Triethoxy(vinyl)silane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

NOAEL (reproductive toxicity) ≥750 mg/kg bw/day

NOAEL (systemic toxicity) = 62.5 mg/kg bw/day

RA CAS 2768-02-7:

Extended One-Generation Reproductive Toxicity Study (OECD 443), rat:

NOAEL(P0, P1 (cohort 1B) and F1 (cohort 1A and 1B), reproductive toxicity) ≥300 mg/kg bw/day

NOAEL(P0, P1 (cohort 1B) and F1 (cohort 1A), general systemic toxicity) = 40 mg/kg bw/day

NOAEL(F1 (cohort 1A and 1B) and F2 (cohort 1B), developmental toxicity) ≥300 mg/kg bw/day

NOAEL(F1 (cohort 3), developmental immunotoxicity) ≥300 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and developmental immunotoxicity (Cohorts 1A, 1B with extension, and 3)

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:

NOAEL

Remarks:

Parental animals / reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive function observed.

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Dose descriptor:

NOAEL

Remarks:

Parental animals / general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

P1 (Cohort 1B) / reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive function observed

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Dose descriptor:

NOAEL

Remarks:

P (Cohort 1B) / general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Generation:

F1 (cohort 1A)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

other: F1 (Cohort 1A) & F1 (Cohort 1B)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive organs observed

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

other: F1 (Cohort 1A) & F1 (Cohort 1B)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental immunotoxicity

Generation:

F1 (cohort 3)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse immunotoxic effects observed.

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F2 (cohort 1B)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed.

Remarks on result:

other: Source CAS 2768-02-7 (BSL, 2021)

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In the extended one-generation reproductive toxicity study with CAS 2768-02-7, conducted according to OECD Test Guideline 443 and in compliance with GLP, the NOAEL for general systemic toxicity for P and F1 (Cohort 1A) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on male and female reproductive organs, on oestrous cycle and sperm parameters, and male and female fertility and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

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

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 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

Details on mating procedure:

- M/F ratio per cage: Mating was performed using a ratio of 1:1 (male to female).
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage.

Analytical verification of doses or concentrations:

yes

Remarks:

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

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

Details on study schedule:

Not applicable

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

Parental animals: Observations and examinations:

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: No

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

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.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Additionally, any abnormal behavior of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Bood samples from 2 pups/litter (1 male and 1 female, if possible) at termination on PND 13 were assessed for serum levels for thyroid hormones. Assessment of T4 in blood samples from day 4 pups was not carried out.

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) by using anaesthesia (ketamine/xylazin).
- Maternal animals: Females were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazin).

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.

Postmortem examinations (offspring):

SACRIFICE
All surviving pups were killed by cervical dislocation on PND 13.

GROSS NECROPSY
- Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

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 non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.

Reproductive indices:

Indices calculated were the copulation index, delivery index and fertility index.

Offspring viability indices:

Viability index between PND 0 to PND 4 was calculated.

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 beas 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.

Dermal irritation (if dermal study):

not examined

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.

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).

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.

Food efficiency:

not examined

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

not examined

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.

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.

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.

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of parental males. No statistically significant differences for T4 levels and thyroid/parathyroid glands weight were found for any dose group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related effect of the test item on pre-coital interval and duration of gestation observed.

The statistically significantly increased precoital interval (70.8% above control) and duration of gestation (2.6% above control) in the high-dose group was not considered to be a treatment-related effect as no histomorphological changes of toxicological relevance were found in the reproductive organs including testes, epididymides, prostate glands, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina from the high-dose group males or females. Additionally, the mean pre-coital interval and duration of gestation were within the range of historical control data.

Mean post implantation loss was observed to be higher in the mid-dose group (post implantation loss: 10.34%) and high-dose group (post implantation loss: 11.97%) compared to the control (post implantation loss: 6.06 %). In the high-dose group, the pre implantation loss was found to be slightly higher (16.20%) than in the control group (13.16%).

Mean percentage of pre and post implantation loss was found to be within the historical control data range for all dose groups and the control group and additionally, no statistical significance was found. An effect of the treatment with test item was therefore not concluded. The pre-natal and post-natal parameters including the number of corpora lutea, implantation sites and alive pups on PND 0, 4 and 13 showed no statistically significant differences between test item-treated groups and the controls.

The copulation index and delivery index was 100% in the control group and in all dose groups.

Fertility index was 100% in the control group, 100% in the low-dose group, 90% in the mid-dose group and 80% in the high-dose group. The slightly lower fertility index in the mid-dose group was caused by 1/10 animals in the mid-dose group and 2/10 animals in the high-dose group, which were not pregnant. At histopathological evaluation no special findings were observed in ovaries, uterus with cervix and vagina. Additionally, no special findings were noted in testes, epididymides, prostate, seminal vesicles and coagulating glands for the high-dose males, which were paired with these females.

