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

Endpoint summary

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, the NOAEL was concluded to be 300 mg/kg bw/day based on changes in liver, kidney and heart in animals treated with 1000 mg/kg bw/day (Eurofins, 2019).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

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 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 (if applicable) 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 females 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: ad libitum, 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

Details on route of administration:

The test item and vehicle were administered at a single daily dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Vehicle:

corn oil

Details on oral 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 (pre-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 was given daily.

Dose / conc.:

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

Remarks:

Group 1 (control group)

Dose / conc.:

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

Remarks:

Group 2 (low dose group LD)

Dose / conc.:

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

Remarks:

Group 3 (mid dose group MD)

Dose / conc.:

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

Remarks:

Group 4 (high dose group 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
- Rationale for selecting recovery groups: Two recovery groups were included which were given vehicle only or high dose test substance.
- Post-exposure recovery period in recovery groups: 15 days
- Section schedule rationale (if not random): random

Positive control:

Not included

Observations and examinations performed and frequency:

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, 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 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 prior 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.

OPHTHALMOSCOPIC EXAMINATION: Yes. Performed as part of neurobehavioural examination
- Time schedule for examinations: Before the first treatment and during the last week of the treatment in males and during the last week of the lactation period in females (only lactating females were evaluated) of each group. Recovery groups were examined once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: 5 randomly selected males and females of treatment groups and all males and females of recovery groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice and at the end of the recovery period.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group and all recovery animals
- Parameters checked in table No 3 were examined: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice and at the end of the recovery period.
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group and all recovery animals.
- Parameters checked in table No 3 were examined: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment prior to or as part of the sacrifice and at the end of the recovery period from 5 randomly selected males and females (only lactating females were evaluated) from each group and all recovery animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table were examined. Yes

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

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 4)

HISTOPATHOLOGY: Yes (see table No 4)

Other examinations:

THYROID GLAND ASSESSMENT - The weight of thyroid gland was evaluated at the end of the treatment. Serum thyroxine hormone (T4) levels were evaluated in male and female rats.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry 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. 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).

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 moderate 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.

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 postmating 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 during 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 perid 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 ASAT 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 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.
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

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):

Statistically significant organ weight changes observed in liver and kidneys were most likely related to the treatment with the test item and correlated with the observed histopathology changes in males from the HD group. A significant decrease in body weight in male animals of the HD group affected relative organ weights normalized to body weight. Further, in the absence of a histopathological correlation, all other recorded organ weight changes were not considered toxicologically relevant.
Moderate, statistically significant increased kidney weight (relative) was observed in male animals of the HD group at the end of treatment (82 % above controls) and recovery (41 % above controls) and a statistically significant increase in absolute kidney weight was observed in male animals of the HD group at the end of the recovery period (34 % above controls). 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).
Slight, statistically significant increased liver weight (relative) was observed in males of the HD group at the end of treatment period (24 % above controls) and at the end of the recovery period (13 % above controls). 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.
Moderate, statistically significant increased spleen weight (relative) was observed in males of the HD group at the end of treatment (50 % above controls) and recovery periods (41 % above controls) and statistically significant increased absolute spleen weight was observed in male animals of the HD group at the end of the recovery period (35 %).
Slight, statistically significant increased heart weight (relative) was observed in male animals of the HD group at the end of the recovery period (10 % above controls).
Slight, statistically significant decreased absolute and relative prostate glands weights were observed in males of the HD group (29 % and 19% below controls, respectively) at the end of treatment. Slight, statistically significant increased adrenal gland weight (relative) was observed in males of the HD group at the end of treatment (24 % above controls).
Slight, statistically significant increased testes/epididymis weight (relative) was observed in males of the HD group at the end of treatment (13 % and 15 % above controls, respectively).

