1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, conducted according to OECD Test Guideline 422 and in compliance with GLP, concluded a NOAEL of 1000 mg/kg bw/day for reproduction based on no effects observed at the highest dose tested (Eurofins, 2019).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th June 2018 to 05th September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC)

Version / remarks:

No. 440/2008, L 142, Annex Part B, May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: homogenous and stability was demonstrated for 10 days at room temperature
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were prepared with dried corn oil and thereafter administered within the stability time frame of 10 days. The test item was weighe d into a tared plastic vial on a suitable precision balance and the vehicle was added to give the a ppropriate final concentration of the test item further vortexing it for 2-3 minutes and/or stirring until v isual homogeneity was achieved.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: appr. 12-13 weeks old
- Weight at study initiation: males: 344 - 398 g (mean: 365.31 g, ± 20 % = 292.25 – 438.37 g) females: 204 - 243 g (mean: 225.58 g, ± 20 % = 180.46 – 270.69 g)
- Fasting period before study: not specified
- Housing: Animals were 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 femal es were housed together in ratio 1:1 (male to female). 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. In each cage Altromin saw fibre was used as bedding
- Diet: ad libitum: free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: adequate acclimatisation period (at least 5 days) under laboratory conditions

DETAILS OF FOOD AND WATER QUALITY: Food and water quality are certified by the test facility every two years.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): : 22 +/- 3 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared with dried corn oil and thereafter administered within the stability time frame of 10 days. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item further vortexing it for 2-3 minutes and/or stirring until visual homogeneity was achieved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item formulation was prepared with dried corn oil. The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): MKCC9871, MKCF8882
- Purity: not specified

Details on mating procedure:

- M/F ratio per cage: ratio of 1:1 (male to female)
- Length of cohabitation: 14 days of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Due to unsuccessful mating pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): After the confirmation of mating, females were kept individually during gestation/lactation period
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For verification of dose concentrations samples were taken of formulation samples in study week 1 (p re-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).

Duration of treatment / exposure:

The test item was administered 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. The males of the recovery groups were treated for 28 days and were subjected to necropsy 15 days after the last administration (end of recovery period). The females of the recovery groups were treated up to the first scheduled necropsy of dam and were subjected to necropsy 15 days thereafter (end of recovery period).

Frequency of treatment:

A single dose given daily.

Details on study schedule:

- P0 parental animals not mated during the 14-day pre-mating period.
- Selection of parents from F1 generation when pups were [...] days of age. Not applicable
- Age at mating of the mated animals in the study: [...] weeks: 14-15 weeks of age

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose (LD)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose (MD)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose (HD)

No. of animals per sex per dose:

10 males and 10 females per test group plus additional 10 recovery group males and 10 recovery group females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selected dose levels were based on findings of a dose range-finding study conducted with the registered substance and in consultation with the sponsor.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no
- Other:

Positive control:

Not included.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once per day for general health condition and twice daily for mortality and morbidity
- Cage side observations checked included: mortality, morbidity and general health condition of the animals

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 of the main groups and the recovery groups outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, rectum bent 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 gestation days (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 animal prematurely sacrificed was weighed pr ior to the sacrifice.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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.

Oestrous cyclicity (parental animals):

All 70 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were selected in the main study and 10 females in recovery groups.

Sperm parameters (parental animals):

Parameters examined in all male parental animals: testis weight, epididymis weight, checked on completeness of cell populations, completeness of stages and degenerative changes.

Litter observations:

The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the 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 littering (PND 0) and on PND 4 and PND 13.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrified on study day 28
- Maternal animals: All surviving animals were sacrified on post-natal day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table No 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations macroscopic and/or microscopic examination.

