period) in comparison to the control animals. Females of the high dose group had a slightly reduced mean body weight on day 14 and days 21-29 (10 animals, experimental period) and days 29-31 (5 animals, recovery period) of the recovery period.

FOOD CONSUMPTION:
Males from high and mid dose groups on days 3-29 (experimental period) and females on days 3-29 (experimental period) and days 29-31 (recovery period) had slight reductions in feed intake.

WATER CONSUMPTION:
In high dose males there was an increased water consumption on day 7 until the end of the experimental period. Females of this group had elevated water consumption on days 7-29 (experimental period). In the mid dose group males, water consumption was increased from day 14 until start of recovery period, and in females of this group from day 14-29 (experimental period).

OPHTHALMOSCOPIC EXAMINATION:
No effects on the eyes.

HAEMATOLOGY:
Statistically significant effects one day after the end of exposure were as follows: increased albumin in mid dose females, increased alpha2 globulin in high dose males, decreased alpha3 globulin in mid and high dose females, increased albumin to globulin ratio in mid dose females.
Statistically significant effects 15 days after the end of exposure (recovery group) were as follows: increased albumin in low dose males and high dose males and females, decreased beta1 globulin in low and high dose males, decreased g1 globulin in low dose male and females and high dose females, and increased albumin to globulin ratio in low and high dose males. However, all values were in the normal range and were not considered to be toxicologically relevant.

URINALYSIS:
No treatment-related effects.

NEUROBEHAVIOUR:
Not examined but there were no neurological disorders observed in the clinical observations.

ORGAN WEIGHTS:
There were statistically significant increases in lung weight in mid dose males one day after exposure, and lung weight in high dose females sacrificed 15 days after exposure. However, all values were within the normal range, and there were no corresponding histopathological findings. Therefore, the effect was considered non-adverse.

GROSS PATHOLOGY:
There were no treatment-related macroscopic changes identified.

HISTOPATHOLOGY:
There were no treatment related findings in the control, low and mid dose groups. In the high dose group three males and one female, which were sacrificed one day after the last exposure, had signs of minimal intense irritation in the alveoli (isolated and small clusters of foam cells). There were no other treatment-related effects.

Dose descriptor:

NOAEC

Effect level:

ca. 3 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.

Dose descriptor:

NOAEC

Effect level:

2 890 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.

Critical effects observed:

no

Conclusions:

The test item was tested for subacute inhalation toxicity according to OECD TG 412 and in compliance with GLP. The NOAEC was determined to be 3000 mg/m³ (nominal). No specific target organ toxicity was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 890 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was according an aapropriate OECD test guideline, and in compliance with GLP.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

There are two oral long-term studies available for trimethoxy(2, 4, 4-trimethylpentyl)silane: a combined repeated dose toxicity and reproduction/developmental toxicity screening test according to OECD TG 422 and a 90-day toxicity study in rats according to OECD TG 408.

In the GLP-compliant combined repeated dose oral toxicity and reproduction/developmental toxicity screening test, male and female Wistar rats received the test substance trimethoxy(2, 4, 4-trimethylpentyl)silane via gavage at dose levels of 50, 150, and 300 mg/kg bw/day (BSL, 2020). The vehicle corn oil was administered to the control group. Satellite control and high-dose animals were included to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects after a 15-day recovery period.

No mortality occurred during the treatment period or recovery period of this study. No consistent clinical signs of toxicity were seen in this study. Moving the bedding and salivation were seen transiently in timely relation to dose administration and were considered as local effects of the test item formulation, but not systemic toxicity. There were no ophthalmoscopic findings in this study. Trimethoxy(2, 4, 4-trimethylpentyl)silane had no effect on functional behavioural parameters evaluated at the end of the treatment and recovery period. The test item had no effect on body weight and food consumption in this study. At the end of the treatment and recovery period there were no relevant alterations in haematological and clinical biochemistry parameters of parental animals in any of the dose groups. Serum T4 levels of males were not affected by the treatment. Trimethoxy(2, 4, 4-trimethylpentyl)silane had no effect on urinary parameters in this study. At the high dose level, liver and kidney weights of male animals were increased moderately, when compared to controls. No relevant changes were observed in liver or kidney weight of females of this dose group. Changes in kidney and liver weight coincided with histopathological findings. Slightly higher adrenal gland weight in females of the high dose group coincided with minimal to slight hypertrophy of the fasciculata cortical layer with cytoplasmic vacuolation and is assumed to be stress-related. At necropsy of the parental animals no test item related - but only incidental macroscopic alterations were noted.