Overall, as no histomorphological changes to be of toxicological concern were found in the reproductive organs including ovaries, uterus, cervix and vagina in the two high-dose group and the one mid-dose animals, the lower fertility and viability indexes in mid-dose and/or high-dose females were assumed not to be an effect related to treatment with test item.

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

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse reproductive effects observed.

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:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The survival of pups from PND 0 to PND 4 and from PND 4 to PND 13 was not statistically significantly affected in the test item groups when compared to the control group.
Observed total mortality in the high-dose group of 13.54% between PND 0-4 was achieved by the death of 1/12 pups of dam no. 72 and 7/7 pups of dam no. 80, but no mortality was seen for pups in the high-dose group between PND 4-13. As the mortality was observed for pups of two single dams from the high-dose group and only during the first observation days without statistical significance, no effect of the treatment with test item is considered.
No mortality was observed in the control, low-dose and mid-dose group from PND 0 to PND 4 and from PND 4 to PND 13.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup weight on PND 13 was statistically significantly lower in the high-dose group (17.24% below control) when compared to the control group, but no statistically significant difference was found on PND 0 and PND 4. The mean values for mean pup weight on PND 0, 4 and 13 were within the historical control data range for the high-dose group and all other dose groups as well as the control group. Therefore, an effect of the treatment with test item was not assumed.

Total litter mean weight on PND 13 was statistically significantly lower in the high-dose group (26.30% below control) whereas no statistically significant difference to the control was noted for mean male and female litter weight on PND 13. Male litter weight was 38.53% but not statistically significantly below control on PND 13. The litter weight data on PND 0, 4 and 13 were within the historical control data range. An effect of treatment with test item was therefore not assumed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Male pups of the high-dose group had a statistically significantly higher absolute (2.63 mm in the high-dose group compared to 2.40 mm in the control group) and relative (1.43 mm in the high-dose group compared to 1.29 mm in the control group) anogenital distance compared to males of the control group. A slight and statistically significantly higher absolute and relative anogenital distance was also seen for female pups (absolute: 1.06 mm in the high-dose group compared to 0.95 mm in the control group; relative: 0.59 mm in the high-dose group compared to 0.52 mm in the control group).
All mean values were within the range of historical control data. Therefore, a relation to treatment with test item on the increase of absolute and relative AGD in high-dose males and females was not assumed.

In the absence of dose dependency the statistically significant increase of cube root pup weight for the male pups (low-dose 1.90, control 1.85) and female pups (low-dose and mid-dose 1.85, control 1.81) is considered to be toxicologically not relevant.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Nipple retention of male pups on PND 12 was seen with a tendency towards a higher mean value between the dose groups, but no statistical significance was found for all dose groups when compared to control (mean high dose up to 0.5, mean control 0.26).

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death.

External findings seen in the mid-dose group were a black tail tip (2/10 pups between PND 11-12 and 4/10 pups on day of death from dam number 70). In the high-dose group, for 8/10 pups from one dam hairless area on the back was seen between PND 8-12 and the day of death and additionally, haematoma on the mouth was observed for 1/10 pups on PND 0. No indication of suckling was observed for 2/10 pups from another high-dose female on PND 0 and haematoma on the mouth, neck or back for 4/7 pups of a different high-dose dam on PND 0. The external findings were observed for pups of single dams in the dose groups and therefore considered to be of no toxicological relevance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of pups sacrificed on day 13. Additionally, there were no considerable changes between thyroid/parathyroid glands weight of 13 day old male and female pups in all dose groups and the corresponding control group.

Mean T4 level of female pups on day 13 showed a decrease of 13.8% below control in the mid-dose group and 25.6% in the high-dose group, without achieving statistical significance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 750 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

Reproductive effects observed:

no

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 and 750 mg/kg bw/day for reproduction/developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

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 X, 8.7.3, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

As described in the repeated dose toxicity chapter systemic toxicity was observed based on reduced body weight development (mid- and high-dose) and food consumption (high-dose), increased urea and creatinine levels (high-dose) at clinical chemistry, increased kidney weights (high-dose) and adverse macro- and microscopic findings in kidney, urinary bladder and ureter (mid- and high-dose). Based on the test item-related findings, the NOAEL was determined to be 62.5 mg/kg bw/day for general toxicity.

With regard to reproductive toxicity, 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. These alterations were considered to be secondary to low body weight or due to stress related to the deterioration of general condition and not to be of an adverse nature.

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 was no treatment-related effect of the test item on pre-coital interval and duration of gestation observed. The test item had no effect on the pre and mean post implantation loss. The pre-natal and post-natal parameters including the number of corpora lutea, implantation sites and alive pups on PND 0, 4 and 13 showed no statistically significant differences between test item-treated groups and the controls. The copulation index and delivery index was 100% in the control group and in all dose groups. No test-item related changes were noted on the fertility and viability index, respectively.