CLINICAL CHEMISTRY: Blood samples were collected from from 2 female (if possible) pups/litter on postnatal day 4 and from 2 pups/litter (1 male and 1 female, if possible) at termination on day 13 . All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups were assessed for serum levels for thyroid hormones (T4).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

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

Reproductive indices:

copulation, fertility, viability and delivery indices

Offspring viability indices:

Viability indices

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following clinical signs were observed in the HD group and are assumed to be related to systemic toxicity of the test item: Haematuria was observed in male animal no. 33 which euthanized for animal welfare reasons on day 25 and also in male animal no. 38 transiently. Hypothermia was observed in male animals nos. 31 and 33. General signs of impaired health condition, like reduced spontaneous activity, piloerection and half eyelid closure observed only or mainly in the HD group are also likely to be test item- related.
Females of LD, MD and HD group showed clinical signs like moving the bedding and slight to mode rate salivation and males of the HD group showed moving the bedding immediately after administration during the treatment period. All these findings indicate local reactions to the oral gavage and are not assumed to be a sign of systemic toxicity.
No clinical signs were observed in C and HD recovery groups during the recovery period. Clinical signs like scratch, broken left incisor, cut on tongue, hairless area, regurgitation of the test material, sunken flanks and crust (ear) were observed mostly transiently or on single days and were within the normal background frequency.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found during the treatment or recovery periods.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item-related mortality was observed at a daily dose of 1000 mg/kg body weight/day in 5/15 male animals. Three animal (nos. 47, 40 and 48) were found dead on days 7, 10 and 12, respectively. Two animal (nos. 33 and 32) were euthanized for animal welfare reasons on days 11 and 25, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The test item had no effect on body weight development in this study up to the MD level.
Moderate, but statistically significantly lower body weight gain in male animals was observed at HD during the treatment period when compared to controls and is considered to be toxicologically relevant. At the end of the treatment period, body weight was approximately 19.7 % (premating to post-mating day 14) and 17.4% (premating to terminal) below controls in this group and had even decreased slightly when compared to pre-treatment level.
A statistically significant decrease in body weight gain was also observed in HD recovery males du ring the treatment period (day 7-28). In this group a slight and statistically non-significant decrease in body weight (day 35 and 42) was seen during the recovery period.
Test item had no effect on body weight development in females of the HD group during premating, gestation and lactation period. However, slight and statistically significant lower body weight was observed in HD recovery females (day 7-14) during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The test item had no effect on food consumption up to the MD level. A tendency towards lower food consumption was observed in HD males during the treatment period. A slight decrease in food consumption was observed on day 1-7 during the recovery period in HD recovery males. Slight, but statistically significant lower food consumption was observed in females of the HD group towards end of gestation/lactation and. A slight decrease in food consumption was observed on day 1-14 during the recovery period in HD recovery females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period a statistically significantly higher white blood cells count was found in male animals of the HD group (127 % above controls). This coincided with statistically non-significant decrease in the rate of lymphocytes (26 % below controls) and increase in the rate of neutrophils (122 % above controls). Moreover, monocytes were statistically significantly higher than controls (78 % above controls). Taken together these changes likely reflect inflammation in the renal system. In the HD group moderate, statistically significantly higher reticulocytes were observed at the end of the treatment period (males 100 % and females 60% above control) and in males at the end of the recovery period (130 % above control). This was not associated with changes in red blood cell parameters.
At the end of the recovery period also moderate and statistically significantly higher white blood cell count was observed in male but not female animals of the HD group (70% above controls). The rate of neutrophils and monocytes of HD group males was statistically significant higher (+62 % and +129 %, respectively) and that of lymphocytes statistically significant lower (-12 %). In HD females monocytes were also moderately statistically significant elevated (119 % above controls) at the end of the recovery period. Moreover, reticulocytes of male animals of the HD group were statistically significant higher (130 % above controls).
Slightly but statistically significant higher platelet count in the HD group at the end of the treatment period in males (39 % above controls) and females (25 % above controls) and at the end of the recovery period in males (30 % above controls) are not considered toxicologically relevant as values were within the range of historical control data. Coagulation was not affected by the test item. Minimal but statistical differences in PT in recovery HD male (23 % above control) are not considered to be biologically relevant, as the individual values were within the normal range of variation for this strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A moderate to marked, dose dependent and statistically significant higher serum ALAT level (approx. 6-7-fold above control) and ASAT level (approx. 2-4-fold above control) was observed in males of the HD group at the end of treatment. Slight and statistically non-significant changes were also observed in these animals in serum AP, Chol, TBA levels. At the end of the recovery period ALAT and ASA T levels were markedly increased (>5-fold and >3-fold of controls, respectively) in these animals, however without statistically significance. ALAT and ASAT were also moderately but statistically nonsignificiant increased in female animals of the HD group (>4-fold and >2-fold of controls, respectively) at the end of the recovery period. This was accompanied by a slight, non-significant increase in TBA (>2-fold of controls). All above-mentioned findings are assumed to be related to hepatotoxicity observed histopathologically.
A single male animal of the LD group (no. 14) showed elevated ALAT, ASAT, TBA and Chol levels which coincided with necrosis in the liver.
In male animals of the HD group a slight, non-significant increase was found in UREA levels (31 % above controls) at the end of the treatment period. This is assumed to be related to histopathological findings in the renal system.
Slight and statistically significant increases in TP and Alb were observed in female animals of the HD group at the end of the recovery period (9 and 13 % above controls, respectively). These slight changes are not considered toxicologically relevant as there were no corresponding changes in globulin fraction or creatinine levels and they were not associated with histopathological findings