Test-item exposure at 300 mg/kg bw/day – but not at 50 or 150 mg/kg bw/day – caused an adverse symmetric polyneuropathy in both sexes, but more pronounced in females. After the 14-day-recovery period, this lesion persisted. The most striking lesions were observed at the dorsal spinal nerve root L4. Given the lack of lesions within the ganglia, spinal cord, cranial nerves and brain, considering that demyelination was the most prominent histological feature, the lesion was diagnosed as a peripheral myelinopathy. The test item is considered to cause Wallerian-type demyelination (either by direct myelin damage, by interfering with myelin synthesis or by selective toxicity to Schwann cells) in peripheral nerves, followed by secondary axonal degeneration. No changes compatible with myofiber atrophy by denervation were observed in the studied muscles, therefore suggesting a most likely damage of the sensory tracts. In the urinary tract, the diffuse urothelial hyperplasia at ≥150 mg/kg bw/day was considered a dose-dependent adverse induced effect caused by the test-item, which did not show any significant improvement after the recovery period. Urothelial damage was most likely related to direct toxic damage to the renal urothelial cells or to induced changes in urine physical properties with subsequent urothelial hyperplastic reaction. The excessive presence of hyaline droplets in the renal cortex is also deemed to be item-related and might represent intratubular accumulation of protein, and based on the presence only in males most likely an increase inα2-microglobulin. Tubular basophilia is associated with renal tubular regeneration. The slight urothelial hyperplasia seen in one control male rat was a focal lesion, considered to be a background finding.

Presence of lipid accumulation in the intestine, Peyer’s patches, mesenteric lymph nodes, and thymus from high dose male rats suggests a test-item related impairment in the process of lipid transport through the lacteals (small intestinePeyer’s patchesmesenteric lymph nodes). Very similar histopathological findings have been recorded in few previous studies in rats associated with interference in the intestinal lipid uptake, re-synthesis, or transport through the lacteals caused by treatments with puromycin, ethionine or a glucose inhibitor. 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 adaptive change to alteration in lipid transport from the intestine to the lymphatic vessels. In the lungs from rats of the high dose group, minor and scattered alveolar histiocytosis and perivascular/peribronchiolar granulocytic infiltrates were found that are commonly reported as a background finding in untreated control animals. In the present study, there was an increased incidence and severity of these changes in the treated group, especially in females, most likely associated with accidental deviation of the product from oral gavage and is not considered to be an adverse effect of trimethoxy(2, 4, 4-trimethylpentyl)silane. The increase in frequency and severity of hepatic hypertrophy compared to normal range levels seeing control animals, observed at ≥ 50 mg/kg bw/day, 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. The lymphoid depletion in lymph nodes, thymic atrophy, and hypertrophy of the adrenal gland detected in treated animals at 150 and 300 mg/kg bw/day is considered a stress related finding, which in turn might be indirectly linked to the test item. In females, the latter two findings were also observed both in control and treated females, which might be related to stress or to pregnancy. It is unclear whether there is an association between the test item and the thrombus and endarteritis found in the brachial plexus of rat No. 15 and No. 35. A traumatic origin of the vascular damaged cannot be ruled out. Under the conditions of this study the NOAEL for systemic toxicity was established at 50 mg/kg bw/day for males and females.  

 

In the 90-day toxicity study according to OECD TG 408 and in compliance with GLP, trimethoxy(2, 4, 4-trimethylpentyl)silane was given orally by gavage for at least 90 days to Wistar Han rats at dose levels of 20, 50 and 150 mg/kg bw/day (CRL, 2021). The control animals received the vehicle dried and de-acidified corn-oil. To evaluate the potential reversibility of any findings, a 28-day treatment-free period was included for the control and high-dose groups.

No toxicity was observed up to the highest dose level tested (150 mg/kg bw/day).
No mortality occurred and no toxicologically relevant clinical signs were observed. Body weights and body weight gain were considered to have been unaffected by treatment with the test item. No toxicologically significant changes were noted for food consumption, ophthalmology, functional observations and hematology and coagulation parameters. Increased total protein, albumin and calcium concentrations were observed in females at 150 mg/kg bw/day at the end of the dosing period, which normalized by the end of the recovery period. As the changes were only minimal, recovered after a dosing free period, and occurred in absence of any corroborative findings, these were considered not to be test item-related. There were no test item related alterations in organ weights and no test-item related gross observations.

At the microscopic level, test item-related diffuse hyperplasia of the urothelium of the urinary bladder was noted starting at 50 mg/kg bw/day in females (minimal) and at 150 mg/kg bw/day in both sexes (up to mild). Since the growth pattern was simple, the severity was up to mild only, there was partial recovery after a 28-day treatment-free period, and there was no evidence of cellular atypia, this finding was regarded non-adverse.

Based on the earlier OECD 422 study results, where test item-related polyneuropathy had occurred at 300 mg/kg bw/day an extensive examination of skeletal muscles and peripheral nerves was performed in this 90-day study. There was no evidence of any test item-related finding in these organs. This was also the case for the other target organs (i.e. kidneys of males, liver, small intestines, mesenteric lymph node and thymus) identified in the previous OECD 422 study.
In conclusion, based on the results, the NOAEL for trimethoxy(2, 4, 4-trimethylpentyl)silane was established as being at least 150 mg/kg bw/day, since no adverse effect was observed.