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 animals at the end of treatment and recovery period.
Animal no. 32 and 33 were euthanized for animal welfare reasons on days 25 and 11 respectively. These animals showed abnormal coloured liver (red/white/spotted), right kidney pelvis and ureters of both sides were dilated. The urinary bladder was filled with an abnormal content, blood and thymus were small and red spotted in animal no. 32. Animal nos. 40, 47 and 48 were found dead on days 10, 7 and 12, respectively. All animals were autolyzed and animal no. 48 showed enlarged kidneys of both sides, dilated ureters on both sides and clear, watery fluid in the abdominal cavity.
White spotted kidneys were observed in male HD animals at the end of the treatment period (5/10; animal nos. 31, 35, 36, 37, 38) and at the end of the recovery period (2/5; animal nos. 46, 50). Kidneys with abnormal content/fluid were observed in one HD male (animal no. 31). Enlarged kidneys on both sides were observed in male animals of the HD group at the end of the treatment period (6/10; animal nos. 31, 34, 35, 38, 40) and at the end of the recovery period (3/5; animal nos. 46, 50). The pelvis (right) was dilated in 3/10 male HD animals at the end of treatment and 1/5 at the end of recovery. Kidney pelvis dilatation on both right and left sides was observed in one animal (animal no. 37).
Ureters were dilated in 2/10 male animals of the MD group (animal nos. 28, 30) and in 6/10 male animals (animal nos. 31, 34, 35, 36, 37, 40) of the HD group and also in 2/5 (animal nos. 46, 50) HD males at the end of the recovery period. The urinary bladder contained blood in 1/10 male animals (animal no. 32) of the HD group. 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.
The thymus was red spotted in 1/10 male animals of the LD group (animal no. 16) and 2/10 male animals of HD group (animal nos. 21, 30) and was small in HD male animal no. 32. Reduced thymus size is possibly stress-related and therefore considered to be a secondary effect of the test item.
Single incidental findings were: enlarged axillary lymph node observed in control animal no. 1; 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

Details on results:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period during each occasions (study weeks 1, 3, 5 and in the last week of the study) as measured concentrations were within acceptance criterion of 10%.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: hepatobiliary, cardiovascular and urinary

Organ:

heart

kidney

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

NOAEL for general toxicity was established at 300 mg/kg body weight/day. In the absence of adverse effects on reproductive parameters the NOAEL for reproduction toxicity was established at 1000 mg/kg body weight/day in this study.

Table 1- Statistically significant changes (p<0.05)organ and body weights

Liver
Groups / Sex	C/M	LD/M	MD/M	HD/M
No. of weights	5	5	5	4
Mean Organ / Body [%]	3.0518u	3.0886	3.1301	3.7801*
SD [g]	0.1141	0.1979	0.1879	0.2122
Kidneys
Groups / Sex	C/M	LD/M	MD/M	HD/M
No. of weights	5	5	5	4
Mean Organ / Body [%]	0.6124u	0.6415	0.6125	1.1164*
SD [%]	0.0347	0.0142	0.0265	0.3914
Body weight
Groups / Sex	C/M	LD/M	MD/M	HD/M
No. of weights	10	10	10	7
Mean weight [g]	403.8000u	398.9000	405.0000	350.1429**
SD [%]	-	-1.2135	0.2972	-13.2880

SD =StandardDeviation

** =p <0.01;u =KRUSKALL-WALLIS-DUNN

Table 2 - Test item-related changes - correlation table necropsy - microscopy

Group/Subset	Animal Number	Organ	Correlation necropsy - histopathology
			Necropsy observation	Histopathology findings
HD	31	Kidneys	Abnormal color spotted	Multifocal inflammatory cell infiltrates
HD	32	Liver	Abnormal color spotted	Hepatocytes vacuolation
HD	33	Liver	Abnormal color, spotted	Hepatocytes vacuolation
HD	34	Kidneys	Enlarged, both sides, spotted	Pelvic dilatation, inflammatory cell infiltrates and fibrosis
HD	35	Kidneys	Abnormal color, spotted	Multifocal inflammatory cell infiltrates
HD	36	Kidneys	Abnormal color, spotted	Multifocal inflammatory cell infiltrates
HD	37	Kidneys	Abnormal color, spotted	Multifocal inflammatory cell infiltrates and fibrosis
HD	38	Kidneys	Abnormal color, spotted	Multifocal inflammatory cell infiltrates
HD recovery	50	Kidneys	Abnormal color, white	Interstitial fibrosis

C = control, LD = low dose, MD = medium dose, HD = high dose

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 300 mg/kg bw/day based on changed in liver, kindey and heart in animals treated with 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: hepatobiliary, urinary and cardiovascular

Organ:

heart

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

Test item-related mortality occurred only in the HD group. Three male animal (no. s. 47, 40 and 48) were found dead on days 7, 10 and 12, respectively. Two male animals (nos. 33 and 32) were euthanized for animal welfare reason on days 11 and 25, respectively. No mortalities were recorded in females of this group at the lower dose levels or in the control group. Histopathologically, the cause of mortality was found to be hepatic microvesicular fatty change. Hepatic microvesicular fatty changes and myocardial and coronary vessel mineralization contributed to morbidity in the two male animals.