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed at the end of the treatment period and recovery period.
Blood and a high count of leukocytes were found in the urine of male animal no.14 of the LD group. This is assumed to be related to histopathological findings in the renal system.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In males and females, no test item-related effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period and also at the end of recovery period.

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):

Liver: Test item-related changes were observed in decedent and surviving animals from the HD group only. In all moribund and decedent animals minimal to marked microvesicular fatty change was observed. Additionally, slight hepatocellular necrosis was noticed in the decedent Animal No. 33. In surviving animals from the HD group (treatment and recovery periods), minimal microvesicular fatty change was observed. Minimal hepatocellular necrosis (single cell necrosis) was also observed in Animal No. 39.
Further, in Animal No. 14 (LD group), hepatocellular necrosis was observed. The above-mentioned hepatic change was associated with severe congestion and moderate hemorrhage and was not considered to be a test item-related finding, but rather an incidental finding.
Kidney: Test item-related changes were observed in decedent and surviving animals from the HD group only. In decedent animals from the HD group, slight to moderate tubular dilatation and minimal tubular necrosis were observed. Furthermore, severe tubular dilatation, minimal to slight tubular necrosis, slight tubular basophilia and slight multifocal mineralization (dystrophic change) were observed.
In surviving males from the HD group (end of treatment period), moderate to severe tubular dilatation, minimal to moderate mononuclear and mixed cell infiltrates in the tubular lumen and renal interstitium, slight to moderate interstitial fibrosis, slight tubular necrosis, slight tubular basophilia and minimal to slight mineralization were observed. Additionally, moderate mixed cell infiltrates and minimal tubular dilatation were noticed in females from the same group,. In recovery males from the HD group, minimal to slight tubular dilatation, slight to moderate mixed cell infiltrates, minimal to slight mononuclear cell infiltrates, slight to moderate fibrosis, slight tubular necrosis and slight to moderate tubular basophilia were observed, whereas in females no histopathological changes were noticed.

Heart: Cardiac lesions that could be attributed to the treatment with the test item were observed in decedent recovery animals from the HD group only. These cardiac changes consisted of moderate myocardial mineralization, slight vascular mineralization (e.g. tunica media of aorta and few coronary vessels), slight cardiomyocytes necrosis and slight mononuclear infiltrates.
All other recorded findings were considered incidental or were within the range of spontaneous background alterations that may be recorded in Wistar rats at these ages. No histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina could be detected. The tubular stages of the spermatogenic cycle were checked for completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Moreover, no treatment-related effect on interstitial cell structure was noticed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

no biologically significant effect on the estrous cycle in treatment groups when compared to the controls. There were no considerable changes in the length or sequence of cycle stages between the dose groups and the control group

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item- related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group. In the HD group, a reduced copulation index of 90 % was observed compared to 100 % in the control group. This effect on copulation index was attributed to 1/10 in non-pregnant females and therefore, considered to be biological variation.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Description (incidence and severity):

Pre- and Post-Natal Data - There were no test item treatment- related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent pre-implantation loss and post implantation loss in LD, MD and HD groups when compared with the control group.

