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Diss Factsheets

Administrative data

Description of key information

Oral:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), rat: NOAEL (systemic) = 62.5 mg/kg bw/day

Inhalation:

RA CAS 2768-02-7

Subchronic Inhalation Toxicity 90-Day Study (OECD 413), rat: NOAEC (systemic) = 100 ppm (equivalent to 605 mg/m³ air)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jan - 30 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
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. 14-15 weeks old
- Weight at study initiation: males: 337 - 395 g; females: 189 - 233 g
- Housing: housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of food and water were available and archived.

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 vehicle corn oil was dried and de-acidified. The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added (w/v) to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared at least every 11 days. The prepared formulation was stored protected from light and at room temperature.
Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15.63, 62.5 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no.: MKCH1635, MKCH6411, 2IC0148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before 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.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous (after at least 30 min without stirring), 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).
Concentration analysis: The recoveries observed for the low-dose group was between 93.7% and 99.7% of the nominal value, between 95.6% and 98.2% for the mid-dose dose group and between 94.3% and 99.8% of the nominal value for high-dose group. The mean recoveries observed in the low-, medium- and high-dose groups were 96.9%, 96.6%, and 97.3% 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:
Maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females during the gestation period and up to post-natal day (PND) 12 in females; Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a previous dose range finding study. In the dose-range finding study, 3 males and 3 females per group were treated orally with the test item at doses of 100, 300 and 1000 mg/kg bw/day for 14 days. Mortality was observed in 1/3 male animals treated with 1000 mg/kg bw/day. Additionally, main test item-related findings at 1000 mg/kg bw/day were macroscopic findings at the kidney/ureter (enlarged/dilated), a tendency for increased mean kidney weight and increased clinical biochemical kidney parameters (Crea, Urea, TP, Alb). No adverse findings were seen in the dose groups treated with 100 and 300 mg/kg bw/day.
Based on this, the highest dose in this study was set as 750 mg/kg bw/day. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Inadvertently, the detailed clinical observation was not performed in week 4 for the five randomly selected female animals.
- Clinical observations 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 were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed 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 GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to their sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption: Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and during the last week of lactation in females
- Dose groups that were examined: all dose groups (5 randomly selected animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: adult males

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females. Inadvertently, functional observations of the 5 randomly selected animals were not performed before treatment for female animals, as well as for male animals of the low-, mid- and high-dose groups.
- Dose groups that were examined: all dose groups
- Sensory reactivity to different modalities was assessed: 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 and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

OTHER:
OESTROUS CYCLE
Oestrous cycles were monitored before treatment started to select for the study females with regular oestrous cyclicity. Vaginal smears were examined daily from the beginning of the treatment period until evidence of mating.
Vaginal smears were also examined on the day of necropsy to determine the stage of oestrous cycle.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy consisted of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.

ORGAN WEIGHTS: Yes
- The wet weight of the organs (see Table 4) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each main group, as well as of all males and females of the recovery groups was recorded as soon as possible. In addition reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) of all animals were weighed.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/ parathyroid glands was measured after fixation.

HISTOPATHOLOGY: Yes
The tissues indicated in Table 5 from five randomly selected males and females were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
A full histopathology was carried out on the preserved organs and tissues (Table 5) of the selected animals of the control and high dose group which were sacrificed at the end of the treatment period. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Any gross lesion macroscopically identified was examined microscopically in all animals. Possible discoloration due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Optional endpoint(s):
None
Other examinations:
None
Statistics:
A statistical analysis 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 were 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 nonparametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The male animal of the control group which was euthanized in moribund condition on treatment day 18 showed moderately reduced spontaneous activity, abnormal breathing and severely increased salivation on the day of sacrifice.

Moving the bedding was observed transiently in 1/10 males and 5/10 females in the high-dose group and increased salivation was noted transiently in 1/10 males in the mid dose, 5/10 males in the high dose and 1/10 females in the low dose and 6/10 females in the high dose. The clinical signs of moving the bedding and increased salivation were observed in a close timely relation to the dose administration and therefore considered to be a sign of discomfort or a local reaction of the test item. They were not considered to be adverse systemic effects.

The clinical signs hairless area/scratch at different body locations (1/10 males of the low-dose group, 1/10 females of the control, 3/10 females of the low-dose and 3/10 females of the high-dose group), diarrhoea (1/10 males of the high-dose group) and abnormal breathing (2/10 females of the mid-dose group) were seen in single animals without dose dependency or on single observation days and were not considered to be test item-related.

