Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL (reproductive, rat) = ≥ 100 mg/kg bw/day (OECD 422, Anon. 2010)

NOAEL (general toxicity, rat) 30 mg/kg/day (OECD 422, Anon. 2010)

NOAEL (reproductive, rat) = ≥ 100 mg/kg/day (OECD 443, Anon. 2019)

NOAEL (general toxicity, rat) 30 mg/kg/day (OECD 443, Anon. 2020)

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 September 2009 -10 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study has been performed according to OECD 422 guidelines and GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
Exposure period of female rats (41-48 days) is less than the suggested 54 days in the guideline with the stated lactation period being significantly less than the stated duration.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Age at study initiation: Approximately 9-10 weeks- Fasting period before study: no- Housing: Pre-matingAnimals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).MatingFemales were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).Post-matingMales were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).LactationPups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).GeneralSterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cageenrichment was provided during activity monitoring.- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). - Water (e.g. ad libitum): Free access to tap-water. - Acclimation period: At least 5 days prior to start of treatment.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 3°C (actual range: 20.2 – 21.9°C)- Humidity (%): 40 - 70% (actual range: 28 - 76%) - Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 22 September 2009 (allocation) To: 10 November 2009 (end of in-life phase)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for the density of the test substance (1.0202), the specific gravity of the vehicle (1.036) and the water content (66.44%).VEHICLE- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.- Concentration in vehicle: 2, 6 and 29 mg/mL (expressed as mas neat mg Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine/kg b.w.- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation: A maximum of 13 days was allowed for mating.- Proof of pregnancy: sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy- Further matings after two unsuccessful attempts: no- After successful mating each pregnant female was caged (how): individually- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase (28 September and 08 October 2009), according to a validated method (NOTOX project 492054). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 11-12 weeks
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the dose range finding study (NOTOX Project 491831)
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: At least twice daily (early morning/late afternoon).
DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating or housed individually. BODY WEIGHT: Yes- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.
FOOD CONSUMPTION: Yes- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
WATER CONSUMPTION: No, Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
HAEMATOLOGY: Yes - Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. - Anaesthetic used for blood collection: Yes iso-flurane- Animals fasted: yes, but water was available - How many animals: 5 males/group (random) and all females with live pups- Parameters examined were: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated Partial thromboplastin time.
CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. - Animals fasted: yes, but water available- How many animals: 5 males/group (random) and all females with live pups- Parameters examined were: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling). - Dose groups that were examined: all- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity
Oestrous cyclicity (parental animals):
not determined (not required)
Sperm parameters (parental animals):
Parameters examined in all male parental animals:testis weight, epididymis weight.in addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.
Litter observations:
PARAMETERS EXAMINEDThe following parameters were examined in F1 offspring:number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.GROSS EXAMINATION OF DEAD PUPS: YesIf possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).- Maternal animals: All surviving animals on lactation Days 5-6.- Females which failed to deliver: Post-coitum Day 27 (evidence of mating) or 22 days after the last day of the mating period (without evidence of mating).GROSS NECROPSYAll animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The number of former implantation sites and corpora lutea was recorded for all paired females.From all animals, samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):Identification marks (not processed), Cervix, Clitoral gland, Epididymides*, Ovaries, Preputial gland, Prostate gland, Seminal vesicles including coagulating gland, Testes*, Uterus, Vagina, All gross lesions.From 5 males/group (random) and all females with live pups, samples of the following tissues and organs were collected and fixed in addition to the abovementioned list:Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Colon, Duodenum, Eyes (including optic nerve and Harderian gland)*, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Lacrimal gland (exorbital), Larynx, Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), Nasopharynx, Oesophagus, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Thymus, Thyroid including parathyroid (if detectable), Tongue, Trachea, Urinary bladder.*Fixed in modified Davidson's solution and transferred to formalin after fixation for at least 24 hours.ORGAN WEIGHTS: YesThe following organ weights and terminal body weight were recorded from 5 males/group (random) and all females with live pups on the scheduled day of necropsy:Adrenal glands, Brain, Epididymides (all males), Heart, Kidneys, Liver, Ovaries Spleen, Testes (all males), Thymus, Uterus (including cervix), Prostate*, Seminal vesicles including coagulating glands*, Thyroid including parathyroid** weighed when fixed for at least 24 hours.HISTOPATHOLOGY: Yes The following slides were examined by a pathologist:- The preserved organs and tissues of 5 selected animals/sex of Groups 1 and 4.- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.- All gross lesions of all animals (all dose groups).- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy pups.
Postmortem examinations (offspring):
SACRIFICEPups surviving to planned termination were killed by decapitation on lactation Days 5-6.GROSS NECROPSYAll pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.HISTOPATHOLOGY / ORGAN WEIGTHSno
Statistics:
The following statistical methods were used to analyse the data:- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Dunnett C.W., 1955)- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Miller R.G., 1981)- The Fisher Exact-test was applied to frequency data. (Fisher R.A., 1950)The following additional methods of statistical analysis were used:The numbers of corpora lutea and implantation sites were transformed by using log x and x2, respectively, to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.No statistical analysis was performed on histopathology findings.
Reproductive indices:
For each group, the following calculations were performed:Percentage mated females: Number of females mated/Number of females paired x 100Fertility index: Number of pregnant females/Number of females paired x 100Conception index: Number of pregnant females/Number of females mated x 100Gestation index: Number of females bearing live pups/Number of pregnant females x 100Duration of gestation: Number of days between confirmation of mating and the beginning of parturition For each group, the following calculations were performed:Percentage mated females: Number of females mated/Number of females paired x 100Fertility index: Number of pregnant females/Number of females paired x 100Conception index: Number of pregnant females/Number of females mated x 100Gestation index: Number of females bearing live pups/Number of pregnant females x 100Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Checkx 100Number of live pups at First Litter Check Viability index (%) Number of live pups on Day 4 of lactation x 100Number of pups born alive
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg body weight/day, salivation was noted for all animals from Week 4 of treatment onwards. Rales (slight to moderate) were noted for two males and one female treated at 100 mg/kg for 2, 5 or 12 days. The male that showed rales for 12 days, also showed piloerection for 2 days. Rales (slight) were also noted for one male treated at 10 mg/kg for 2 days and one female treated at 30 mg/kg for 2 days. At this low incidence, these observations were not considered toxicologically significant. Alopecia was noted for three females. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Minor statistically significant decreases arising between controls and animals receiving 100 mg/kg bw/d (decreased haemoglobin and decreased APTT for males and increased APTT for females) were considered not to represent a change of biological significance as the changes were slight and the values remained within the range considered normal for rats of this age and strain.Individual increases of neutrophil counts with concurrently reduced lymphocyte counts was noted for one male (no. 3) of the control group. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg, total protein and albumin were slightly decreased for both sexes. Any statistically significant changes at 30 mg/kg (bile acids for males) and at 100 mg/kg (chloride for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution or remained within the range considered normal for rats of this age and strain. The increased values for bile acids (not statistically significant) noted for females of all dose groups were considered to have occurred by chance and to be of no toxicological relevance as these changes were caused by individual animals only.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related morphologic alterations in Wistar (Han) rats subjected to a combined 28-Day study with Hexahydro-1,3,5-Trimethyl-S-Triazine via oral gavage at doses up to 100 mg/kg in stomach of both sexes and possibly eyes of females. There were no morphologic alterations at 30 mg/kg Hexahydro-1,3,5-Trimethyl-S-Triazine. In the stomach, minimal lymphogranulocytic inflammation of the glandular stomach was recorded in 5/5 males and 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males and 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy. There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related. At 100 mg/kg body weight/day, salivation was noted for all animals from Week 4 of treatment onwards. Rales (slight to moderate) were noted for two males and one female treated at 100 mg/kg for 2, 5 or 12 days. The male that showed rales for 12 days, also showed piloerection for 2 days. Rales (slight) were also noted for one male treated at 10 mg/kg for 2 days and one female treated at 30 mg/kg for 2 days. At this low incidence, these observations were not considered toxicologically significant. Alopecia was noted for three females. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
BODY WEIGHT AND WEIGHT GAIN: Mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
FOOD CONSUMPTION: Food consumption before or after allowance for body weight was similar between treated and control animals.
NEUROBEHAVIOUR: No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The variation in motor activity did not indicate a relation with treatment.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No toxicologically significant changes.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No toxicologically significant effects on reproductive parameters were noted.
ORGAN WEIGHTS: No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg body weight/day. The statistical significant decreased values for testes and epididymides weights (absolute and relative) at the high dose group were not considered toxicologically significant as the changes were very slight, no corroborative findings were noted at microscopic examination, and reproduction was unaffected.
GROSS PATHOLOGY: At 100 mg/kg, five males and eight females showed a thickened limiting ridge of the stomach. This finding was also noted for one female treated at 30 mg/kg body weight/day. However, at this single occurrence and without microscopic correlate it was not considered toxicologically significant. For the male at 100 mg/kg/day that died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling, incomplete exsanguination was noted. One female of the control group showed one dead fetus in the right uterus horn. Other incidental findings included soft yellowish or greenish nodule at the epididymides, isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid.The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.
HISTOPATHOLOGY: NON-NEOPLASTIC: In the stomach, minimal lymphogranulocytic inflammation of the glandular stomach was recorded in 5/5 males and 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males and 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy. There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined. No abnormalities were seen in the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy offspring which could account for infertility. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No Effects on reproduction and fertility
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
No toxicologically significant effects on developmental parameters were observed.No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant femalesGestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.One pup of Group 2 was found dead on Day 2 of lactation. This single occurrence was considered to have occurred by chance.Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level.
Reproductive effects observed:
not specified