The litter parameters including the total number of pups, still births and runts born on PND 0, the number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group. No treatment-related effect on these parameters is concluded. Mean pup weight, total litter mean weight and survival of pups was not affected by the test item. Anogenital distance and nipple retention values were within the range of historical control data. No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death. Treatment with the test item had no effect on serum T4 levels of parental males and of pups sacrificed on day 13. Additionally, there were no considerable changes between thyroid/parathyroid glands weight of 13 day old male and female pups in all dose groups and the corresponding control group.

Thus, based on the results of this study, the NOAEL for reproductive toxicity was determined to be ≥750 mg/kg bw/day.

No extended-one generation reproductive toxicity study on triethoxy(vinyl)silane (CAS 78-08-0) is available. Therefore, the hazard assessment for that endpoint was performed based on the available data from the source substance trimethoxy(vinyl)silane (CAS 2768-02-7). 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. 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) read across from the analogue substance has been applied to support the human health hazard assessment of triethoxy(vinyl)silane (CAS 78-08-0).

Details on read across justification can be found in the justification for grouping of substances attached in IUCLID Section 13.

 

In the extended one-generation reproductive toxicity study (BSL, 2021), conducted according to OECD TG 443 and in compliance with GLP, 25 male and 25 female Parental (P0) Wistar rats per group, were exposed to 0, 40, 100, 300 mg/kg bw/day trimethoxy(vinyl)silane (CAS 2768-02-7) in corn oil by oral gavage for 2 weeks during pre-mating (males and females), for up to 2 weeks during mating (males and females), for 6 weeks during post-mating and up to termination after weaning of F1 (males; 10 weeks total treatment) or during pregnancy and lactation up to termination after weaning of F1 (females; 8-10 weeks total treatment), respectively. At weaning, selected F1 offspring were assigned to specific cohorts for investigations consisting of sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. In F1 males and females, the direct exposure to test item was started at weaning until the scheduled termination, i.e., until an age of 13 weeks (Cohort 1A, 20 animals per sex and group) or until study termination (weeks 20-25: Cohort 1B, 20 animals per sex and group). Furthermore, Cohort 3 animals underwent evaluation of developmental immunotoxicity and were sacrificed at an age of 8-10 weeks (10 animals per sex and group). The dose levels were selected based on the findings of the 28-day oral repeated dose toxicity study (BSL Bioservice, 2020).

During the administration period the animals (P0, F1 and F2) were observed closely each day for signs of toxicity. Detailed clinical observations were made once before the first exposure, and once a week thereafter, in all P0 animals and all cohorts.

Body weight and food consumption were measured in all animals at specific intervals, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 2 weeks pre-mating period, P0 male and female rats from the same dose group were mated (1:1 pairing). F1 (P1) males and females from Cohort 1B were bred (1:1 pairing) after minimum treatment up to PND 90 to obtain a F2 generation.

Each F1 and F2 litter was examined as soon as possible after the delivery of the dam (PND 0) to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on days 4, 7, 14 and 21 post-partum.

The anogenital distance (AGD) of each F1 and F2 (Cohort 1B extension) pup was measured on PND 0 and all male pups were checked for the presence of nipples/areolae on PND 12.

All selected F1 male and female pups from all cohorts (except surplus not selected for cohorts) were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females.

Vaginal smears of P0 females were examined 2 weeks before the beginning of the treatment period, during the 2-week premating period and until confirmation of mating. Vaginal smears were examined daily for all F1 females in Cohort 1A after the onset of vaginal patency until the first cornified smear was recorded. Vaginal smear in Cohort 1A was also examined for 2 weeks starting from PND 75. The vaginal smear in Cohort 1B was examined during the mating period to confirm evidence of mating.

Haematological, coagulation, thyroid hormone analysis (T4 and TSH) and clinical biochemistry parameters were determined with blood samples obtained from 10 randomly selected P0 males and females and 10 randomly selected F1 Cohort 1A male and female animals at terminal sacrifice. Urinalysis was also performed on samples collected from these animals prior to or as part of their terminal sacrifice. Thyroid hormone analysis (T4 and TSH) was also performed on 10 pups/sex/group at PND 4 and after weaning (pups not allocated to cohorts) on PND 22.

To identify possible toxic effects on male fertility, sperm motility and testicular sperm head count an evaluation was performed at the end of the treatment period from all Parental generation males and all Cohort 1A males of each group by using a Hamilton Thorn sperm analyser. Sperm morphology was evaluated at the end of the treatment period from all P0 generation males and all F1 Cohort 1A males from control and HD group.

Cohort 3 animals consisting of 10 males and 10 females (on PND 56±3 days) from each treatment group were used for a T-cell dependent antibody response assay to measure KLH-specific IgM antibodies. Pre- and postnatally induced immunotoxic effects at termination from 10 male and 10 female F1 Cohort 1A animals from each treatment group were evaluated by analysis of splenic lymphocyte subpopulations (lymphocytes, T cells, CD4+ (helper T cells) CD8+ (cytotoxic T cells), B lymphocytes and natural killer (NK) cells using one half of the spleen.