Anogenital Distance and Nipple Retention - In male and female pups, no test item- related effects in absolute and relative anogenital distance were observed in the dose groups when compared to the controls. No statistically significant effect or toxicological relevance was observed on nipple retention i n the male pups of any of the groups when compared with the controls.

Pup Survival Data - No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in LD and HD groups. However, a slightly and statistically significant higher mean mortality of pups on PND 0-4 was observed in the MD group (5.41 %). As this effect was not dose-dependent, it is not considered to be an adverse effect of the test item.

Pup External Findings - No test item-related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. A few specific findings like flattened abdomen (pup no. 13 from dam no. 56) and missing tail tip (pup no. 1 from dam no. 60) of control group were observed. No indication of suckling on PND4 for the pups 1-15 from dam no. 68 of LD was observed. No indication of suckling on PND0 for pup 10 and dark head for the pup 4, 10 from dam no. 73 of MD werewas observed. All these findings were considered to be spontaneous and not related to test item treatment.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in LD and HD groups. However, a slightly and statistically significant higher mean mortality of pups on PND 0-4 was observed in the MD group (5.41 %). As this effect was not dose-dependent, it is not considered to be an adverse effect of the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related or statistically significant effect on pup mean weight, total litter weight, male litter weight, or female litter weight on PND 0, PND 4 and PND 13 observed in LD, MD and HD groups when compared with the controls.

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:

no effects observed

Description (incidence and severity):

No statistically significant effect or toxicological relevance was observed on serum T4 levels of PND 13 pups.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight (male and female).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.
A few specific findings like flattened abdomen (pup no. 13 from dam no. 56) and missing tail tip (pup no. 1 from dam no. 60) of control group were observed. No indication of suckling on PND4 for the pups 1-15 from dam no. 68 of LD was observed. No indication of suckling on PND0 for pup 10 and dark head for the pup 4 from dam no. 73 of MD were observed. All these findings were considered to be spontaneous and not related to test item treatment.

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

Summary result rables and individual animal data result tables will be added at a later date when they are available.

Conclusions:

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 2,4,6-tris[3-(trimethoxysilyl)propyl] isocyanurate, conducted according to OECD Test Guideline 422 and in compliance with GLP, concluded a NOAEL of 1000 mg/kg bw/day for reproduction based on no effects observed at the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, conducted according to OECD Test Guideline 422 and in compliance with GLP, the test item was administered at dose levels of 100 (LD), 300 (MD) and 1000 (HD) mg/kg bw/day in corn oil to 10 male and 10 female rats per dose and to additional 10 male and female rats used as recovery group in the control and high dose groups. Corn oil was given to the negative control animals. Male animals were treated for at least 28 days. Female animals were treated until post-natal day 13. Male and female animals were mated by cohabitation in ratio 1:1 for at least 14 days. Positive vaginal smear was considered as day 0 of gestation. Animals in the recovery groups were not mated and dosed for 28 days (males) or until the first scheduled kill of dams (females). They were observed for a period of 14 days following the last administration.

During the treatment period, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the termination of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, 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.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment for all animals and in the last week of treatment in five randomly selected males and females of each group.

After parturition 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. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 30 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The males of the recovery groups were subjected to necropsy 15 days after the last administration (end of recovery period). The females of the recovery groups were treated up to the first scheduled necropsy of dam and were subjected to necropsy 15 days thereafter (end of recovery period).  

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues of the control and groups which were sacrificed at the end of the treatment period was completed. 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. Further, liver, kidney and heart from all available animals (treatment and recovery period) were processed and evaluated. In addition, reproductive organs from all males and females from the control and groups were processed and evaluated. For the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff)stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

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

There were no test item treatment-related or statistically significant effects observed in LD, MD and HD groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, number of male and female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

There was no test item-related or statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in LD, MD and HD groups when compared with the controls.