Results of examinations: parental animals

Clinical signs (parental animals)

yes

Body weight and food consumption (parental animals)

no effects

Test substance intake (parental animals)

not examined

Reproductive function: estrous cycle (parental animals)

no effects

Reproductive function: sperm measures (parental animals)

no effects

Reproductive performance (parental animals)

no effects

Organ weights (parental animals)

no effects

Gross pathology (parental animals)

yes

Histopathology (parental animals)

yes

Details on results (parental animals)

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related. At 100 mg/kg body weight/day, salivation was noted for all animals from Week 4 of treatment onwards. Rales (slight to moderate) were noted for two males and one female treated at 100 mg/kg for 2, 5 or 12 days. The male that showed rales for 12 days, also showed piloerection for 2 days. Rales (slight) were also noted for one male treated at 10 mg/kg for 2 days and one female treated at 30 mg/kg for 2 days. At this low incidence, these observations were not considered toxicologically significant. Alopecia was noted for three females. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

BODY WEIGHT AND WEIGHT GAIN Mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION Food consumption before or after allowance for body weight was similar between treated and control animals.

NEUROBEHAVIOUR: No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The variation in motor activity did not indicate a relation with treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) No toxicologically significant changes.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) No toxicologically significant effects on reproductive parameters were noted.

ORGAN WEIGHTS No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg body weight/day. The statistical significant decreased values for testes and epididymides weights (absolute and relative) at the high dose group were not considered toxicologically significant as the changes were very slight, no corroborative findings were noted at microscopic examination, and reproduction was unaffected.

GROSS PATHOLOGY At 100 mg/kg, five males and eight females showed a thickened limiting ridge of the stomach. This finding was also noted for one female treated at 30 mg/kg body weight/day. However, at this single occurrence and without microscopic correlate it was not considered toxicologically significant. For the male at 100 mg/kg/day that died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling, incomplete exsanguination was noted. One female of the control group showed one dead fetus in the right uterus horn. Other incidental findings included soft yellowish or greenish nodule at the epididymides, isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related microscopic findings were present in stomach (males and females) and eyes (females) at 100 mg/kg:

- Stomach: Lymphogranulocytic inflammation of the glandular stomach (minimal) was recorded in 5/5 males and 4/8 females. Hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males (minimal) and 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy.

- Eyes (females): Degeneration of the retina (atrophy) was recorded in 2/5 females (minimal-slight). All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar Han rats of this age and strain. No abnormalities were seen in the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy offspring which could account for infertility. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Results of examinations: offspring

Viability (offspring)

no effects

Clinical signs (offspring)

no effects

Body weight (offspring)

no effects

Sexual maturation (offspring)

not examined

Organ weights (offspring)

not examined

Gross pathology (offspring)

no effects

Histopathology (offspring)

not examined

Details on results (offspring) No toxicologically significant effects on developmental parameters were observed. No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females Gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment. One pup of Group 2 was found dead on Day 2 of lactation. This single occurrence was considered to have occurred by chance. Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

Conclusions:
Formulation analysis showed that the formulations were prepared accurately and homogenously and were stable for at least 6 hours at room temperature. At 100 mg/kg body weight/day, parental toxicity consisted of clinical signs (salivation, rales and/or piloerection),slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge. No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, body weight, food consumption, and organ weights) at 100 mg/kg. No parental toxicity was observed at 10 and 30 mg/kg/day. No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg body weight/day. Based on these results, the following No Observed Adverse Effect Level (NOAEL) was derived: Parental NOAEL: 30 mg/kg/day and reproductive and developmental toxicity of NOAEL: ≥100 mg/kg/day.
Executive summary:

OECD 422 (2010) - In a combined repeat dose toxicity study with reproductive/developmental toxicity screening (OECD 422), Hexahydro-1,3,5-Trimethyl-S-Triazine in rats by oral gavage at dosage of 10, 30 and 100 mg/kg bw/day. Male were exposed for 28 days i.e. 2 weeks prior to mating, during mating and up to termination. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, post coitum and during at least 4 days of lactation. A summary of adult responses to the test item are described below;

No mortality occurred during the study period that was considered to be related to treatment with the test.  At the highest dose group, clinical signs included salivation, rales and/or piloerection, slightly decreased total protein and albumin. A thickening limiting ridge of the stomach at macroscopic examination and microscopic finding such as lymphogranulocytic inflammation of the glandular stomach was recorded in 5/5 males and 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males and 6/8 females (minimal-slight).

There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy)  in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.

Food consumption was unaffected and the mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.  

No toxicologically significant effects on reproductive parameters were observed; percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites were unaffected by the treatment.

No difference in maternal care were noted. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among pregnant females.

Gestation index duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index and early postnatal pup development (mortality, clinical signs, bodyweight and external macroscopy) were unaffected by the treatment.

On pup of group 2 (pup 8 of litter 55) was found dead on day 2 of lactation. This single occurrence was considered to have occurred by chance.

Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.  

The No Observed Adverse Effect Level (NOAEL) was derived as:

Parental NOAEL = 30 mg/kg bw/day

Reproductive and developmental toxicity of NOAEL ≥100 mg/kg/day

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Remarks:
Cohort 1C and surplus cohorts were included. Surplus and chort 2B were not dosed.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2018 - 31 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance to international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
Deviations:
yes
Remarks:
Minor deviations listed in attachment under overall remarks. The deviations do not impact the validity of the study.
Qualifier:
according to
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151
Version / remarks:
July 2013
Deviations:
yes
Remarks:
Minor deviations listed in attachment under overall remarks. The deviations do not impact the validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The design of this study was based on the final decision on a compliance check of Hexahydro-1,3,5-trimethyl-1,3,5-triazine by ECHA (Decision no. CCH-D-2114371729-35-01/F, Date: 22 Sep 2017).
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han rat
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. the testing facility has general and reproduction/developmental/neurological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, neurological toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: Not stated.
- Age at study initiation: 6 weeks old
- Weight at study initiation: Males: 138 - 167g & Females: 113 -142 g
- Fasting period before study: No
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
-Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals had no access to water for a maximum of 2 hours.
- Acclimation period: 5 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 ºC (actual mean daily temperature was 20 to 22 ºC)
- Humidity (%): 40 to 70 % (actual mean daily Humidity was 43 -74%)
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 18th April 2018 To 05 Nov 2018. 