At the conclusion of the treatment period of P0 animals and various cohorts, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. Animals that died or were sacrificed in a moribund condition were examined macroscopically and histopathologically.

A full histopathological evaluation of the collected tissues was performed on HD and control P0 and F1 Cohort 1A animals. The histopathological examination of F1 Cohort 1A female ovaries included quantitative evaluation of primordial and small growing follicles and corpora lutea. From F1 Cohort 1A males, 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. These examinations were not extended to animals of the other dosage groups as no treatment-related changes were observed in the HD group.

Any gross lesion macroscopically identified was examined microscopically in all animals including found dead or moribund sacrificed animals. Additionally, based on the histopathology findings of the HD group, lower dose levels (LD and MD groups) were evaluated in the P0 generation including kidneys, ureters, urinary bladder, duodenum, jejunum and ileum and F1 Cohort 1A with kidneys, ureters, urinary bladder and jejunum.

In the P0 generation, 1/25 LD group female was found dead during the study period. In F1 Cohort 1A, 1/20 LD male, 1/20 MD female and 1/20 HD female were moribund sacrificed and 1/20 LD female was found dead during the study period. In F1 Cohort 1B, 2/20 control, 4/20 LD group, 1/20 MD group females and 1/20 HD male were found dead during the study period. No mortality was observed in F1 Cohort 3. The cause of the remaining unscheduled deaths was deemed to be accidental due to gavage error, and none were considered to be treatment-related.

In terminally sacrificed P0 and F1 male and female animals, predominant clinical signs transiently observed in the majority of the HD group were increased salivation (slight/moderate) and/or moving the bedding. Low incidences of clinical signs including hairless area, crust, scratch/cut, eyelid closure, piloerection, chromodacryorrhea, corneal opacity and diarrhoea (single incidence), exophthalmos (left), wound, overgrown teeth and spontaneous reduced activity (slight) were observed in few animals on few days in all groups including control. As these findings showed no dose-dependency and were transient, they were not considered to be toxicologically relevant.

 

The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect. None of the P0 or F1 Cohort 1B females showed signs of abortion or premature delivery. During the weekly detailed clinical observation, no toxicologically relevant differences between the treated groups and the control group were observed in P0 and F1 Cohorts (1A, 1B and 3) during the entire study period. In all P0 and F1 cohorts, mean body weight and mean body weight gain remained unaffected by the treatment with the test item. There were no test item-related effects on food consumption for P0 and F1 animals. The test item had no biologically or statistically significant effect on the oestrus cycle of P0 and F1 Cohort 1A females.

 

In P0 and Cohort 1B females, there were no test item-related or statistically significant effects on litter parameters including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, or runt on PND 0, as well as number of alive pups, alive male and female pups and sex ratio on PNDs 4, 7, 14 and 21 when compared to the control. Statistical analysis of litter data revealed no significant effects in treatment groups when compared with the control. There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in P0 and F1 Cohort 1B treatment groups when compared with the controls. There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the P0 and F1 Cohort 1B treatment group females when compared to the control.

 

There were no test item-related effects observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent preimplantation loss and post implantation loss in P0 and F1 Cohort 1B treatment group females when compared with the corresponding control group. There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in P0 and F1 Cohort 1B treatment group animals when compared to the respective control group. No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in P females and from PND 0 to 4, PND 4 to 21 in F1 Cohort 1B female treatment groups was observed when compared to the control group. There was no test item-related effect on anogenital distance and nipple retention in F1 and F2. No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F1 and F2 pups. No test item-related adverse effects were observed in haematological parameters in the dose groups when compared to the control group.

 

In HD group males, statistically significantly higher mean WBC, RBC, HGB and HCT were observed when compared to the control. In MD group males, higher mean HGB and HCT and lower RET percent were observed when compared to the control. In females, no statistical significance was observed when compared with the control. All these values were without any dose dependency or consistency; hence they were not considered to be adverse. No test item-related effect was observed on coagulation parameters in P0 males and females when compared with the control, except for slight but statistically significantly higher mean PT value in HD group males and MD and HD group females when compared to the control. All other group mean and most of the individual values for haematological parameters in males and females were comparable to controls and within the normal range of variation, and they were also in line with historical control data of reproductive and developmental toxicity studies in this strain.