Detailed clinical observation showed no toxicological relevant differences in all male and female groups when compared to the control group.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study a control male was euthanized in moribund condition on treatment day 18.In the histopathological examination, inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The microscopic findings suggest that there was unexpected influx of the dosing solution into the respiratory tract. From these findings, the cause of the animal’s morbidity was considered to be a technical error that happened during the oral gavaging procedure.
All remaining animals survived the scheduled study period.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item had an effect on the body weight in the male and female high-dose and male mid-dose group during the treatment period.
A statistically significant decrease of body weight was found in high-dose males in the last week of the premating phase and the entire mating/postmating period (up to 14% below control). The body weight at terminal sacrifice showed a statistically significant decrease of 12% below control. The weekly body weight gain was statistically significantly decreased in high-dose males during the treatment period with exception in the second week of mating/postmating. Additionally, a statistically significant decrease in weight gain was noted in the mid-dose male group between premating day 1 to terminal sacrifice (40.57% below control) and the first week of premating and premating day 1 to postmating day 14 (86.84% and 46.31% below control).

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).

In high-dose females, the weekly measurement of body weight showed a decrease between 3.8% and 7.8% compared to control during the lactation phase with statistical significance on lactation day 13 (7.8% below control). The weekly body weight gain was 42.47 % and 39.69% lower than in the control group during lactation, but no statistical significance was found.

No toxicological effect was found in the male and female low-dose and the female mid-dose groups. Body weight development and body weight gain was comparable between test item-treated groups and their respective control group throughout the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to treatment with the test item.

During the premating phase, the food consumption in high-dose males showed a decrease of 31.11% below control in the first week and 32.55% in the second week of the treatment period. The food consumption in the high-dose female group was statistically significantly decreased during the entire lactation phase when compared to the control group (between 22.97% and 26.63% below control).

The statistically significantly increased mean food consumption between day 0 and day 4 in low-dose females (19.65% above control) was considered to be incidental and of no toxicological relevance.

No test item-related effect on food consumption was found in the male and female low- and mid-dose groups. Mean values were comparable to the respective control group and no specific differences were found.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on haematological and coagulation parameters of selected male and female animals analysed at the end of the treatment period of this study.

Mean values of mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly decreased in the low-dose and high-dose male group when compared to the control group (low-dose group: 4.62% and 4.99% below control, high-dose group 5.74% and 5.57% below control), but no dose dependency was found between all dose groups. Therefore, no toxicological relevance is considered.

The statistically significant increase of 40.4% above control group for reticulocytes observed in the high-dose male group is considered to be of no toxicological relevance as no dose dependent changes were found between the dose groups and no toxicological effect was seen for red blood cell parameters.

In the absence of dose dependency, the slightly but statistically no significant decreased mean white blood cells value in the male low-dose group of 28.03% and in the high-dose group of 10.80% below the control group is considered to be incidental and without toxicological relevance.

In differential blood cell count, the statistically significant increase of 108.99% in the high-dose male group for monocytes and the statistically significant decrease of 53.84% in the low-dose male group for large unstained cells followed no dose dependency and are therefore considered to be of no toxicological relevance. Additionally, the increased and no statistically significant group mean values for neutrophils in high-dose males (115.95% above control) and 63.12% above control in low-dose males followed no dose dependency and are considered not to be toxicological relevant.

The coagulation parameter prothrombin time, was found with a statistically significant increase in mid-dose males (10.05% above control) and high-dose males (18.08% above control) and is assumed to be of no toxicological relevance as no statistical significant increase for prothrombin time was seen in the female dose groups.

No statistical significant changes were observed for any parameter of haematology and coagulation parameters for all female dose groups. The group mean values were comparable to the respective control group and no differences with toxicological relevance were found.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, the test item showed effects on clinical biochemical parameters related to the kidney in the male (Urea, Crea) and female (Urea) high-dose group analysed in the selected animals.

Urea was statistically significantly increased in the high-dose male group (41.41% above control), but no dose dependent increase was noted. Additionally, the parameter creatinine (Crea) showed an increase of 47.% above control in high-dose males but without statistical significance. A dose dependent increase was not observed for this parameter. In females, no dose dependency between the low-, mid-, and high-dose group or an increase in the high-dose group were noted for creatinine. Urea showed an increase of 8.47% in the high-dose group, but without statistical significance. The changes observed for the renal clinical chemical parameters correlate to the complex lesions found at histopathological evaluation. Therefore, the slightly increased mean value for Urea in high-dose females cannot fully be excluded as test item-related.

The slight and statistically significant increase of 4.73% above control for albumin in the male low-dose group was considered not to be an effect of the treatment with test item as no such increase was noted in the mid- and high-dose group.