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours adding the vehicle to the test item, from 23 Apr 2018 until 25 Jun 2018 and from 17 Aug 2018 onwards.

From 26 Jun 2018 until 16 Aug 2018, the dosing formulations were also prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light; the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. Correction was made for the purity/composition of the test item. A factor of 2.435 was used to correct for the purity/composition (i.e. water content) of the test item.

DIET PREPARATION: N/A 
 - Rate of preparation of diet (frequency):
 - Mixing appropriate amounts with (Type of food): 
 - Storage temperature of food:
 
 VEHICLE
 - Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure according to the pertaining Standard Operating Procedure. The test item was soluble in propylene glycol and stable for at least 6 hours at room temperature and 8 days in the refrigerator over concentration range of 2 to 20 mg/mL
- Concentration in vehicle: 2 to 20 mg/mL
- Amount of vehicle (if gavage): 2 to 20 mg/mL
- Lot/batch no. (if required): Not stated 
 - Purity: Not stated
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.

- Further matings after two unsuccessful attempts: yes as detailed deviation

- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated, males were housed in type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
- Any other deviations from standard protocol:
Detection of mating was not confirmed in first instance for two females at 10 mg/kg/day (No. 141 and 142). For female No. 142, evidence of mating was obtained by palpation and indirectly by delivery of a litter. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. For female No. 141, evidence of mating was obtained by implantation sites recorded at necropsy; a mating date of this animal could not be estimated. Apparently, mating was overlooked in the assessment of the vaginal lavage of these animals, which explains the continuation of di-estrus during the mating in these females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20146537). Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 6 hours at room temperature under normal light conditions and 8 days in a refrigerator.
Duration of treatment / exposure:
F0-female = 16 -18 weeks
F0-female which failed to deliver : 16 weeks
F0-male = 17 -18 weeks
F0-female no. 190& 194= Not dosed one occasion
F1- Cohort 1A = 10
F1- Cohort 1B= 11 weeks
F1- Cohort 1C = 4 weeks
F1- Cohort 2A = 8 Weeks
F1- Cohort 2B = Not Dosed
F1- Cohort surplus = Not Dosed
Frequency of treatment:
Once daily seven days a week.
Details on study schedule:
F0-males were treated for 17-18 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating, up to and including the day before scheduled necropsy. F0-females were treated for 16-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 16 weeks.
The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
F0-female nos. 190 and 194 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 1C and 2A were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 4 weeks). The F1-animals of Cohort 2B and Cohort Surplus were not dosed.
Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
For more information refer to table 6 in the additional information on methology section.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
10 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
F0 = 25
F1 Cohort 1A & B = 20
F1 Cohort 1C, 2A, 2B + surplus = 10 with exception of Group 2 Cohort 1C 5 M ale only
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:

The dose levels in this study were selected based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in the rat (Test Facility Study No. 491807). In this OECD 422 study, rats received 0, 10, 30 or 100 mg/kg/day test item by daily oral gavage. Parental toxicity was noted at 100 mg/kg/day body weight/day and consisted of clinical signs (salivation, rales and/or piloerection), slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge. No parental toxicity was observed at 10 and 30 mg/kg, and no reproductive/developmental toxicity was observed at any dose level.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is intended to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Rationale for animal assignment (if not random):

F0-animal was identified using earmark and tattoo.
F1-pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurs the identification, the pups were identified by tattoo on the feet.
From weaning (PND 21) onwards, the animals of Cohorts 1A, 1B, 1C and 2A were identified by ear and tail mark, and from approximately PND 42 onwards, these animals were identified by earmark and tattoo. The animals of Cohort 2B and Cohort Surplus were identified by tail mark.

Fasting period before blood sampling for clinical biochemistry:
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals1 housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

- Other:
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. If the target volume of 0.5 mL could not be reached by poling two pups, a third culled pup was sampled, if available.
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes 
 - Time schedule: During clinical observations conducted prior to dosing and 0-30 minutes after dosing then twice daily up to the day prior to necropsy.
- Cage side observations checked in table [No.?] were included. Yes

 DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
 - Time schedule: Observations conducted prior to dosing and 0-30 minutes after dosing then twice daily up to the day prior to necropsy.

 BODY WEIGHT: Yes 
 - Time schedule for examinations:Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 
 - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes 
 - Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

 OTHER: Not specified

Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
For all surviving males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, food and water consumption, clinical chemistry, coagulation, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae count in male offspring, sperm analysis, splenic lymphocyte subpopulation analysis, macroscopic pathology / abnormalities. Histopathology. Thyroid Hormone Analysis, Urinalysis, Functional Observation Battery, acoustic Startle response, Vaginal patency (vaginal opening), balanopreputial separation, Estrous stages.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 Males: Around weaning (a minimum of 10 weeks of treatment).
- Maternal animals:Females which delivered;LD 23-25
-F0 females failing to produce a viable litter:
With evidence of mating: Post-coitum Days 25-27 (Nos. 105, 120, 132, 134, 136, 144, 146, 156, 172, 175, 176, 179, 182 and 199).
Without evidence of mating: Approximately 24-26 days after the last day of the mating period (No. 141; evidence for mating was only obtained at necropsy when this females was found to have implantation sites).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent).
Postmortem examinations (offspring):
SACRIFICE
- Culled Pups (PND 4) – F1 -Generation: Post natal day (PND) 4
- Cohort 1A: PND 96 (nos. 456-459, 776-778) PND 100 (nos. 442-445) & PND 89-95 (other animals).
-Cohort 1B: PND ≥ 97
-Cohort 1C:After VP/BPS positive (VP = viaginal paptency, BPS = balanopreputial separation)
-Cohort 2A: PND 76-90
-Cohort 2B: PND 21-22
-Cohort Surplus: PND 22
-Spare F1-animals: PND 22-24

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, lymph node, muscle skeletal, nerves (optic, sciatic & tibial), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferens.
Specific tissues: Basal ganglia, Cerebellum, cerebral cortex, hippocampus, hypothalamus, medulla oblongata, midbrain, olfactory bulbs, pons, retina, root fibres, thalamus and tibial nerve calf muscle branches.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, lymph node, muscle skeletal, nerves (optic, sciatic & tibial), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferens.
Specific tissues: Basal ganglia, Cerebellum, cerebral cortex, hippocampus, hypothalamus, medulla oblongata, midbrain, olfactory bulbs, pons, retina, root fibres, thalamus and tibial nerve calf muscle branches.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
Group 2 Vs. Group1
Group 3 Vs. Group1
Group 4 Vs. Group1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Reproduction and Developmental Variables (indices): Mating index males & females (%), Precoital time, Fertility index males and females (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%),
Offspring viability indices:
Offspring Variables (indices): Viability index (%), Weaning index (%) and Percentage live males and females at weaning (%).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was seen after dosing among animals of all groups, including controls, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste and/or irritating properties of the test item rather than a sign of systemic toxicity.