 

In F1 Cohort 1A males, marginally but statistically significantly higher mean RBC was observed in MD group males when compared with the control. Marginally but statistically significantly higher mean RBC, HGB and HCT were observed in MD group females when compared with the control. As the differences were marginal and all values were within the range of historical control data, these findings were not assumed to be toxicologically relevant. All other group means and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation. In the absence of test item-related histopathological findings and effect on splenic lymphocyte subpopulation in the study, the above-mentioned increase or decrease in a few haematology parameters was not considered to be adverse. No test item-related effect was observed on coagulation parameters in Cohort 1A males and females when compared with the respective control. There were a few marginal but statistically significant differences in clinical biochemistry parameters of male and female animals of P0 and F1 Cohort 1A. In Parental animals, lower creatinine was observed in HD group males and in MD and HD group females. In F1 Cohort 1A animals, differences in potassium (LD group males), protein (HD group males), and ASAT and Cholesterol (HD group females) were observed. As they were within the range of historical control data, not dose-dependent, and did not coincide with histopathological findings, these findings were not considered toxicologically relevant. No test item-related changes were observed in urinary parameters in P0 and F1 Cohort 1A groups. No effects were noted in thyroid hormone analysis for P0, F1 Cohort 1A and F1 pups on PND 4 and PND 21. No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in F1 (Cohort 1A, 1B and 3) treatment groups when compared to the control. There were no test item-related effects on sperm analysis in P0 and F1 Cohort 1A animals. There was a slight increase in mean lymphocytes and T cell populations in both male and female F1 Cohort 1A animals when compared to the control. There was no test item-related or statistically significant change in the number of splenic B cells. They were found to be comparable between all dose groups and control group in both males and females. There was no significant indication of immunosuppressive effect of the test item on lymphocyte subpopulations.

 

The results of the TDAR in F1 Cohort 3 indicate a functional immune system. KLH-specific IgM levels fold difference in the HD group was found to be variable but LD and MD groups were comparable to the negative control and did not show any sign of effect on the specific immune response. Macroscopic findings were not recorded in animals with unscheduled death. In the decedents, there were no gross lesions that could be attributed to treatment with the test item. In animals that survived their scheduled treatment period, gross lesions that were considered to be treatment-related were recorded in the urinary bladder of females from P0-generation and in the urinary bladder and/or ureters of both sexes of animals from F1-generation Cohort 1B. Test item-related gross findings were not recorded in F1-generation Cohort 1A animals. There were no statistically significant differences in the terminal body weights (TBW) in both sexes of both generations (P0-generation and F1-generation Cohort 1A). Test item-related changes were found in the kidney of P0-generation and F1-generation Cohort 1A animals. In the histopathological examination, for both the P0- and F1-generations, microscopic changes that could be attributed to treatment with the test item were observed in the kidneys and urinary bladder of both sexes of animals treated with 100 mg/kg bw/day and higher, and in the ureters and small intestine of both sexes of animals treated with 300 mg/kg bw/day.

 

In the kidneys of  P0-generation the following changes were observed: diffuse urothelial hyperplasia and mixed inflammatory cell infiltration in the suburothelium in both sexes; fibrosis at/around the fornix, focal to multifocal interstitial fibrosis, pelvic luminal precipitates, and foreign body giant cells at/around the fornix in males; and increased incidence and/or severity of pelvic dilation, focal to multifocal mononuclear cell infiltration, tubular basophilia and tubular dilatation in males. In the urinary bladder of P0-generation the following changes were observed: diffuse urothelial hyperplasia, mixed inflammatory cell infiltration in the mucosa, increased incidence and/or severity of mononuclear cell focus/foci in lamina propria, submucosal oedema, congestion, and haemorrhage in both sexes. In the ureters of P0-generation the following changes were observed: diffuse urothelial hyperplasia in both sexes; increased incidence and severity of luminal dilatation in males; and mixed inflammatory cell infiltration and submucosal oedema, as well as diffuse urothelial hyperplasia and luminal dilatation, in the ureters present by chance on the prostate sections. In the small intestine of P0-generation the following changes were observed: lipid accumulation in lamina propria was observed in the duodenum, jejunum and ileum of males and in the jejunum and ileum of females.

 

In the kidneys of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia and mixed inflammatory cell infiltration in the suburothelium in both sexes; tubular dilatation, granuloma at/around the fornix and increased incidence of pelvic dilation in males; and pelvic luminal precipitates in female. In the urinary bladder of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia, mixed inflammatory cell infiltration in the mucosa, mononuclear cell focus/foci in lamina propria, haemorrhage, and increased incidence of submucosal oedema in both sexes; and congestion in males. In the ureters of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia and increased incidence of luminal dilatation in both sexes; mixed inflammatory cell infiltration and submucosal oedema, as well as diffuse urothelial hyperplasia and luminal dilatation, were identified in the ureters present by chance on the prostate sections. In the small intestine of F1-generation Cohort 1A the following changes were observed: lipid accumulation in lamina propria was observed in the jejunum of both sexes. There was no test item-related histomorphological changes in the male and female reproductive organs in either generation. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in either generation.

 

The NOAEL for general systemic toxicity for P0 and F1 (Cohort 1A and 1B [P1]) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P0 and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on reproductive organs and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response.