Higher doses could not be examined in this study based on adverse findings observed in the OECD 422 study in which adverse polyneuropathy, hyperplasia of the bladder and hyperplasia of the renal pelvis were seen at 300 mg/kg bw/day, and hyperplasia of the bladder was also observed at 150 mg/kg bw/day. The dose levels in the current study had been based on the expectation that the prolonged exposure to the test item in the current study could lead to adverse polyneuropathy at 150 mg/kg bw/day.

 

 

Inhalation

Trimethoxy(2, 4, 4-trimethylpentyl)silane was tested for repeated dose toxicity after subacute inhalative treatment according to OECD TG 412 and in compliance with GLP (Hoechst, 1986). Groups of ten male and ten female Wistar rats were exposed to a respirable aerosol of the test item at concentrations of 0.32, 1.54 and 2.89 mg/L (mean actual concentration) for 28 days (6 h/day, 5 days/week). After the exposure period five male and five female rats of each group were kept during a 14-day recovery period before necropsy. Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. There were sporadic changes to body weights in comparison to the controls. The low dose and control group animals did not have any clinical signs. There were no treatment-related effects on haematology, clinical chemistry, urinalysis, organ weights, and gross pathology. There were signs of minimal intense alveolar irritation (isolated and small clusters of foam cells) in several high dose animals, which is considered to be the predominant effect. Overall, the NOAEC for this study was considered to be 2.89 mg/L.

Justification for classification or non-classification

The results of the combined repeated dose oral toxicity studies, with a reproduction/developmental toxicity screening test (OECD 422, BSL 2020) and the oral 90-day repeated dose toxicity study (OECD 408; CRL 2021) as well as the 28-day inhalation toxicity study (OECD 412; Hoechst 1986) have been reviewed with regards to STOT RE 2 classification for trimethoxy(2, 4, 4-trimethylpentyl)silane.

 

The review of the repeated dose data revealed that repeated exposure to trimethoxy(2, 4, 4-trimethylpentyl)silane caused the following adverse treatment-related lesions based on the results of the OECD 422 study:

1.   In the peripheral nerves: an adverse chronic polyneuropathy consisting mainly of demyelination (myelinopathy), affecting mostly females. (at 300 mg/kg bw/day)

2.   In urinary bladder and kidney (≥150 mg/kg bw/day): Adverse diffuse, urothelial hyperplasia in the urinary bladder (≥150 mg/kg bw/day) and renal pelvis (at 300 mg/kg bw/day).

 

The observed effects in the OECD 422 study have been reviewed in order to determine whether there are any effects that rise to the level of a toxic effect within the guidance value range of >30 mg/kg bw/day and ≤300 mg/kg bw/day (Category 2, 28-day study). Given that the exposure duration for females was up to 65 days, Haber’s rule can be used to adjust the standard guidance values for females, resulting in an upper value of the guidance range ≤200 mg/kg bw/day (Category 2).

 

1) Adverse effects in the peripheral nerves

Since the adverse findings in the peripheral nerves were only observed at 300 mg/kg bw/day which exceeds the upper guidance value for females, the findings do not require classification as STOT RE 2 in females. For males, which have been exposed to up to 28 days, the finding is exactly on the cut-off value for STOT RE 2. However, only one to two male animals of in total 10 males in the high-dose group were affected at the lowest severity score possible (1=minimal) and no effects have been observed in the male recovery group. Moreover, there are no signs of marked organ dysfunction, no clinical signs, no findings in the functional observations battery in this OECD 422 study. Thus, the requirements for classification as STOT RE 2 are not met for either sex. This is supported by the recently performed oral 90-day study, in which an extensive microscopic examination of skeletal muscles and peripheral nerves did not reveal any test-item related findings. 

 

2) Adverse effects on the urinary system

For females, the hyperplasia in the urinary bladder as given below, in the renal pelvis is above the upper guidance values for a STOT RE 2 classification. For males, the hyperplasia in the urinary bladder as reported below, in the renal pelvis is exactly on the cut-off value for a STOT RE 2 classification.

However, the findings of urothelial hyperplasia in either sex only reached a severity score of minimal to slight (1 and/or 2). As no indication of a marked organ dysfunction in the urinary tract and the kidneys was observed, these findings do not fulfil the requirements for STOT RE 2 classification. Additionally, in the oral 90-day study only non-adverse morphologic changes in the urinary bladder of females at 50 mg/kg bw/day and in both sexes at 150 mg/kg bw/day were noted.

In the 28-day subacute inhalation study, no effects were identified up to the maximum exposure dose of 2890 mg/m³.

In conclusion, the available data on repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.