For the liver parameters alanine aminotransferase, aspartate-aminotransferase and alkaline phosphatase, a decrease was noted for all male dose groups when compared to controls with exception of alanine aminotransferase and alkaline phosphatase in the low-dose group. In high-dose males the decrease was up to 21.73% for alanine aminotransferase, 21.41% for aspartate-aminotransferase and 28.24% for alkaline phosphatase (mid-dose group: 36.82% for alkaline phosphatase). A decrease in the mentioned parameters is not considered to be related to the treatment with test item and furthermore, no dose dependent changes with respect to the control group were seen within the male and additionally the female dose groups for (alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase).

Total bile acids was found with a percentage of 152.76% above control in high-dose and 29.57% above control in mid-dose males. No such increase was found between the female dose groups. Individual total bile acids values in males and females were comparable to the respective control group, with exception of one high level in high-dose male animal, which is considered to have resulted in the high mean value. The differences between the dose groups and the control groups are therefore assumed not to be an effect of treatment with the test item.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study.
Findings in single male animals such as ketone in two high-dose animals and slightly higher levels of glucose in two mid-dose animals and one high-dose animal when compared to the control group are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no test item-related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the respective control group.
Body temperature was observed with a slightly and statistically significant increase in males of the high-dose group in the last week of the treatment period when compared to the control group (38.7 °C in the high-dose group compared to 37.9 °C in the control group). As the increase was slight and no test item-related findings were observed during clinical observation, the statistical difference is considered to be of no toxicological relevance.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher absolute and relative kidney weight was recorded in males of the high-dose group (absolute 62.52%, relative to body weight 92.59% above control). An increased absolute and relative kidney weight in the female high-dose group did not reach statistical significance (absolute 9.9%, relative to body weight 22.63% above control). The weight changes in the kidneys were considered to be associated with the test item-related complex renal lesions such as tubular basophilia, mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, pyelitis, pelvic dilatation and hyperplasia of urothelial cells.

Statistically significant changes were found for the prostate including seminal vesicle and coagulating glands in the male dose groups. Absolute and relative weight to body weight of the accessory reproductive organs were statistically significantly decreased in the low- (absolute 15.59%, relative 17.56% below control) and high-dose group (absolute 30.63%, relative 21.48% below control), whereas in the mid-dose group the decrease in mean absolute and relative weight was found without statistical significance. The absolute and relative thymus weight in males showed a tendency towards a decrease between the dose groups and was statistically significantly decreased in the high-dose group (absolute 40.47% below control). In females, no statistical significant changes were found for all dose groups, but the absolute and relative mean weight showed a tendency to decrease when compared to the control (absolute 29.58% and relative 20.80% below control) in the female high-dose group. Following consideration of the histopathological evaluation, the weight changes of the thymus and the accessory male reproductive organs were deemed to be an alteration secondary to low body weight (statistically significantly decreased in high-dose males and high-dose females (12.28% and 7.8% below control at necropsy) or due to the stress related to the deterioration of general conditions.

Statistically significant changes from the control were found for pituitary glands in the mid-dose male group (relative weight to body weight: 19.50% above control), heart in the high-dose male group (relative weight to body weight: 14.84% above control) and in high-dose females for brain weight (absolute weight 7.77% below control). In the absence of dose dependent changes and test item-related findings observed at histopathological evaluation, it was considered that the changes for pituitary glands, heart and brain were of no toxicological relevance.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were gross lesions observed in the male and female high-dose groups and the female mid-dose group, which were considered to be an effect of the test item. Adverse test item-related findings were noted in kidney, urinary bladder and ureter in the high-dose groups and the female mid-dose group (enlarged ureter in 1/10 mid-dose females).
For one control male, which was euthanized for animal welfare reasons in a moribund condition on treatment day 18, was observed with dark red fluid in the trachea, red coloured lung and dark thymus. Histopathologically, mixed inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The findings in the respiratory tract indicate a technical error during the oral gavage as the cause of the moribund condition.

Test item-related findings were seen in the high-dose male group in the kidney, ureter and urinary bladder. Findings of the kidney were fluid yellow content in 1/10 high-dose males, dilatation of the right kidney in 1/10 high-dose males, enlarged kidney for 5/10 animals and pelvis dilatation for 1/10 males. The ureter were filled with yellow or white fluid (for 2 high-dose males) and enlarged/dilated in 5/10 high-dose males. For 5/10 high-dose males the urinary bladder was found with a thickened wall. In females, test item-related findings were noted in the kidney of 1/10 high-dose females (right enlarged, green), for the ureter of 2/10 high-dose females (dilatation) and 1/10 mid-dose female animals (enlarged). The thickened wall of the urinary bladder was noted in 3/10 high-dose females and the organ was enlarged for 2/10 high-dose females.