The type and incidence of any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post-coitum and lactation phase.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male at 100 mg/kg/day (No. 94) was euthanized in extremis on Day 99 of treatment, due to a swelling and wound (with scabs) at the left inguinal region and pale appearance. Veterinary examination on Day 91 and 95 of treatment confirmed an open, partly movable wound/swelling. Macroscopy confirmed a wound in the left inguinal region, which correlated histopathologically with a basal cell carcinoma of the skin of the left inguinal region. This neoplastic lesion was considered to be a spontaneous alteration unrelated to the treatment with the test item.

One female at 30 mg/kg/day (No. 168) was euthanized in extremis on Day 109 (Day 13 of Lactation) due to exophthalmos and enlargement of the left eye. Veterinary examination on Day 12 of lactation additionally showed corneal vascularisation, yellowish contents of the posterior chamber of the eye and vitreous humor, and consistent pupillary constriction. Necropsy confirmed findings of exophthalmos and enlargement and correlated histopathologically with increased fluid in the anterior and posterior chamber. This early sacrifice was regarded to be unrelated to treatment with the test item.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males and/or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased absolute neutrophil count in males (1.55x; within historical control range). White blood cell counts (WBC) were also marginally higher than controls (1.22x; not statistically significant and within historical control range).
- Increased absolute eosinophil count in females (2.00x; exceeding the historical control range).
- Increased red blood cell count in females (1.08x; within historical control range).
- Increased absolute reticulocyte count in males (1.27x; concurrent control mean was at the lower range of the historical control range and the mean at 100 mg/kg/day was well within this range). In contrast, absolute reticulocyte counts in females were reduced (0.68x), and were also reduced at 10 (0.80x; not statistically significant) and 30 mg/kg/day (0.78x); the concurrent control mean was at the upper range of the historical control data range, and means at 10, 30 and 100 mg/kg/day remained within this range.
Other statistically significant differences in haematology parameters were considered not to represent a change of biological relevance as values remained within the normal range and/or occurred in the absence of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses
- Decreased total protein level in males (0.93x; within historical control range).
- Decreased urea level in males (0.84x; within historical control range).
- Decreased creatinine level in males (0.92x; within historical control range).
- Increased potassium level in females (1.08x; within historical control range).
- Increased chloride level in males (1.02x; exceeding the historical control range).
- Decreased inorganic phosphate level in males (0.90x; within historical control range).
In addition, higher bile acid levels (4.64-5.77x) were noted in female Nos. 152 and 159 (at 30 mg/kg/day), and No. 180 (at 100 mg/kg/day), that were above the historical control range. In the absence of any corroborating findings, this was considered to be of no toxicological significance.
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males at 30 and 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume (1.63x; within historical control range).
- Decreased specific gravity (0.99x; within historical control range, not statistically significant at 30 mg/kg). This was considered secondary to the higher urinary volume.
The statistically significantly lower mean ketone level in males at 100 mg/kg/day was of no toxicological relevance as an increase is expected in case of target organ toxicity, and since all individual values remained within the historical control range.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg/day in males an increased incidence of lymphogranulocytic infiltrate was recorded at mini mal degree and in a few females hemorrhage (minimal-slight), erosion/ulceration (minimal-slight) and degeneration/regeneration mucosa (minimal in a single female).

At 100 mg/kg/day most animals of both sexes showed lymphogranulocytic infiltrate (minimal-modera te). This was accompanied by hemorrhage (minimal-moderate), erosion/ulceration (males: minimal- moderate, females: minimal-slight), degeneration/regeneration mucosa (up to slight) and/or edema (up to slight). In addition, in males decreased number of parietal cells (minimal-slight) was seen in
some animals.
The degeneration/regeneration of the glandular mucosa was an alteration which was mainly seen in the area close to the limiting ridge and was characterized by increased basophilia of the mucosal c ells.
The findings recorded for the glandular stomach in the remaining dose groups including controls (lymp hogranulocytic infiltrate, hemorrhage and/or erosion/ulceration) were considered to be within backg round pathology for rats of this age and strain subjected to a repeated oral gavage study.
Adrenals glands: An increased incidence of vacuolation of the zona glomerulosa was recorded at 100 mg/kg/day. A background level of vacuolation of the zona glomerulosa was seen in the remaining do se groups including controls.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalen ce, severity, or histologic character of those incidental tissue alterations.
Male No. 94 (100 mg/kg) sacrificed after 109 days on test due to a basal cell carcinoma of the skin showed increased severity (moderate) of extramedullary hematopoiesis in the spleen. Microscopic
alterations which were in line with the microscopic findings in the remaining animals of this dose gro up consisted of moderate erosion/ulceration of the glandular mucosa of the stomach with edema and lymphogranulocytic inflammatory cell infiltrate. The basal cell carcinoma was regarded to be the cause of morbidity for this animal and this neoplastic lesion in a single animal was considered to be a spontaneous alteration and unrelated to the treatment with the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females had regular cycles of 4 to 5 days, except for one female at 10 mg/kg/day (No. 141), for which the estrous cycle could not be determined. At necropsy, this female was found to have 3 implantation sites only. Given the incidental nature and the absence of a dose-related incidence, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
At 100 mg/kg/day, a statistically significantly lower number of cells with a normal morphology was recorded (0.93x). As mean and individual values of abnormal sperm cells at this dose level generally remained within the control range and/or within the available historical control range , this slight difference was considered not to be related to treatment. At 10, 30 and 100 mg/kg/day, the mean number of cells with a coiled tail appeared higher than the concurrent control mean (16, 17 and 21, vs 11 in the control). Means remained well within the historical control range, did not achieve a level of statistical significance, occurred in the absence of a dose-related trend, and the concurrent control mean was slightly low compared to the historical control range.
Reproductive performance:
no effects observed
Description (incidence and severity):
Histopathological examination of reproductive tissues revealed no evidence of a treatment- related effect on reproduction.
Of the 25 couples of each dose group, two control couples, six couples at 10 mg/kg/day, three couples at 30 mg/kg/day and four couples at 100 mg/kg/day did not succeed in producing healthy offspring. The reproductive organs from one additional couple at 30 mg/kg/day (168/68) were examined because the female was euthanized in extremis on Day 13 of the lactation period. For male No. 5 (control) and male No. 75 (30 mg/kg), massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides was regarded the cause of infertility of couples 105/5 and 175/75. No abnormalities were seen in the reproductive organs of the remaining couples, which could account for their lack of healthy offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Except for male No. 5 (control) and No. 75 (30 mg/kg/day), the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
F0-generation Data

Mortality: One male at 100 mg/kg/day (No. 94) was euthanized in extremis on Day 99 of treatment, due to a swelling and wound (with scabs) at the left inguinal region and pale appearance. Veterinary examination on Day 91 and 95 of treatment confirmed an open, partly movable wound/swelling. Macroscopy confirmed a wound in the left inguinal region, which correlated histopathologically with a basal cell carcinoma of the skin of the left inguinal region. This neoplastic lesion was considered to be a spontaneous alteration unrelated to the treatment with the test item.
One female at 30 mg/kg/day (No. 168) was euthanized in extremis on Day 109 (Day 13 of Lactation) due to exophthalmos and enlargement of the left eye. Veterinary examination on Day 12 of lactation additionally showed corneal vascularisation, yellowish contents of the posterior chamber of the eye and vitreous humor, and consistent pupillary constriction. Necropsy confirmed findings of exophthalmos and enlargement and correlated histopathologically with increased fluid in the anterior and posterior chamber. This early sacrifice was regarded to be unrelated to treatment with the test item.

Clinical Observations: No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation was seen after dosing among animals of all groups, including controls, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste and/or irritating properties of the test item rather than a sign of systemic toxicity. The type and incidence of any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose- related trend. At the incidence observed, these were considered to be unrelated to treatment.