There was no test item-related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the LD MD and HD groups and precoital interval in all treatment groups when compared to the control group.

There were no test item treatment-related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent pre-implantation loss and post implantation loss in LD, MD and HD groups when compared with the control group.

There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group was noted.

In male and female pups, no test item-related effects in absolute and relative anogenital distance were observed in the dose groups when compared to the controls.

No statistically significant effect or toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.

No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight. Serum thyroxine hormone (T4) levels of males of the LD, MD and HD groups at the end of the treatment period, of males of the HD group at the end of the recovery period or of PND 13 pups were not considerably different when compared to controls.

No test item-related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups on PND 0-12.

There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.

Testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

In the absence of adverse effects on reproductive parameters the no-observed-adverse-effect-level (NOAEL) for reproduction toxicity was established at 1000 mg/kg body weight/day in this study.

Effects on developmental toxicity

Description of key information

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, conducted according to OECD Test Guideline 422 and in compliance with GLP, concluded a NOAEL of 1000 mg/kg bw/day for developmental toxicity based on no effects observed in F1 at the highest dose tested (Eurofins, 2019).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th June 2018 to 05th September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / De velopmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulations (EC)

Version / remarks:

440/2008, L 142, Annex Part B, May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: homogenous and stability was demonstrated for 10 days at room temperature
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were prepared with dried corn oil and thereafter administered within the stability time frame of 10 days. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item further vortexing it for 2-3 minutes and/or stirring until visual homogeneity was achieved.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Crl: WI(Han)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared with dried corn oil and thereafter administered within the stability time frame of 10 days. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item further vortexing it for 2-3 minutes and/or stirring until visual homogeneity was achieved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item formulation was prepared with dried corn oil. The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): MKCC9871, MKCF8882
- - Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For verification of dose concentrations samples were taken of formulation samples in study week 1 (p re-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).

Details on mating procedure:

- M/F ratio per cage: ratio of 1:1 (male to female)
- Length of cohabitation: 14 days of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Due to unsuccessful mating pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): After the confirmation of mating, females were kept individually during gestation/lactation period
- Any other deviations from standard protocol: none

Duration of treatment / exposure:

The test item was administered 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. The males of the recovery groups were treated for 28 days and were subjected to necropsy 15 days after the last administration (end of recovery period). The females of the recovery groups were treated up to the first scheduled necropsy of dam and were subjected to necropsy 15 days thereafter (end of recovery period).

Frequency of treatment:

A single dose was given daily.

Duration of test:

Up to 63 days

Dose / conc.:

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

Remarks:

low dose (LD)

Dose / conc.:

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

Remarks:

mid dose (MD)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose (HD)

No. of animals per sex per dose:

10 males and 10 females per test group plus additional 10 recovery group males and 10 recovery group females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selected dose levels were based on findings of a dose range-finding study conducted with the registered substance and in consultation with the sponsor.
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once per day for general health condition and twice daily for mortality and morbidity
- Cage side observations checked included: mortality, morbidity and general health condition of the animals

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 of the main groups and the recovery groups outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, rectum bent 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 gestation days (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 animal prematurely sacrificed was weighed pr ior to the sacrifice.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-natal day 13
- Organs examined: See table No 1

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, 5 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 homogenei ty and normality tests. Statistical comparisons of data acquired during the recovery period werewill be performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Indices:

viabiliyt, copulation, fertility, viability and delivery indices

Clinical signs:

no effects observed

Description (incidence and severity):