Further findings in the female high-dose group were red coloured salivary glands and dark red/brown coloured axillary lymph nodes for one high-dose female. These findings were considered not to be related to the treatment with test item, as they were seen in one single animal from the high-dose group or were also found in all other female dose groups (1/10 low-dose animals, 2/10 mid-dose animals) and the control group (2/10 animals). The findings of brown coloured liver (one low-dose female), slightly enlarged adrenal glands (one low-dose female) and small right uterus (one control female) were considered to be incidental.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed that the test item produced histomorphological changes in kidneys, urinary bladder, ureters, duodenum, jejunum, liver, thyroid glands, bone marrow, thymus and/or adrenal glands of animals that received 750 mg/kg bw/day (high-dose group). This included changes due to primary and secondary effects of the treatment. Qualitatively comparable changes were also observed in kidneys, ureters, urinary bladder of both sexes and in thymus and adrenal glands of males from the mid-dose group (250 mg/kg bw/day).

There were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item at a dose of 750 mg/kg bw/day (high-dose group), although increased incidence and group mean severity grade of reduced secretion were found in prostate glands, coagulating glands and seminal vesicles. Reduced secretion was considered to be an alteration secondary to low body weight or due to the stress related to the deteriorlation of general conditions. There were no morphologic abnormalities (such as cellular denegeration or atrophy) in cell- and tissue-construction in these organs, and hence, the reduced secretion without any further indicators of tissue and cellular injury was not considered to be adverse.

In the kidney, urinary bladder and ureter, several degenerative, inflammatory and/or reactive changes were observed in both sexes of the ,mid- and high-dose groups. The treatment related changes observed in the kidney were complex lesions composed of the following changes: increased incidence and severity of tubular basophilia, as well as mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, papillary necrosis, pyelitis, pelvic dilatation and hyperplasia of urothelial cells (surface epithelial cells covering the papilla and transitional cells lining the other region of renal calix). Intratubular precipitates and pelvic luminal precipitates were also identified in animals of the high-dose group. In addition, granuloma formed mainly at/around the fornices region was observed in some animals, and foreign body giant cells containing precipitates/ precipitates-like substace were involved in this lesion. The renal lesion was also present in both sexes of the mid-dose group, however, that was of less complexity and severity compared to that of the high-dose group and restricted to the pyelocaliceal region. The degenerative and inflammatory changes, as well as the reactive changes, were also observed in the urinary bladder and ureters of both sexes of the mid- and high-dose group. These included transitional cell hyperplasia accompanied by infiltration of mixed inflammatory cells or mononuclear cells, with occasional hemorrhage, in the the urinary bladder, and transmural inflammation, transitional cell hyperplasia and luminal dilatation in the ureter. In the present study, increased granulopoiesis was observed in both sexes of the high-dose group. This was considered to be a reactive increase associated with inflammatory changes in the urinary system including kidneys, urinary bladder and uteters, and it was not considered to be of an adverse nature.

In the duodenum and jejunum, lipid accumulation in the lamina propria mucosa was observed for both sexes of animals from the high-dose groups. This was recognized as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.

In the liver, centrilobular hepatocellular hypertrophy was observed for both sexes of the high-dose group. There were no further indicators of liver injury, hence, this change was considered to be of an adaptive nature, most likely to be related to enzyme induction.

In the thyroid glands, diffuse follicular cell hypertrophy was observed for both sexes of the high-dose group. A possible relationship between the liver change was considered, and the changes recorded in the liver and thyroid glands were deemed not to be adverse.

In the adrenal glands and thymus, increase in the incidence and/or severity of vacuolation in zona fasciculata of the adrenal cortex and thymic atrophy was observed in males of the mid-dose group and for both sexes of the high-dose group. Both findings recorded were not considered to be of an adverse nature, but these were considered to be stress-related secondary changes associated with deteriorlation of general conditions, reduction of body weight gain or body weight decrease, or resulting from the lesions in the urinary system.

In conclusion, due to presence of treatment-related changes recorded in kidneys, ureters and urinary bladder for both sexes at 250 mg/kg bw/day and higher, the no-observed-adverse-effect-level (NOAEL) can be established at 62.5 mg/kg bw/day under the conditions of this study. Meanwhile, there were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item up to a maximum dose of 750 mg/kg bw/day.

No detailed description of the histopathology findings are available in the draft report. Further details will be provided once the final report is available.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of parental males.