Body Weights and Body Weight Gains: Body weights and body weight gain of treated animals were considered not affected by treatment. Any statistically significant changes in body weights and body weight gain were considered to be unrelated to treatment as no trend was apparent regarding dose and duration of treatment.

Food Consumption: Food consumption before or after correction for body weight was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as no trend was apparent regarding dose and duration of treatment.

Haematology: The following statistically significant changes were noted in treated males and/or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are 4 indicated between parentheses. Relevant internal control data are provided by footnote .
- Increased absolute neutrophil count in males (1.55x; within internal control range). White blood cell counts (WBC) were also marginally higher than controls in males (1.22x; not statistically significant and within internal control range).
-  Increased red blood cell count in females (1.08x; within internal control range).
-  Increased absolute reticulocyte count in males (1.27x; concurrent control mean was slightly below the internal control range and the mean at 100 mg/kg/day was well within this range). In contrast, absolute reticulocyte counts in females were reduced (0.68x), and were also reduced at 10 (0.80x; not statistically significant) and 30 mg/kg/day (0.78x); the concurrent control mean was at the upper range of the internal control data range, and means at 10, 30 and 100 mg/kg/day remained within this range.
Increased absolute eosinophil count in females (2.00x; exceeding the internal control range) was primarily attributed to a high value for one female (No. 189), and therefore considered not to be related to treatment with the test item. Other statistically significant differences in haematology parameters were considered not to represent a change of biological relevance as values remained within the normal range and/or occurred in the absence of a dose-related response. 

Coagulation: Coagulation parameters of treated rats were considered not to have been affected by treatment.
The statistically significantly higher platelet counts in males at 100 mg/kg/day occurred in the absence of a clear dose-related trend, and the mean remained within the internal control range . This variation was therefore considered not to be related to treatment.
The low platelet count in one male at 30 mg/kg/day (No. 53) was considered not related to treatment given its incidental occurrence and absence of a dose-relationship.

Clinical Chemistry: The following statistically significant changes were noted in treated males or females at 100
mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
- Decreased total protein level in males (0.93x; within internal control range).
- Decreased urea level in males (0.84x; within internal control range).
- Decreased creatinine level in males (0.92x; within internal control range).
- Increased potassium level in females (1.08x; within internal control range).
- Increased chloride level in males (1.02x; exceeding the internal control range).
- Decreased inorganic phosphate level in males (0.90x; within internal control range). 
In addition, higher bile acid levels (4.64-5.77x) were noted in female Nos. 152 and 159 (at 30 mg/kg/day), and No. 180 (at 100 mg/kg/day), that were above the internal control range. In the absence of any corroborating findings, this was considered to be of no toxicological significance. Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose- related response. As such, these slight differences were considered not related to treatment. 
Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by 
treatment. 


Urinalysis: The following statistically significant changes were noted in treated males at 30 and 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
- Increased urinary volume (1.63x; within internal control range).
- Decreased specific gravity (0.99x; within internal control range, not statistically significant at 30 mg/kg). This was considered secondary to the higher urinary volume. 
The statistically significantly lower mean ketone level in males at 100 mg/kg/day was of no toxicological relevance as an increase is expected in case of target organ toxicity, and since all individual values remained within the internal control range. 
Urinalysis parameters of females were considered not affected by treatment. 


Organ Weights: There were no test item-related alterations in organ weights in the F0 animals.

Macroscopic Findings: Test item-related macroscopic alterations at the end of the treatment period were recorded for the stomach and were observed in the 30 and 100 mg/kg/day group males and females. Among surviving animals, these alterations consisted of:
- Thickening of the limiting ridge in 8/25 males and 4/24 females at 30 mg/kg/day and 23/24 males and 21/25 females at 100 mg/kg/day. 

- Dark red/reddish/black-brown/black focus/foci in the glandular mucosa in 4/24 females at 30 mg/kg/day and in 17/24 males and 17/25 females at 100 mg/kg/day. 
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/25 males at 10 mg/kg/day and 3/25 females of the control group and thickening of the limiting ridge in 2/25 males at 10 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain. 


Histopathology: Test item-related microscopic findings were noted in the stomach of both sexes starting at 30 mg/kg/day and the adrenal glands of males at 100 mg/kg/day; Stomach: Microscopic alterations in the stomach were seen in the glandular part and consisted of a combination of findings. At 30 mg/kg/day in males an increased incidence of lymphogranulocytic infiltrate was recorded at minimal degree and in a few females hemorrhage (minimal-slight), erosion/ulceration (minimal-slight) and degeneration/regeneration mucosa (minimal in a single female).
At 100 mg/kg/day most animals of both sexes showed lymphogranulocytic infiltrate (minimal-moderate). This was accompanied by hemorrhage (minimal-moderate), erosion/ulceration (males: minimal-moderate, females: minimal-slight), degeneration/regeneration mucosa (up to slight) and/or edema (up to slight). In addition in males decreased number of parietal cells (minimal-slight) was seen in some animals.
The degeneration/regeneration of the glandular mucosa was an alteration which was mainly seen in the area close to the limiting ridge and was characterized by increased basophilia of the mucosal cells.
The findings recorded for the glandular stomach in the remaining dose groups including controls (lymphogranulocytic infiltrate, hemorrhage and/or erosion/ulceration) were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study. Adrenal glands: An increased incidence of vacuolation of the zona glomerulosa was recorded at 100 mg/kg/day. A background level of vacuolation of the zona glomerulosa was seen in the remaining dose groups including controls. There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Male No. 94 (100 mg/kg) sacrificed after 109 days on test due to a basal cell carcinoma of the skin showed increased severity (moderate) of extramedullary hematopoiesis in the spleen. Microscopic alterations which were in line with the microscopic findings in the remaining animals of this dose group consisted of moderate erosion/ulceration of the glandular mucosa of the stomach with edema and lymphogranulocytic inflammatory cell infiltrate. The basal cell carcinoma was regarded to be the cause of morbidity for this animal and this neoplastic lesion in a single animal was considered to be a spontaneous alteration and unrelated to the treatment with the test item.

Reproductive performance: Histopathological examination of reproductive tissues revealed no evidence of a treatment- related effect on reproduction
Of the 25 couples of each dose group, two control couples, six couples at 10 mg/kg/day, three couples at 30 mg/kg/day and four couples at 100 mg/kg/day did not succeed in producing healthy offspring. The reproductive organs from one additional couple at 30 mg/kg/day (168/68) were examined because the female was euthanized in extremis on Day 13 of the lactation period. For male No. 5 (control) and male No. 75 (30 mg/kg/day), massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides was regarded the cause of infertility of couples 105/5 and 175/75. These males also had no motile/progressive sperm cells. No abnormalities were seen in the reproductive organs of the remaining couples, which could account for their lack of healthy offspring. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Except for male No. 5 (control) and male No. 75 (30 mg/kg/day) the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Sperm Analysis: Sperm motility, concentration and morphology were considered not affected by treatment. At 100 mg/kg/day, a statistically significantly lower number of cells with a normal morphology was recorded (0.93x). As mean and individual values of abnormal sperm cells at this dose level generally remained within the control range and/or within the available internal control range , this slight difference was considered not to be related to treatment. At 10, 30 and 100 mg/kg/day, the mean number of cells with a coiled tail appeared higher than the concurrent control mean (16, 17 and 21, vs 11 in the control). Means remained well within the internal control range, did not achieve a level of statistical significance, occurred in the absence of a dose-related trend, and the concurrent control mean was slightly low compared to the internal control range. Male No. 5 (control) and male No. 75 (30 mg/kg/day), both of which had massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides, had no motile or progressive sperm. This was regarded the cause of infertility of couples 105/5 and 175/75.