Effects on developmental toxicity

Description of key information

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

NOAEL (developmental toxicity)≥750 mg/kg bw/day

NOAEL (systemic toxicity) = 62.5 mg/kg bw/day

 

RA CAS 2768-02-7:

Inhalation:

Prenatal Developmental Toxicity Study (EPA OTS 798.4350), rat:

NOAEC (maternal toxicity) = 25 ppm (equivalent to approx. 144 mg/m³),

NOAEC (developmental toxicity) ≥300 ppm (equivalent to approx. 1730 mg/m³)

 

Oral:

Prenatal Developmental Toxicity Study (OECD 414), rabbit:

NOAEL (maternal toxicity) = 7.5 mg/kg bw/day,

NOAEL (developmental toxicity) ≥75 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal 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

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

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 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%.

Details on mating procedure:

- M/F ratio per cage: Mating was performed using a ratio of 1:1 (male to female).
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage.

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

Duration of test:

28 days in males and maximum 63 days in females

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.

Maternal examinations:

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

POST-MORTEM EXAMINATIONS: Yes; tables 4 and 5
SACRIFICE
- Male animals: All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) by using anaesthesia (ketamine/xylazin).
- Maternal animals: Females were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazin).

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Blood sampling:

Blood was collected at necropsy and haematological, coagulation and clinical biochemistry parameters examined (for details please refer to "Maternal examinations").

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

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 non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.

Indices:

Indices calculated were the copulation index, delivery index and fertility index. Viability index between PND 0 to PND 4 was calculated.

Historical control data:

Yes

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.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

All dams survived the scheduled study period.

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.

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).

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.

Food efficiency:

not examined

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

not examined

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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

IAt 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.

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.

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.

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.

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.

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.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of parental males.

Number of abortions:

no effects observed

Description (incidence and severity):

The delivery index was 100% in the control and in all dose groups.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean post implantation loss was observed to be higher in the mid-dose group (post implantation loss: 10.34%) and high-dose group (post implantation loss: 11.97%) compared to the control (post implantation loss: 6.06%). In the high-dose group, the pre implantation loss was found to be slightly higher (16.20%) than in the control group (13.16%).

Mean percentage of pre and post implantation loss was found to be within the historical control data range for all dose groups and the control group and additionally, no statistical significance was found. An effect of the treatment with test item was therefore not concluded.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Observed total mortality in the high-dose group of 13.54% between PND 0-4 was achieved by the death of 1/12 pups of dam no. 72 and 7/7 pups of dam no. 80, but no mortality was seen for pups in the high-dose group between PND 4-13. As the mortality was observed for pups of two single dams from the high-dose group and only during the first observation days without statistical significance, no effect of the treatment with test item is considered.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There was no treatment-related effect of the test item on pre-coital interval and duration of gestation observed.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The delivery index was 100% in the control group and in all dose groups.
Fertility index was 100% in the control group, 100% in the low-dose group, 90% in the mid-dose group and 80% in the high-dose group. The slightly lower fertility index in the mid-dose group was caused by 1/10 animals in the mid-dose group and 2/10 animals in the high-dose group, which were not pregnant. At histopathological evaluation no special findings were observed in ovaries, uterus with cervix and vagina. Additionally, no special findings were noted in testes, epididymides, prostate, seminal vesicles and coagulating glands for the high-dose males, which were paired with these females.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

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

Abnormalities:

effects observed, treatment-related

Localisation:

other: urinary (kidney, bladder, ureter)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup weight on PND 13 was statistically significantly lower in the high-dose group (17.24% below control) when compared to the control group, but no statistically significant difference was found on PND 0 and PND 4. The mean values for mean pup weight on PND 0, 4 and 13 were within the historical control data range for the high-dose group and all other dose groups as well as the control group. Therefore, an effect of the treatment with test item was not assumed.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

One out of 11 pups of a control dam and 2/7 pups of another control dam were found dead on PND 0. Two out of 10 pups of a mid-dose dam were found dead on PND 0. In the high-dose group, 1/12 pups from one dam and and 5/7 pups from another dam (number 80) were not found on PND 0 and additionally 2/7 pups of dam no. 80 on PND 3sex. Death of pups was noted in dose groups as well as the control group and considered to be of no toxicological relevance.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group. No treatment-related effect on these parameters is concluded.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The litter parameters including the total number of pups was not statistically significantly different in test item groups compared to the control group. No treatment-related effect on this parameter is concluded.
Total litter mean weight on PND 13 was statistically significantly lower in the high-dose group (26.30% below control) whereas no statistically significant difference to the control was noted for mean male and female litter weight on PND 13. Male litter weight was 38.53% but not statistically significantly below control on PND 13. The litter weight data on PND 0, 4 and 13 were within the historical control data range. An effect of treatment with test item was therefore not assumed.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Male pups of the high-dose group had a statistically significantly higher absolute (2.63 mm in the high-dose group compared to 2.40 mm in the control group) and relative (1.43 mm in the high-dose group compared to 1.29 mm in the control group) anogenital distance compared to males of the control group. A slight and statistically significantly higher absolute and relative anogenital distance was also seen for female pups (absolute: 1.06 mm in the high-dose group compared to 0.95 mm in the control group; relative: 0.59 mm in the high-dose group compared to 0.52 mm in the control group).
All mean values were within the range of historical control data. Therefore, a relation to treatment with test item on the increase of absolute and relative AGD in high-dose males and females was not assumed.
In the absence of dose dependency the statistically significant increase of cube root pup weight for the male pups (low dose 1.90, control 1.85) and female pups (low-dose and mid-dose 1.85, control 1.81) is considered to be toxicologically not relevant.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