Females of LD, MD and HD group showed clinical signs like moving the bedding and slight to mode rate salivation immediately after administration during the treatment period. All these findings indicate local reactions to the oral gavage and are not assumed to be a sign of systemic toxicity.
No clinical signs were observed in C and HD recovery groups during the recovery period. Clinical signs like scratch, broken left incisor, cut on tongue, hairless area, regurgitation of the test material, sunken flanks and crust (ear) were observed mostly transiently or on single days and were within the normal background frequency.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found during the treatment or recovery periods.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Mortality was observed in 5/15 male animals at 1000 mg/kg bw/day. No mortality occurred among female test animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Test item had no effect on body weight development in females of the HD group during premating, gestation and lactation period. However, slight and statistically significant lower body weight was observed in HD recovery females (day 7-14) during the recovery period. The test item had no effect on body weight development in this study up to the MD level

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight, but statistically significant lower food consumption was observed in females of the HD group towards end of gestation/lactation and. A slight decrease in food consumption was observed on day 1-14 during the recovery period in HD recovery females. The test item had no effect on food consumption up to the MD level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the HD group moderate, statistically significantly higher reticulocytes were observed at the end of the treatment period (females 60% above control). This was not associated with changes in red blood cell parameters. In HD females monocytes were moderately statistically significant elevated (119 % above controls) at the end of the recovery period. Slightly but statistically significant higher platelet count in the HD group at the end of the treatment period in females (25 % above controls) are not considered toxicologically relevant as values were within the range of historical control data. Coagulation was not affected by the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALAT and ASAT were moderately but statistically non-significiant increased in female animals of the HD group (>4-fold and >2-fold of controls, respectively) at the end of the recovery period. This was accompanied by a slight, non-significant increase in TBA (>2-fold of controls). All above-mentioned findings are assumed to be related to hepatotoxicity observed histopathologically. Slight and statistically significant increases in TP and Alb were observed in female animals of the HD group at the end of the recovery period (9 and 13 % above controls, respectively). These slight changes are not considered toxicologically relevant as there were no corresponding changes in globulin fraction or creatinine levels and they were not associated with histopathological findings.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed at the end of the treatment period and recovery period.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In females, no test item-related effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period and also at the end of recovery period.

Immunological findings:

not examined

Description (incidence and severity):

The study method may give a basic indication of immunological effects, however, no particular investigation were performed for this purpose

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slightly to moderately increased kidney weight (relative and absolute) was observed in female animals of the HD group at the end of the treatment (13 % and 17 % above controls, respectively) and was statistically significant also at the end of the recovery period (8 % and 15 % above controls, respectively). A slight, statistically significant decreased liver weight (relative) was observed in female animals of the MD group (9 % below controls) at the end of treatment

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings correlating with histopathology changes were observed in kidney and livers of the HD group males at the end of treatment and recovery period. Ureters were dilated in 1/10 female animals of the LD group (animal no. 61) and in 2/10 female animals (animal nos. 81, 82) of the HD group. A fluid-filled dilated uterus (both horns) was observed in control recovery female no. 91; white spotted adrenal glands were observed in MD female no. 74; a red spotted thymus was observed in control female no. 57.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Test item-related changes were observed in decedent and surviving animals from the HD group only. In all moribund and decedent animals minimal to marked microvesicular fatty change was observed. Additionally, slight hepatocellular necrosis was noticed in the decedent Animal No. 33. In surviving animals from the HD group (treatment and recovery periods), minimal microvesicular fatty change was observed. Minimal hepatocellular necrosis (single cell necrosis) was also observed in Animal No. 39.
Further, in Animal No. 14 (LD group), hepatocellular necrosis was observed. The above-mentioned hepatic change was associated with severe congestion and moderate hemorrhage and was not considered to be a test item-related finding, but rather an incidental finding.
Kidney: Test item-related changes were observed in decedent and surviving animals from the HD group only. In decedent animals from the HD group, slight to moderate tubular dilatation and minimal tubular necrosis were observed. Furthermore, severe tubular dilatation, minimal to slight tubular necrosis, slight tubular basophilia and slight multifocal mineralization (dystrophic change) were observed.
In surviving males from the HD group (end of treatment period), moderate to severe tubular dilatation, minimal to moderate mononuclear and mixed cell infiltrates in the tubular lumen and renal interstitium, slight to moderate interstitial fibrosis, slight tubular necrosis, slight tubular basophilia and minimal to slight mineralization were observed. Additionally, moderate mixed cell infiltrates and minimal tubular dilatation were noticed in females from the same group,. In recovery males from the HD group, minimal to slight tubular dilatation, slight to moderate mixed cell infiltrates, minimal to slight mononuclear cell infiltrates, slight to moderate fibrosis, slight tubular necrosis and slight to moderate tubular basophilia were observed, whereas in females no histopathological changes were noticed.