Estrous Cycle: Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 to 5 days, except for one female at 10 mg/kg/day (No. 141), for which the estrous cycle could not be determined. At necropsy, this female was found to have 3 implantation sites only. Given the incidental nature and the absence of a dose- related incidence, this finding did not indicate a relation with treatment.

Mating Index: Mating index was not affected by treatment. All females showed evidence of mating.

Precoital Time: Precoital time was considered not affected by treatment. All females showed evidence of mating within 4 days, except for one female at 10 mg/kg/day (No. 131) and two females at 100 mg/kg/day (Nos. 190 and 194), for which mating took 5, 8 and 12 days, respectively. As this variation in precoital time remained within the normal range of biological variation9 and occurred at low incidence, this was considered to be unrelated to treatment.

Number of Implantation Sites: Number of implantation sites was not affected by treatment. One female at 10 mg/kg/day (No. 141), for which mating was not detected, had 3 implantation sites only. As this occurred in absence of a dose response relationship, this was considered to be unrelated to treatment.

Fertility Index: Fertility index was considered not to be affected by treatment. The fertility indices were 92, 80, 88 and 84% for the control, 10, 30 and 100 mg/kg/day groups, respectively. The number of non-pregnant females versus mated females was 2/25, 5/25, 3/25 and 4/25 in the control, 10, 30 and 100 mg/kg/day groups, respectively. As these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive toxicity, this was considered not to be related to treatment.

Developmental
Data
 Gestation Index and Duration: Gestation index and duration of gestation were unaffected by treatment. All pregnant females had live offspring, except for one female at 10 mg/kg/day (No. 141), that had 3 implantation sites only. The gestation index was 100% for the control and 30 and 100 mg/kg/day groups, and 95% for the 10 mg/kg/day group. The failed pregnancy of female No. 141, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Parturition/Maternal Care: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95, 89, 89 and 93% for the control, 10, 30 and 100 mg/kg/day groups, respectively. For one female at 30 mg/kg/day (No. 154), the number of pups was slightly higher than the number of implantations. . This phenomenon is observed from time to time, and no toxicological relevance was attached to this finding in the current study.

Litter Size: Litter size was not affected by treatment. Live litter sizes were 11.7, 11.8, 11.2 and 12.1 living fetuses/litter for the control, 10, 30 and 100 mg/kg/day groups, respectively.

Sex Ratio: Sex ratio was considered not affected by treatment.

Live Birth Index:The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for the control, 30 and 100 mg/kg/day groups, and 99% for the 10 mg/kg/day group. One pup at 10 mg/kg/day (litter No. 142) was sacrificed in extremis on PND 1, based on a pale appearance and a wound at the right hindleg. One pup of the control group (litter No. 112) and two pups at 10 mg/kg/day (one in each of litter Nos. 126 and 131) were found dead at first litter check. These dead/missing pups were considered unrelated to treatment since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered not affected by treatment. Viability indices were 99, 99, 100 and 98% for the control, 10, 30 and 100 mg/kg/day groups, respectively.
Two pups of the control group (one in each of litter Nos. 101 and 111), three pups at 10 mg/kg/day (one in each of litter Nos. 126 and 133), one pup at 30 mg/kg/day (litter No. 161) and four pups at 100 mg/kg/day (one in each of litter Nos. 180, 185, 186 and 195) were found dead or missing between PND2 and 4. In addition, one pup at 100 mg/kg/day (litter No. 188) was sacrificed in extremis on PND 1, based on absence of milk in the stomach and fissures at the flews (cleft lip). Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Weaning Index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. One pup at 10 mg/kg/day (litter No. 126) was found dead on PND 21 (cannibalised) and all 8 pups of litter No. 168 (30 mg/kg/day) were necropsied together with the dam that was sacrificed in extremis on PND 13 , resulting in weaning indices of 100, 99, 95 and 100% for the control, 10, 30 and 100 mg/kg/day groups, respectively. No toxicological relevance was attributed to the dead/sacrificed pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on repoductive parameters
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was seen after dosing among animals of all test item-treated groups, in a dose-related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste of the test item and/or irritating properties of the test item rather than a sign of systemic toxicity.
One male at 100 mg/kg/day (No. 449) was recorded to have overgrown teeth on a single day. Hunched posture, piloerection and lean appearance recorded for this animal on the same day were considered secondary to overgrown teeth. As such, these findings were considered not to be related to treatment.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A total 13 F1 animals that were dosed in the post-weaning phase were found dead or were sacrificed in extremis during the first 4 days of treatment, mostly after dosing. This concerned 2/120 control animals, 3/105 animals at 10 mg/kg, 3/120 animals at 30 mg/kg, and 5/120 animals at 100 mg/kg.
Based on the nature of the necropsy findings recorded for these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of these animals showed clinical signs prior to death/sacrifice. For two of these females (Nos. 601 and 689), tissues were examined histopathologically. For female no. 689, the cause of death was not evident from the sections examined microscopically. For female No. 601, a histopathological cause of death was indicated as marked ulceration of the cecum (see also 9.5.16 Histopathology).
For one male at 10 mg/kg/day (No. 301), a possible cause of death could not be established due to cannibalism, but it could not be excluded that this animal had died due to similar respiratory lesions. No clinical signs were shown by this animal prior to death.
One female at 100 mg/kg/day (No. 789) was sacrificed due to clinical signs (pallor, piloerection, labored respiration) and since its cage mates were biting this animal. Its deteriorated condition may also have been influenced by an incident occurring on the previous day where this animal had dropped on the floor. Necropsy findings for this female (many dark-red foci on the glandular mucosa of the stomach) was distinct to what was previously seen for other sacrificed animals.
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Overall, the mortality incidence recorded over the dose groups did not show a dose-related trend. None of the F1 animals that survived until their scheduled necropsy showed similar lesions in the respiratory tract.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased white blood cell (WBC), neutrophil and monocyte count in males (1.31x, 1.50x and 2.00x, respectively; within historical control range).
- Increased reticulocyte counts in males (1.23x; within historical control range) and in females (1.37x; within historical control range).
Haematological values at 10 and 30 mg/kg/day were considered not affected by treatment.
Coagulation parameters of treated rats were considered not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses
- Decreased total protein levels in males (0.96x; within historical control range).
- Increased cholesterol levels in females (1.58x; within historical control range).
Any other statistically significant changes in clinical chemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend (lower total bilirubin level in males at 30 mg/kg) or since an increase would be expected in case of target organ toxicity (lower alkaline phosphatase activity (ALP) in females at 100 mg/kg).
Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted at 30 and/or 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume in males at 100 mg/kg/day (1.71x; within historical control range).
- Decreased specific gravity in males at 100 mg/kg/day (0.99x; within historical control range). This was considered secondary to the higher urinary volume.
- Increased urinary pH in females at 30 and 100 mg/kg/day (1.08x and 1.10x, respectively; within historical control range, and control mean at the lower range of the historical control range).
The statistically significantly lower protein level in males at 10 mg/kg/day occurred in the absence of a dose-related trend and an increase is expected in case of target organ toxicity. Therefore, this change was considered not related to treatment.
Any statistically significant changes in urinary parameters were considered to have arisen as a result of slightly low control values, were only minor in magnitude and/or lacked a dose relationship. These were therefore considered not to be toxicologically relevant.
Sexual maturation:
no effects observed
Description (incidence and severity):
Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus and time between vaginal opening and first estrus in females was considered not to be affected by treatment.
Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher than controls (PND 33 vs. PND 32 in controls), but the mean was similar as recorded at 10 and 30 mg/kg. Mean body weight on the day of vaginal opening was also statistically significantly higher at 100 mg/kg, as well as at 10 mg/kg/day (without a dose-related trend). Individual values remained within the concurrent control range and mean values remained within the historical control range . The day of acquiring first estrous was statistically significantly higher at 10 and 30 mg/kg. However, means did not show a dose-related trend and remained within the available control range . Therefore, these variations in vaginal opening time and day of acquiring first estrous were considered not to be related to treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 100 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (PND 89-95): At 100 mg/kg/day, a statistically significantly higher liver weight was recorded for females (absolute and relative to body weight, 1.13x and 1.07x, respectively). Means remained within the internal control data range. For males at 100 mg/kg/day, a statistically significant marginally higher liver to body weight ratio was recorded (1.06x), but a clear dose-related trend (also in absolute body weights) was absent. Therefore, liver weights in males were considered not affected by treatment. There were no other test item-related organ weight changes. The statistically significant higher absolute ovary weight at 30 mg/kg/day occurred in the absence of a dose-related trend and as such was considered to be unrelated to treatment..