Two out of 7 pups from a high-dose dam were not found on PND 3. This was considered to be of no toxicological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death.
External findings seen in the mid-dose group were a black tail tip (2/10 pups between PND 11-12 and 4/10 pups on day of death from dam number 70). In the high-dose group, for 8/10 pups from one dam hairless area on the back was seen between PND 8-12 and the day of death and additionally, haematoma on the mouth was observed for 1/10 pups on PND 0. No indication of suckling was observed for 2/10 pups from another high-dose female on PND 0 and haematoma on the mouth, neck or back for 4/7 pups of a different high-dose dam on PND 0. The external findings were observed for pups of single dams in the dose groups and therefore considered to be of no toxicological relevance.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Nipple retention of male pups on PND 12 was seen with a tendency towards a higher mean value between the dose groups, but no statistical significance was found for all dose groups when compared to control (mean high dose up to 0.5, mean control 0.26).

Treatment with the test item had no effect on serum T4 levels of pups sacrificed on day 13. Additionally, there were no considerable changes between thyroid/parathyroid glands weight of 13 day old male and female pups in all dose groups and the corresponding control group.
Mean T4 level of female pups on day 13 showed a decrease of 13.8 % below control in the mid-dose group and 25.6 % in the high-dose group, without achieving statistical significance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effects observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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 maternal toxicity and 750 mg/kg bw/day for developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

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

Species:

rat

Key result

Dose descriptor:

NOAEC

Remarks:

maternal toxicity

Effect level:

25 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: Based on concentration-dependent reductions in gestational body weight gain (gestation days 6-9); equivalent to approx. 191.1 mg/m³

Remarks on result:

other: Source CAS 2768-02-7 (BRRC, 1993)

Abnormalities:

effects observed, treatment-related

Localisation:

other: body weight gain

Dose descriptor:

NOAEC

Remarks:

developmental

Effect level:

>= 300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Evidence of slightly delayed development in fetuses from the 300 ppm group as indicated by delayed ossification in several skeletal districts in the presence of maternal toxicity; equivalent to approx. 2423.67 mg/m³

Remarks on result:

other: Source CAS 2768-02-7 (BRRC, 1993)

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Exposure of pregnant CD® rats during organogenesis to the test material by inhalation resulted in a NOAEL (maternal) of 25 ppm (equivalent to approx. 191.1 mg/m³) and a NOAEL (developmental) ≥300 ppm (equivalent to approx. 2423.67 mg/m³), respectively.

Reference 3

Endpoint:

developmental toxicity

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

Species:

rabbit

Key result

Dose descriptor:

LOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Source CAS 2768-02-7 (BSL Bioservice, 2020)

Abnormalities:

effects observed, treatment-related

Localisation:

other: urinary tract

Dose descriptor:

NOAEL

Remarks:

foetal

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: Source CAS 2768-02-7 (BSL Bioservice, 2020)

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In a prenatal developmental toxicity study in rabbits, conducted according to OECD Test Guideline 414 and in compliance with GLP, the LOAEL for maternal toxicity was concluded to be 7.5 mg/kg bw/day based on degenerative changes characterized by tubular dilatation and tubular basophilia in all dose groups; the NOAEL for developmental toxicity was concluded to be equal to or greater than 75 mg/kg bw/day based on no observed adverse effects in any of the foetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

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 analogue justification for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.7.2, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 730 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

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 analogue justification for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7.2, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

As described in the repeated dose toxicity chapter systemic toxicity was observed based on reduced body weight development (mid- and high-dose) and food consumption (high-dose), increased urea and creatinine levels (high-dose) at clinical chemistry, increased kidney weights (high-dose) and adverse macro- and microscopic findings in kidney, urinary bladder and ureter (mid- and high-dose). Based on the test item-related findings, the NOAEL was determined to be 62.5 mg/kg bw/day for general toxicity.