Heart: Cardiac lesions that could be attributed to the treatment with the test item were observed in decedent recovery animals from the HD group only. These cardiac changes consisted of moderate myocardial mineralization, slight vascular mineralization (e.g. tunica media of aorta and few coronary vessels), slight cardiomyocytes necrosis and slight mononuclear infiltrates.
All other recorded findings were considered incidental or were within the range of spontaneous background alterations that may be recorded in Wistar rats at these ages. No histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina could be detected. The tubular stages of the spermatogenic cycle were checked for completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Moreover, no treatment-related effect on interstitial cell structure was noticed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test item treatment-related effects observed on the number of pre-implantation loss and post implantation loss in LD, MD and HD groups when compared with the control group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in LD and HD groups. However, a slightly and statistically significant higher mean mortality of pups on PND 0-4 was observed in the MD group (5.41 %). As this effect was not dose-dependent, it is not considered to be an adverse effect of the test item.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There was no test item-related or statistically significant effect observed on the duration of precoital interval and the duration of gestation in the LD, MD and HD groups when compared to the control group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse reproductive effects were observed in maternal animals

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Based on histopathological changes in liver and kidneys.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related or statistically significant effect on pup mean weight, total litter weight, male litter weight, or female litter weight on PND 0, PND 4 and PND 13 observed in LD, MD and HD groups when compared with the controls.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no test item-related or statistically significant effects observed in LD, MD and HD groups on litter data parameters like group mean total number of pups born number of live pups, still birth, runt on PND 0 as well as number of live pups on PND 4 and PND 13 when compared with the controls.. No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in LD and HD groups. However, a slightly and statistically significant higher mean mortality of pups on PND 0-4 was observed in the MD group (5.41 %). As this effect was not dose-dependent, it is not considered to be an adverse effect of the test item.

Changes in sex ratio:

not examined

Description (incidence and severity):

There were no test item-related or statistically significant effects observed in LD, MD and HD groups on litter data parameters like number of male pups, number of female pups, sex ratio, as well as number of male and female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no test item-related or statistically significant effect on pup mean weight, total litter weight, male litter weight, or female litter weight on PND 0, PND 4 and PND 13 observed in LD, MD and HD groups when compared with the controls.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in LD and HD groups. However, a slightly and statistically significant higher mean mortality of pups on PND 0-4 was observed in the MD group (5.41 %). As this effect was not dose-dependent, it is not considered to be an adverse effect of the test item.

External malformations:

no effects observed

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.
A few specific findings like flattened abdomen (pup no. 13 from dam no. 56) and missing tail tip (pup no. 1 from dam no. 60) of control group were observed. No indication of suckling on PND4 for the pups 1-15 from dam no. 68 of LD was observed. No indication of suckling on PND0 for pup 10 and dark head for the pup 4 from dam no. 73 of MD were observed. All these findings were considered to be spontaneous and not related to test item treatment.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In male and female pups, no test item-related effects in absolute and relative anogenital distance were observed in the dose groups when compared to the controls.
No statistically significant effect or toxicological relevance was observed on nipple retention in the male pups of any of the groups when compared with the controls.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed in F1

Abnormalities:

no effects observed

Developmental effects observed:

no

Summary result rables and individual animal data result tables will be added at a later date when they are available.