There was no effect on the oestrous cycle assessed during the premating period in females of any of the test item-treated groups when compared to the control group. There were no toxicologically relevant considerable differences in the number of normal or abnormal cycles.
Mean cycle length was slightly longer in the high-dose group (5.27 days) when compared to the control (4.25 days). As the increase in the hihg-dose group mean value was slightly above a normal cycle length of 5 days, no test item-related effect is considered.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
ureter
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Under the conditions of this study and based on the test item-related finding, the NOAEL is determined to be 62.5 mg/kg bw/day for general toxicity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
62.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
urinary
Organ:
bladder
kidney
ureter

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached analogue justification in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
urinalysis
Remarks on result:
other: Source CAS 2768-02-7 (BRRC, 1990)
Critical effects observed:
yes
Lowest effective dose / conc.:
400 ppm
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Rats repeatedly exposed to 400 ppm trimethoxy(vinyl)silane for 14 weeks had minimal to mild alterations in body weight, water consumption, urinalysis, organ weights, and bladder and kidney histopathology. At a concentration of 100 ppm there were some body weight gain reductions in females, but they were less than 10% and not always statistically significant, and there were no such finding in males; males had mild effects on urine osmolality and urine volume in week 1 only and there were no associated organ weight changes, macroscopic or microscopic findings. There were no findings at the end of the recovery period. Therefore, the findings at 100 ppm were concluded not to be adverse and the NOAEC in Fischer 344 rats was therefore 100 ppm (605 mg/m³).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
605 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on a common hydrolysis product, similarities in physico-chemical, ecotoxicological and toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6.2, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.
System:
urinary
Organ:
bladder
kidney

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached analogue justification in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
urinalysis
Remarks on result:
other: Source CAS 2768-02-7 (BRRC, 1990)
Critical effects observed:
yes
Lowest effective dose / conc.:
400 ppm
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Rats repeatedly exposed to 400 ppm trimethoxy(vinyl)silane for 14 weeks had minimal to mild alterations in body weight, water consumption, urinalysis, organ weights, and bladder and kidney histopathology. At a concentration of 100 ppm there were some body weight gain reductions in females, but they were less than 10% and not always statistically significant, and there were no such finding in males; males had mild effects on urine osmolality and urine volume in week 1 only and there were no associated organ weight changes, macroscopic or microscopic findings. There were no findings at the end of the recovery period. Therefore, the findings at 100 ppm were concluded not to be adverse and the NOAEC in Fischer 344 rats was therefore 100 ppm (605 mg/m³).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 421 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on a common hydrolysis product, similarities in physico-chemical, ecotoxicological and toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6.2, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

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

Repeated dose toxicity: oral

A combined repeated dose oral toxicity study, with a reproduction/developmental toxicity screening test according to OECD TG 422 and in compliance with GLP is available for triethoxy(vinyl)silane (CAS 78-08-0) (BSL Bioservice, 2021, Draft). The test substance was administered daily in graduated doses (62.5, 250, and 750 mg/kg bw/day) to 3 groups of test animals per gavage for a treatment period of up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 29 days was completed. Animals of an additional control group were handled identically as the dose groups but received the vehicle dried and de-acidified corn oil.

No test-item related mortality and no adverse clinical signs were noted. Treatment with the test item had an effect on the body weight development in the male and female high-dose and male mid-dose groups during the treatment period, which resulted in a lower body weight gain when compared to the control groups. Test item-related effects on food consumption in both high-dose groups were found. The test item had no toxicologically relevant effects on haematological and coagulation parameters, urinary parameters and on any of the parameters of the functional observation battery in selected animals at the end of the treatment period when compared to the respective control group.

The test item showed effects on clinical biochemical parameters related to the kidney (urea, creatinine) in the male and female high-dose groups, which correlate to the complex lesions observed at histopathological evaluation. Adverse test item-related macroscopic findings at necropsy were noted in kidney, urinary bladder and ureter in the high-dose groups and mid-dose groups. Organ weight measurements showed changes that were considered to be directly or indirectly related to treatment with the test item. The changes were a statistically significant increase in absolute and relative (to body weight) weights of kidney in the male high-dose group and higher values of absolute and relative weights compared to control group in the female high-dose group. Furthermore, a statistically significant decrease in absolute thymus weights in high-dose males and a tendency towards a decrease in absolute thymus weight in high-dose females as well as relative thymus weights in high-dose males and females. Prostate glands, seminal vesicles and coagulating glands as a whole showed a statistically significant decrease in absolute and relative weights in high-dose males. 

Histopathological examination revealed that in the kidney, urinary bladder and ureter, several degenerative, inflammatory and/or reactive changes were observed in both sexes of the mid-dose and high-dose groups. Further test-item related histopathological changes were noted in duodenum, jejunum, liver, thyroid glands, bone marrow, thymus and/or adrenal glands of animals from the high-dose groups and in thymus and adrenal glands of mid-dose males.

Following consideration of the histopathological evaluation, the weight changes of the thymus and the accessory male reproductive organs were deemed to be altered secondary to a low body weight (statistically significant decrease in high-dose males and high-dose females (12.28% and 7.8% below control at necropsy) or due to stress related to the deterioration of general condition.

Under the conditions of this study and based on the test item-related findings, the NOAEL is determined to be 62.5 mg/kg bw/day for general systemic toxicity.