Cohort 1B (≥ PND 97): Organ weights and organ to body weight ratios were considered not affected by treatment.
The statistically significantly higher absolute and relative ovary weights at 100 mg/kg/day remained within the historical control data range, and no clear dose-related response was apparent. Moreover, ovary weights did not show a similar change in Cohort 1A animals that were necropsied on a similar time point. Therefore, this change was considered not to be related to treatment

Cohort 2A (PND 76-100): Fixed brain weights were considered not affected by treatment.

Cohort 2B (PND 21-22): Fixed brain weights were considered not affected by treatment.

Cohort Surplus (PND 22): Brain and spleen weight were considered not affected by treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (PND 89-95): Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1A were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
- Thickening of the limiting ridge in 18/20 males and 16/20 females.
- Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 2/20 females.
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/20 males of the control group and thickening of the limiting ridge in 2/20 males at 30 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1B (≥ PND 97): Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1B were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
- Thickening of the limiting ridge in 16/20 males and 19/20 females.
- Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 5/20 females.

Cohort 1C (males: ≥ PND 35; females: ≥ PND 25: There were no gross observations in animals of Cohort 1C.

Cohort 2A (PND 76-100): There were no test item-related gross observations in animals of Cohort 2A.

Cohort 2B (PND 21-22): There were no gross observations in animals of Cohort 2B.

Cohort Surplus (PND 22): There were no test item-related gross observations in animals of Cohort Surplus: Necropsy findings were confined to pale, gray-white discoloration of the lungs in one control male (No. 279) and thickening of the spleen in one male at 100 mg/kg/day (No. 512). These findings were considered not related to treatment as they did not show a clear dose-related trend or were observed in the control group only.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Test item-related microscopic findings in Cohort 1A were noted in the stomach of both sexes at 100 mg/kg/day.
Microscopic alterations in the glandular stomach of the F1 Cohort 1A-animals were in general consistent with the findings in the F0-animals. However, these microscopic alterations in F1 -animals were recorded at a slightly lower incidence and severity and only regarded test item-related at 100 mg/kg/day. These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight).
The inflammatory cell infiltrate recorded for the glandular stomach in the remaining dose groups including controls were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Spermatogenesis-staging (Cohort 1A): Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Ovarian Follicle Counts (Cohort 1A): There were no test item-related effects on the ovarian follicle counts in the F1 females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between
group mean counts represented biological variability and were not statistically significant.
Other effects:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Fixed brain weights of Cohort 2A animals (PND 76-90) and Cohort 2B animals (PND 21-22) were considered not affected by treatment.
Brain dimensions (length and width of brain) of Cohort 2A animals (PND 76-90) and Cohort 2B animals (PND 21-22) were considered not affected by treatment.
In the F1 animals (Cohort 2A) at PND 76-90, there were no test item-related effects on the
H&E stained sections of brain or peripheral nervous system in the control or high-dose group males or females.
In the F1 animals (Cohort 2B) at PND 21-22, there were no test item-related effects on the
H&E stained sections of brain in the control or high-dose group males or females.
Any variations observed during morphometric analysis of the brain of Cohort 2B animals (PND 21-22) and Cohort 2A animals (PND 76-90) were most likely unrelated to treatment with the test item.
Morphometric analysis of the brain at PND 21-22 revealed statistically significantly higher hippocampus thickness measurements in the 100 mg/kg/day group males when compared with the control group. Morphometric analysis of the brain at PND 76-90 revealed statistically significantly lower frontal cortex thickness, parietal cortex thickness, and hippocampus thickness measurements in the 100 mg/kg/day group males and higher frontal cortex thickness measurements in the 100 mg/kg/day group females.
Trends between sexes were lacking, individual animal values were often within the range of the control group, and there were no differences that were observed at both time points or appeared worse with time. Additionally, there were no correlating changes in organ weights, histopathological findings, or clinical signs. Given these circumstances and the inconsistency of differences across brain regions, the differences were most likely unrelated to the test item.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Slightly lower T-cytotoxic cell splenic subpopulations were observed for males and females at 100 mg/kg. A slightly higher Th/Tc ratio was observed for males at 30 and 100 mg/kg/day and slightly lower T-cell splenic subpopulations were observed for females at 100 mg/kg. As these shifts were slight in nature, did not reach statistical significance and occurred in the absence of corroborative findings in the spleen, these shifts were considered to represent biological variability and considered not to represent an effect of the test item.
F1-pups Results:
No clinical signs occurred among pups that were considered to be related to treatment. For some pups of all groups (including control) that were found dead/missing, pallor and/or absence of milk in the stomach was noted. At the incidence observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant.

Body weights of pups were considered not affected by treatment.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment.

Serum T4 levels in male and female pups culled at PND 4 were not affected by treatment. In addition, serum thyroid stimulating hormone (TSH) and T4 levels of male and female pups of Cohort Surplus at PND 22 were considered not affected by treatment.

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment. Brain and spleen weight of surplus pups were considered not affected by treatment.

F1-generation Data (Cohort 1A, 1B, 1C, 2A)
No mortality occurred that was considered to be directly attributable to treatment with the test item. A total 13 F1 animals that were dosed in the post-weaning phase were found dead or were sacrificed in extremis during the first 4 days of treatment, mostly after dosing (see also table below). This concerned 2/120 control animals, 3/105 animals at 10 mg/kg, 3/120 animals at 30 mg/kg, and 5/120 animals at 100 mg/kg. Based on the nature of the necropsy findings recorded for these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of these animals showed clinical signs prior to death/sacrifice. For two of these females (Nos. 601 (control group) and 689 (30 mg/kg)), tissues were examined histopathologically. For female No. 689, the cause of death was not evident from the sections examined microscopically. For female No. 601, a histopathological cause of death was indicated as marked ulceration of the cecum. For one male at 10 mg/kg/day (No. 301), a possible cause of death could not be established due to cannibalism, but it could not be excluded that this animal had died due to similar respiratory lesions. No clinical signs were shown by this animal prior to death. One female at 100 mg/kg/day (No. 789) was sacrificed due to clinical signs (pallor, piloerection, labored respiration) and since its cage mates were biting this animal. Its deteriorated condition may also have been influenced by an incident occurring on the previous day where this animal had dropped on the floor. Necropsy findings for this female (many dark-red foci on the glandular mucosa of the stomach) was different to what was previously seen for other sacrificed animals. One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris. Overall, the mortality incidence recorded over the dose groups did not show a dose-related trend. None of the F1 animals that survived until their scheduled necropsy showed similar lesions in the respiratory tract.