With regard to developmental toxicity, the test item had no effect on the pre and mean post implantation loss. The pre-natal and post-natal parameters including the number of corpora lutea, implantation sites and alive pups on PND 0, 4 and 13 showed no statistically significant differences between test item-treated groups and the controls. No test-item related changes were noted on the viability index. The litter parameters including the total number of pups, still births and runts born on PND 0, the number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group. No treatment-related effect on these parameters is concluded. Mean pup weight, total litter mean weight and survival of pups was not affected by the test item. Anogenital distance and nipple retention values were within the range of historical control data. No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death. Thus, based on the results of this study, the NOAEL was determined to be ≥750 mg/kg bw/day for developmental toxicity.

 

No prenatal developmetnal toxicity studies in rats and rabbits on triethoxy(vinyl)silane (CAS 78-08-0) are available. Therefore, the hazard assessment for that endpoint was performed based on the available data from the source substance trimethoxy(vinyl)silane (CAS 2768-02-7). 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. 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) read across from the analogue substance has been applied to support the human health hazard assessment of triethoxy(vinyl)silane (CAS 78-08-0).

Details on read across justification can be found in the justification for grouping of substances attached in IUCLID Section 13.

 

In the key prenatal developmental toxicity study, conducted according to EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen) and in compliance with GLP, pregnant female rats were exposed to the test concentrations of 25, 100 or 300 ppm via whole body inhalation during gestation days 6 to 15 (Bushy Run Research Center, 1993). The pregnancy rate was equivalent for all groups and ranged from 96 to 100%. No treatment-related mortality occurred during the study. One dam from the 300 ppm group was removed from the study on gestation day 14 due to trauma unrelated to exposure. Another 300 ppm female delivered early on gestation day 21, and was therefore removed from the study and all data summaries with the exception of pregnancy calculations. Two females, one each from the 25 and 300 ppm groups, had only non-viable implants and one female each from the 25 and 100 ppm groups were not pregnant. Assessment of the various reproductive endpoints did not reveal any differences among the groups. Foetal examinations indicated no evidence of treatment-related embryolethality or teratogenicity. Developmental delay in the 300 ppm group was indicated by an increase in the incidence of delayed skeletal ossification of the anterior arch of the atlas, thoracic centra, interparietal, metatarsals, and phalanges. A statistical increase in the number of litters at 100 ppm with unossified anterior arch of the atlas was not considered to be biologically significant because no other endpoints were similarly affected and the incidence (79.2%) was close to that observed in historical controls (29.2-73.9%). Mean foetal body weight/litter were not different among control and treated groups. The NOAEC for maternal toxicity was concluded to be 25 ppm (equivalent to approx. 191.1 mg/m³) based on concentration-dependent reductions in gestational body weight gain and the NOAEC delayed development is greater than or equal to 300 ppm based on evidence of slightly delayed development in foetuses from the 300 ppm (equivalent to approx. 2423.67 mg/m³) group as indicated by delayed ossification in several skeletal districts, but only in the presence of maternal toxicity.

 

In the key prenatal developmental toxicity study in a second species, conducted according to OECD Test Guideline 414 and in compliance with GLP, pregnant female rabbits were exposed to the source substance, trimethoxy(vinyl)silane, at doses of 0, 7.5, 25 or 75 mg/kg bw/day in corn oil via oral gavage during gestation days (GDs) 6 to 27 (BSL Bioservice, 2020). During the period of administration, the animals were observed each day for signs of toxicity and mortality. Animals that died during the study were examined macroscopically. All surviving animals were sacrificed on their respective GDs 28. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), and live and dead foetuses. Foetuses were identified by colour strings, sexed and weighed. The foetuses were observed for external abnormalities, visceral, craniofacial abnormalities and skeletal abnormalities. Body weight and food consumption were measured on GDs 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. The uteri of the non-pregnant females were processed and checked for the early embryonic deaths.

No test item-related mortality was observed in any of the treated groups during the treatment period of this study. Moribund sacrifice of one control female on GD 7 was attributed to a gavaging error. No treatment-related clinical signs or changes in body weight and food consumption were observed in any the treated groups when compared to control.

No treatment-related effect on prenatal and litter data parameters and gross pathology of terminally sacrificed females was observed at any dose tested.

Furthermore, no treatment-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the treatment groups when compared with the controls.

 

In the urinary tract, diffuse transitional cell hyperplasia was observed in kidneys, ureters and urinary bladder of several animals from all groups treated with the test item. In the urinary bladder, the hyperplastic change was accompanied by submucosal congestion, oedema and, in two animals, by mixed cell infiltration. There were degenerative changes in the kidneys of the affected animals, which included tubular dilatation and slightly increased tubular basophilia. The LOAEL for maternal toxicity was concluded to be 7.5 mg/kg bw/day based on the degenerative changes characterized by tubular dilatation and tubular basophilia in all dose groups; the NOAEL for developmental toxicity was concluded to be equal to or greater than 75 mg/kg bw/day based on no observed adverse effects in any of the foetuses.

 

Justification for classification or non-classification

The available data on toxicity to reproduction 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.