 

Repeated dose toxicity: inhalation

In a well conducted, reliable (with restrictions), GLP study (Bushy Run Research Center, 1994) rats were exposed to a vapour of triethoxy(vinyl)silane (CAS 78-08-0) up to concentrations of 50 ppm for 6 hours/day, 5 days/week for 13 weeks. In the range finder for this main study rats were exposed to 100, 500 and 1000 ppm (corresponding to 775, 3936 and 7856 mg/m³ air) for 9 days. The NOAEC was established at 100 ppm (775 mg/m³ air) based on kidney and urinary bladder effects in the higher dose groups and the highest dose for the main study was chosen at 50 ppm (389 mg/m³ air). In the main study no biologically significant or exposure-related effects on clinical signs, body and organ weights, food and water consumption, clinical pathology and ophthalmic evaluations were noted. No exposure-related gross and microscopic lesions were observed in any group. Therefore for this study the NOAEC was set at the highest tested dose of 50 ppm (389 mg/m³ air). 

In a further non-reliable GLP study (Bushy Run Research Center, 1991) rats were exposed to an aerosol of the test substance at a concentration of 145 mg/m³ air for 6 hours per day, 5 days per week (4 days in week 4) for 28 days. No mortality or exposure-related clinical signs were observed throughout the study. There was a statistically significant decrease in body weight gain in treated animals compared with controls during the first week of treatment. However, in the subsequent weeks, body weight gain in the treated animals was comparable to or greater than that for control animals. No gross lesions and microscopic findings attributable to treatment were noted.

As the selected concentraions, in particular the high dose, in the subchronic inhalation study on triethoxy(vinyl)silane (CAS 78-08-0; Bushy Run Research Center, 1994) did not reveal toxicity, the hazard assessment for the registration substance (CAS 78-08-0) is based on data from the source substance trimethoxy(vinyl)silane (CAS 2768-02-7) in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and in accordance with the Read across assessment framework (RAAF, ECHA 2017). Both, the target substance triethoxy(vinyl)silane (CAS 78-08-0) and the source substance trimethoxy(vinyl)silane (CAS 2768-02-7) hydrolyse in contact with water, generating the common hydrolysis product vinylsilanetriol. Details on read across justification can be found in the justification for grouping of substances attached in IUCLID Section 13.

 

A 14-week vapour inhalation study in rats using a protocol similar to OECD TG 413 and in compliance with GLP (Bushy Run Research Center, 1990), rats were repeatedly exposed to nominal concentrations of  10, 100 or 400 ppm of the source substance trimethoxy(vinyl)silane (CAS 2768-02-7) for 6 hours per day over 14 weeks. Minimal to mild alterations in body weight, water consumption, urinalysis, organ weights, and bladder and kidney histopathology were observed. Clinical signs in the 400 ppm group included urogenital area wetness and alopecia. There were no treatment-related eye lesions. Male and female rats of the 400 ppm group had decreased body weights (11 to 16% below control values). Occasional decreases in body weights of the female rats of the 100 ppm group were also observed. Food consumption was not altered. Water consumption was increased in the male rats of the 400 ppm group at study weeks 1, 5, 8, and 14 and for females during the first week. Urinalysis results indicated that male rats of the 400 ppm group had lower osmolality, lower electrolyte concentrations, and a decrease in estimated creatinine clearance. Female rats of the 400 ppm group had similar changes, but at week 14 only. A decrease in urine osmolality with a concomitant increase in urine volume was observed in male rats of the 100 ppm group at week 1. There were no biologically significant changes in haematology or serum chemistries in rats exposed to the test material. At necropsy, there were no exposure-related lesions, and changes in organ weights in rats of the 400 ppm group were considered to result from body weight depression. Noteworthy microscopic lesions in rats of the 400 ppm group were observed in two tissues, the urinary bladder and the kidney. Minimal cystitis in the bladder submucosa was observed at 14 weeks, and submucosal mastocytosis was observed at 18 weeks. Renal lesions in a few of the 400 ppm-exposed rats included papillary necrosis, interstitial oedema, and/or papillary hyperplasia of the transitional epithelium. Electron microscopic examination of the kidneys supported the light microscopic findings. Based on the fact that at 100 ppm (605 mg/m3) there were some body weight gain reductions in females, but they were less than 10% and not always statistically significant, there were no such finding in males, males had mild effects on urine osmolality and urine volume in week 1 only, and there were no adverse findings from serum chemistry, haematology, organ weight, macro- or microscopy and no findings at all at the end of the recovery period for this group, it is concluded that the effects at 100 ppm are non-adverse and the NOAEC is 100 ppm (corresponding to 605 mg/m³ air). [study author opinion - NOAEC and an opinion on adversity of the findings were not stated in the study report].