Haematuria was observed in one male animal euthanized for animal welfare reason on day 25 and also in another male animal transiently. Hypothermia was observed in male animals no. 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 related to systemic toxicity of the test item. Clinical signs like moving the bedding and slight to moderate salivation in females of all dose groups and moving the bedding immediately after administration during the treatment period of males of the HD group indicate slight local reactions after oral gavaging and are not assumed to be a sign of systemic toxicity. There were no clinical signs during the recovery period. During the weekly detailed clinical observation, no relevant differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

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 when compared with the controls. There were no biologically relevant differences observed in between the groups.

The test item had no effect on body weight development up to the MD level. Moderate, but statistically significantly lower body weight gain in male animals was observed at the HD level 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 17-19% below controls in this group. A slight decrease in body weight gain was observed during the recovery period (day 35 and 42), though there was no statistical significance. The test item had no effect on body weight development in female 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 food consumption up to the MD level. A tendency towards lower food consumption was observed in HD males during the treatment period (premating day 1-7 at 26 %, day 7-14 at 35% lower than control). This coincided with the effect on body weight development and considered to be toxicologically relevant. A slight decrease in food consumption was observed on day 1-7 during the recovery period in HD males. Slight, but statistically significant lower food consumption was observed in females of the HD group towards end of gestation/lactation. A slight decrease was also observed on day 1-14 of the recovery period in HD females. All these findings in females are not assumed to be toxicologically relevant.

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.

Higher white blood cell count, rate of neutrophils and monocytes and lower lymphocytes found in HD males at the end of the treatment and recovery period are likely to be related to inflammation in the renal system (see below). This was also was observed at the end of the recovery period. Elevated levels of reticulocytes in the HD group at the end of treatment (males and females) and recovery period (males) were not associated with considerable changes in red blood cell parameters, although they could be a regenerative reaction to slight blood loss trough possible haemorrhage in the renal system (as evidenced by bloody urine observed in single animals) as an effect of test item-related toxicity. Slightly higher platelet count in the HD group at the end of the treatment period in males and females and at the end of the recovery period in females are not considered toxicologically relevant. There were no test item-related changes in haematology and coagulation parameters and all values were within the normal range of variation.

Test item-related elevated serum ALAT and ASAT levels were observed in the HD group at the end of the treatment period in male animals and at the end of the recovery period in male and female animals. Occasionally, other markers of heptatoxicity were also increased (i.e. AP, Chol, TBA). An increase in U level was observed in male animals of the HD group at the end of the treatment period. The above-mentioned clinical biochemistry findings correlated with changes in the organ weights of liver and kidney and hispathological changes.

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

Macroscopically, test item-related changes in liver and kidney in the HD group only (treatment and recovery period) were observed and can be correlated with histopathological findings in these organs (see below). Reduced thymus size in the HD group animals is possibly stress-related and therefore a secondary effect of the test item. Further, there were no test item-related changes in any animal from the LD and MD groups.

Statistically significant organ weight changes observed in liver and kidneys are most likely related to the treatment with the test item and correlate with the observed histopathology changes in animals from the HD group. Further, in the absence of a histopathological correlation, all other recorded organ weight changes were not considered toxicologically relevant.

Histopathologically, test item-related changes were observed in animals from the HD group only.

Liver: In all moribund and decedent animals (treatment and recovery periods), minimal to marked microvesicular fatty change was observed. Additionally, slight hepatocellular necrosis was noticed in the decedent male (animal no. 33) from the treatment period only. In surviving animals from the HD group (treatment and recovery periods), minimal microvesicular fatty change and minimal hepatocellular necrosis (single cell necrosis) was observed in one animal (animal no. 39) only.

Kidney: In decedent animals from the HD group (treatment period), slight to moderate tubular dilatation and minimal tubular necrosis were observed. Further, in the decedent recovery animals from the HD group, severe tubular dilatation, minimal to slight tubular necrosis, slight tubular basophilia and slight multifocal mineralization were observed. In surviving males from the HD group (treatment period), moderate to severe tubular dilatation, minimal to moderate mononuclear and mixed cell infiltrates, slight to moderate interstitial fibrosis, slight tubular necrosis, slight tubular basophilia and minimal to slight mineralization were observed. Further, in females from the same group, moderate mixed cell infiltrates and minimal tubular dilatation were noticed. 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.

Heart: Cardiac lesions consist of moderate myocardial mineralization, slight mineralization of aortic tunica media and a few coronary vessels, slight cardiomyocytes necrosis and slight mononuclear infiltrates were observed in decedent recovery animals from the HD group only.

In summary, treatment with 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione (CAS 26115-70-8) induced changes in liver, kidney and heart of animals at 1000 mg/kg bw/day only. Therefore, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for systemic toxicity was established at 300 mg/kg bw/day.

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 specific target organ toxicity via repeated exposure is required according to Regulation (EC) No 1272/2008.