Conclusions:

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 2,4,6-tris[3-(trimethoxysilyl)propyl] isocyanurate, conducted according to OECD Test Guideline 422 and in compliance with GLP, concluded a NOAEL of 1000 mg/kg bw/day for developmental toxicity based on no effects observed in F1 at the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, conducted according to OECD Test Guideline 422 and in compliance with GLP, the test item was administered at dose levels of 100 (LD), 300 (MD) and 1000 (HD) mg/kg bw/day in corn oil to 10 male and 10 female rats per dose and to additional 10 male and female rats used as recovery group in the control and high dose groups. Corn oil was given to the negative control animals. Male animals were treated for at least 28 days. Female animals were treated until post-natal day 13. Male and female animals were mated by cohabitation in ratio 1:1 for at least 14 days. Positive vaginal smear was considered as day 0 of gestation. Animals in the recovery groups were not mated and dosed for 28 days (males) or until the first scheduled kill of dams (females). They were observed for a period of 14 days following the last administration.

During the treatment period, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the termination of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, 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.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment for all animals and in the last week of treatment in five randomly selected males and females of each group.

After parturition 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. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 30 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The males of the recovery groups were subjected to necropsy 15 days after the last administration (end of recovery period). The females of the recovery groups were treated up to the first scheduled necropsy of dam and were subjected to necropsy 15 days thereafter (end of recovery period).

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues of the control and groups which were sacrificed at the end of the treatment period was completed. 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. Further, liver, kidney and heart from all available animals (treatment and recovery period) were processed and evaluated. In addition, reproductive organs from all males and females from the control and groups were processed and evaluated. For the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff)stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

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

No deaths or test substance-related clinical signs of toxicity were seen in maternal animals. No effects on body weight gain, food consumption or FOB assessment. In the HD group moderate, statistically significantly higher reticulocytes were observed at the end  of the treatment period. This was not associated with changes in red blood cell parameters. In HD females monocytes were moderately statistically significant elevated at the end of the recovery period. Slightly but statistically significant higher platelet count in the HD group at the end of the treatment period in females are not considered toxicologically relevant as values were within the range of historical control data. Coagulation was not affected by the test item. ALAT and ASAT were moderately but statistically non-significiantly increased in female animals of the HD group at the end of the recovery period. This was accompanied by a slight, non-significant increase in TBA. Slight and statistically significant increases in TP and Alb were observed in female animals of the HD  group at the end of the recovery period. These slight changes are not considered toxicologically relevant as there were no corresponding changes in globulin fraction or creatinine levels and they were not associated with histopathological findings. The test item had no toxicologically relevant effect on urinary parameters analysed at the end of the  treatment period and recovery period. A slightly to moderately increased kidney weight (relative and absolute) was observed in female animals of the HD group at the end of the  treatment and was statistically significant also at the  end of the recovery period. A slight, statistically significant decreased liver weight (relative) was observed in female animals of the MD group at the end of treatment. At histopathology moderate mixed cell infiltrates and minimal tubular dilatation were noticed in the kidneys of females. No other histopathological changes were

noticed in females.

There were no test item treatment-related or statistically significant effects observed in LD, MD and HD groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, number of male and female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

There was no test item-related or statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in LD, MD and HD groups when compared with the controls.

There was no test item-related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the LD MD and HD groups and precoital interval in all treatment groups when compared to the control group.

There were no test item treatment-related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent pre-implantation loss and post implantation loss in LD, MD and HD groups when compared with the control group.

There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

No test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group was noted.

In male and female pups, no test item-related effects in absolute and relative anogenital distance were observed in the dose groups when compared to the controls.

No statistically significant effect or toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.

No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight. Serum thyroxine hormone (T4) levels of males of the LD, MD and HD groups at the end of the treatment period, of males of the HD group at the end of the recovery period or of PND 13 pups were not considerably different when compared to controls.

No test item-related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups on PND 0-12.

There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.

Testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

In the absence of adverse effects on developmental parameters the no-observed-adverse-effect-level (NOAEL) for developmental toxicity was established at 1000 mg/kg body weight/day in this study.

Justification for classification or non-classification

Based on the available data for 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, no classification for reproductive or developmental toxicity is required according to Regulation (EC) No 1272/2008.