Justification for classification or non-classification

The results of the combined repeated dose oral toxicity studies, with a reproduction/developmental toxicity screening test (OECD Test Guideline 422) and the extended one-generation reproductive toxicity study (EOGRTS) (OECD Test Guideline 443) with the source substance trimethoxy(vinyl)silane (CAS 2768-02-7) have been reviewed with regards to STOT RE classification.

 

Consideration of STOT RE 2 Classification for triethoxy(vinyl)silane:

The review of the repeated oral exposure data including the EOGRT study on the source substance trimethoxy(vinyl)silane, and the STOT RE 2 classification guidelines, revealed that repeated exposure to triethoxy(vinyl)silane causes a treatment-related effect on the urinary bladder, kidneys, ureters, and the small intestine.  

 

First, the observed effects have been reviewed in order to determine whether there are any test item-related effects that rise to the level of a toxic effect within the guidance value range of >10 mg/kg bw/day and ≤100 mg/kg bw/day:

 • In the combined repeated dose oral toxicity study, with a reproduction/developmental toxicity screening test (OECD Test Guideline 422) (BSL Bioservice, 2021, Draft), test item-related lesions were observed in the kidneys, ureters and urinary bladder at ≥250 mg/kg bw/day and in the duodenum and jejunum at ≥750 mg/kg bw/day.  Given that the exposure duration for females was up to 63 days, the guidance value should be increased by an appropriate factor of two, making the upper value of the guidance range ≤200 mg/kg bw/day.  Since the LOAEL for the study (250 mg/kg bw/day) exceeds the upper guidance value, this study would not support classification for STOT RE in females, no matter what adverse test item-related effects had been observed. The exposure duration for males was 28 days and thus the guidance value should be increased by an appropriate factor of three, making the upper value of the guidance range ≤300 mg/kg bw/day. Therefore, only those test item-related effects observed at 250 mg/kg bw/day in males should be considered as supporting a classification of STOT RE. In examining the results for specific changes at 250 mg/kg bw/day in males, the following results were identified:

- Urinary bladder: minimal mixed inflammatory cell infiltration (mucosa and/or lamina propria) in 2/5 males, minimal focal/multifocal mononuclear cell infiltration (lamina propria) in 3/5 males, minimal hemorrhage (mucosa/lamina propria) in 1/5 males, moderate diffuse transitional cell hyperplasia in 5/5 males

- Ureter: minimal luminal dilatation in 1/5 males, minimal diffuse transitional cell hyperplasia in 2/5 males 

- Kidney: minimal tubular dilatation in 1/5 males, minimal pyelitis in 1/5 males, minimal pelvic dilatation in 1/5 males, minimal diffuse urothelial cell hyperplasia in 5/5 males

 

• In the EOGRTS with the source substance (CAS 2768-02-7) (OECD Test Guideline 443) (BSL Bioservice, 2021), the only test item-related findings observed at ≤100 mg/kg bw/day (i.e., limited to low-dose and mid-dose findings at 40 and 100 mg/kg bw/day, respectively) were:

- Urinary bladder - thickened wall: in 1/24 low-dose and 3/25 mid-dose P-generation females; and in 1/20 F1-generation mid-dose Cohort 1B females;

- Urinary bladder: diffuse urothelial hyperplasia in 20/25 mid-dose males and 12/25 mid-dose females of the P-generation; and in 12/20 mid-dose males and 4/19 mid-dose females of the F1-generation Cohort 1A group;

- Kidney: diffuse urothelial hyperplasia in 1/25 mid-dose males and 2/25 mid-dose females of the P-generation; and in 2/20 mid-dose males and 1/19 mid-dose females of the F1-generation Cohort 1A group.

 

Second, the identified test item-related effects that are within the guidance value range of >10 mg/kg bw/day and ≤100 mg/kg bw/day have been reviewed to determine whether they can be considered as significant toxic effects:  

• Table 3.9.1 of Regulation (EC) No 1272/2008 and Figure 3.9.1 in GHS Regulation specifically refers to “significant toxic effects” in animals as being the basis for STOT RE 2.  

• Section 3.9.2.7.1 of Regulation (EC) No 1272/2008 and GHS Regulation states that “reliable evidence … of a consistent and identifiable toxic effect demonstrates support for classification”. In the reviewed data the effects were limited to findings of hyperplasia in the urinary bladder accompanied by inflammatory reactions, diffuse urothelial hyperplasia in the kidney and luminal dilatation and transitional cell hyperplasia in ureter.

• Section 3.9.2.7.3 of Regulation (EC) No 1272/2008 and GHS Regulation uses modifying terms such as "significant", "mutli-focal" or "diffuse", "marked", or appreciable in describing the types of effects that might trigger classification. The effects observed in the urinary bladder, kidney and ureter of mostly minimal grade are not considered to be indicative of significant organ damage or dysfunction.

• Section 3.9.2.7.3(d) of Regulation (EC) No 1272/2008 and GHS Regulation specifies “significant organ damage.”  The data do not support these criteria.

• Section 3.9.2.7.3(e) of Regulation (EC) No 1272/2008 and GHS Regulation refers to “multi-focal or diffuse necrosis, fibrosis or granuloma formation in vital organs with regenerative capacity.”  The data do not support these criteria.

• Section 3.9.2.7.3(f) of Regulation (EC) No 1272/2008 and GHS Regulation indicates “morphological changes that are potentially reversible but provide clear evidence of marked organ dysfunction.” Although histomorphological changes were observed, overall the data do not support marked organ dysfunction.

     

In addition, the dose-response relationship for the observed effects and influence of the exposure duration have been given consideration:

• Dose-response relationship for the observed effects on urinary bladder, ureter and kidney – the incidence and severity of the observed effects increased with the doses. In the extended one-generation reproductive toxicity study (BSL Bioservice, 2021), diffuse urothelial hyperplasia in the urinary bladder was evident for all male and female P and F1 Cohort 1A animals in the high dose group, while fewer animals were affected in the mid dose group from P and F1 Cohort 1A animals. In addition, higher number of high dose animals from P and F1 Cohort 1A had diffuse urothelial hyperplasia in the kidney when compared to the mid dose animals. Pathology and microscopic findings at low doses do not appear to be early stages of more severe or irreversible tissue damages or tumour formation. The only effect seen at low doses in the extended one-generation reproductive toxicity study is thickened urinary bladder wall in one P female, which was also evident in 3 control P females and no corresponding microscopic changes were detected for these animals. Therefore, these changes can be concluded as non-treatment related.

In the combined repeated dose oral toxicity study, with a reproduction/developmental toxicity screening test (BSL Bioservice, 2021, draft) the renal lesions in high dose-group was also present in both sexes of the mid-dose group, but with less complexity and severity compared to the high dose group. No renal effects were noted in the low dose group. The effect followed a dose-response relationship. In the urinary bladder, transitional cell hyperplasia accompanied by mixed inflammatory cell infiltrates were observed in all animals examined in the high dose group, and hemorrhage in the mucosa and lamina propria was also found in one male in this group. Qualitatively the same lesions were observed in both sexes of the mid-dose group. Specifically, especially diffuse transitional cell hyperplasia was found in all animals of the mid-dose group. However, mixed inflammatory cell infiltrates were not noted in most lesions, and focal/multifocal mononuclear cell infiltration in lamina propria was involved in the lesions instead. Focal/multifocal mononuclear cell infiltration (lamina propria) were observed in each one male animal of the low-dose group and control group, respectively. Transmural inflammation in ureter was observed in two males of the high-dose group. Diffuse transitional cell hyperplasia was observed in all ureters examined in the high-dose group and in two males and one female from the mid-dose group. Luminal dilatation was also found in a male of the mid-dose group and in both sexes of the high-dose. No effects in ureter was noted in the low-dose group.    

• Influence of exposure duration on severity of effects – Similar effects in severity and incidence are seen in the available data for trimethoxy(vinyl)silane and there is no evidence to suggest that longer exposure duration leads to irreversible, or more severe effects.

 

Overall, it was concluded that the adverse test item-related effects observed in the urinary bladder, ureter and kidney do not rise to the level of a “significant toxic effect" as required by Regulation (EC) No 1272/2008 and GHS Regulation. According to Regulation (EC) No 1272/2008 and GHS Regulation, the significant toxic effect must specifically show “significant organ damage”, “multi-focal or diffuse … granuloma formation in vital organs with regenerative capacity”, or “morphologic changes that provide clear evidence of marked organ dysfunction”. The data do not support these criteria.

Furthermore, the treatment-related changes in the urinary bladder and kidney of rats administered trimethoxy(vinyl)silane were evaluated in an Expert Panel Review Report (EPL, 2020) which aimed to determine the pathogenesis of these changes. The Expert Panel concluded that the treatment-related urothelial changes were identical in the urinary bladder, kidney, and ureter (when present) and consisted of a diffuse urothelial hyperplasia without atypia, which was considered to be an adaptive response to a physical or chemical irritant present within the urine. This type of diffuse hyperplasia was not considered to represent a preneoplastic change. For more details on the EPL Report (2020) see attachment to IUCLID Section 13.

Based on the available data, triethoxy(vinyl)silane does not require classification for target organ toxicity following repeated exposure according to Regulation (EC) No 1272/2008.