Clinical Observations: No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation was seen after dosing among animals of all test item-treated groups, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste of the test item and/or irritating properties of the test item rather than a sign of systemic toxicity. One male at 100 mg/kg/day (No. 449) was recorded to have overgrown teeth on a single day. Hunched posture, piloerection and lean appearance recorded for this animal on the same day were considered secondary to overgrown teeth. As such, these findings were considered not to be related to treatment. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Body Weights and Body Weight Gains: No changes in body weight or body weight gain were noted that were considered to be related to treatment. Any statistically significant changes in body weight and/or body weight gain occurring during treatment across all dose groups were considered not related to treatment as a dose-related trend was absent and changes were not consistently recorded with continuing treatment.

Food Consumption: No toxicologically relevant changes in food consumption (before and/or after correction for body weight) were recorded across the dose groups. All statistically significant changes in food consumption (before and/or after correction for body weight) were very minor and did not show a dose-relationship. The initially (statistically significantly) lower food intake may have been caused by variation in delivery dates of dams and subsequent allocation of the pups to the respective cages.

Functional Tests: Acoustic startle response was considered not affected by treatment. Any statistically significant changes in acoustic startle response parameters were considered not related to treatment as these were minimal and/or occurred in the absence of a dose- related trend. Detailed clinical observations revealed no symptoms that were considered to be related to treatment. The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study and were generally also observed in control animals. These findings were therefore considered not to be related to treatment. At 100 mg/kg, mean rectal temperature of males was statistically significantly reduced (0.98x; mean remained within the internal control data range). Mean rectal temperature at 10 and 30 mg/kg/day (both sexes) and at 100 mg/kg/day (females) were similar to the control mean. At 100 mg/kg, a lower motor activity was recorded for females compared to the control means (both for total movements (0.69x) and ambulations (0.66x), statistically significant only for ambulations). Means remained within the internal control data range. Motor activity of males treated up to 100 mg/kg/day and of females up to 30 mg/kg/day was considered not affected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were considered not affected by treatment. A statistically significantly lower mean foot splay value was recorded for males at 30 and 100 mg/kg. However, a dose-related response was absent, i.e. the mean at 100 mg/kg/day was higher than the mean at 30 mg/kg/day and comparable to the mean at 10 mg/kg. As such, this variation was considered to be unrelated to treatment. Hearing ability and pupillary reflex were normal in all examined animals. Grip strength was considered not affected by treatment.

Sexual Maturation: Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrous and time between vaginal opening and first estrous in females was considered not to be affected by treatment. Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher than controls (PND 33 vs. PND 32 in controls), but the mean was similar as recorded at 10 and 30 mg/kg. Mean body weight on the day of vaginal opening was also statistically significantly higher at 100 mg/kg, as well as at 10 mg/kg/day (without a dose-related trend). Individual values remained within the concurrent control range and mean values remained within the internal control range. The day of acquiring first estrous was statically significantly Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrous and time between vaginal opening and first estrous in females was considered not to be affected by treatment. Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher at 10 and 30 mg/kg. However, means did not show a dose-related trend and remained within the available control range. Therefore, these variations in vaginal opening time and day of acquiring first estrous were considered not to be related to treatment.

Estrous Cycle – F1-Generation (Cohort 1A): Length and regularity of the estrous cycle were not affected by treatment. For all females for which estrous cycle regularity could be determined, regular cycles of 4 to 5 days were recorded between PND 75 and 88. No cycle classification was possible for 1/19 females at 10 mg/kg/day (No. 602), 1/19 females at 30 mg/kg/day (No. 695) and 1/20 females at 100 mg/kg/day (No. 763). In addition, one female at 100 mg/kg/day (No. 764) was acyclic. Given the incidental nature and the absence of a dose-related incidence, this single occurrence of an acyclic estrous cycle was considered not to indicate a relation to treatment.

Sperm Analysis – F1-Generation (Cohort 1A): Sperm motility, concentration and morphology were considered not affected by treatment. At 100 mg/kg/day, a statistically significantly lower number of cells with normal morphology was recorded (0.93x). No clear dose-related trend occurred as the mean was similar to that at 10 mg/kg, and the means remained within the internal control range. At both 10 and 100 mg/kg, the number of cells with coiled tail appeared higher than the control mean (not statistically significant), but again no dose-related trend was apparent and means remained within the internal control range. Also, the control mean number of cells with coiled tail was considered to be slightly low compared to the internal control mean. The statistically significantly higher epididymal sperm count at 30 mg/kg/day occurred in the absence of a dose-related trend. Therefore, these variations were considered not to represent an effect of the test item.
Haematology – F1-Generation (Cohort 1A): The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased white blood cell (WBC), neutrophil and monocyte count in males (1.31x, 1.50x and 2.00x, respectively; within internal control range).
- Increased reticulocyte counts in males (1.23x; within internal control range) and in females (1.37x; within internal control range).
Haematological values at 10 and 30 mg/kg/day were considered not affected by treatment.

Coagulation – F1-Generation (Cohort 1A): Coagulation parameters of treated rats were considered not affected by treatment.

Clinical Chemistry – F1-Generation (Cohort 1A): The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
-Decreased total protein levels in males (0.96x; within internal control range).
-Increased cholesterol levels in females (1.58x; within internal control range).
Any other statistically significant changes in clinical chemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend (lower total bilirubin level in males at 30 mg/kg) or since an increase would be expected in case of target organ toxicity (lower alkaline phosphatase activity (ALP) in females at 100 mg/kg).

Thyroid hormone analyses – F1-Generation (Cohort 1A): Serum levels of TSH (thyroid stimulating hormone) and T4 of Cohort 1A were considered not affected by treatment.

Urinalysis – F1-Generation (Cohort 1A): The following statistically significant changes were noted at 30 and/or 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume in males at 100 mg/kg/day (1.71x; within internal control range).
-Decreased specific gravity in males at 100 mg/kg/day (0.99x; within internal control range). This was considered secondary to the higher urinary volume.
-Increased urinary pH in females at 30 and 100 mg/kg/day (1.08x and 1.10x, respectively; within internal control range, and control mean at the lower range of the internal control range).
The statistically significantly lower protein level in males at 10 mg/kg/day occurred in the absence of a dose-related trend and an increase is expected in case of target organ toxicity. Therefore, this change was considered not related to treatment. Any statistically significant changes in urinary parameters were considered to have arisen as a result of slightly low control values, were only minor in magnitude and/or lacked a dose relationship. These were therefore considered not to be toxicologically relevant.

Splenic Lymphocyte Subpopulation – F1-Generation (Cohort 1A): Splenic lymphocyte subpopulations were considered not affected by treatment. Slightly lower T-cytotoxic cell splenic subpopulations were observed for males and females at 100 mg/kg. A slightly higher Th/Tc ratio was observed for males at 30 and 100 mg/kg/day and slightly lower T-cell splenic subpopulations were observed for females at 100 mg/kg. As these shifts were slight in nature, did not reach statistical significance and occurred in the absence of corroborative findings in the spleen, these shifts were considered to represent biological variability and considered not to represent an effect of the test item.

Gross Pathology – F1-Generation:
Cohort 1A - test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1A were recorded for the stomach and were observed in the 100 mg/kg/day group in both males and females. These alterations consisted of:
-Thickening of the limiting ridge in 18/20 males and 16/20 females.
-Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 2/20 females.
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/20 males of the control group and thickening of the limiting ridge in 2/20 males at 30 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B: Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1B were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
-Thickening of the limiting ridge in 16/20 males and 19/20 females.
-Dark red/reddish focus/foci in the glandular mucosa in 8/20 males and 5/20 females.
Cohort 1C: There were no gross observations in animals.
Cohort 2A: There were no gross observations in animals.
Cohort 2B: There were no gross observations in animals.
Cohort surplus: There were no gross observations in animals. Necropsy findings were confined to pale, grey-white discoloration of the lungs in one control male (No. 279) and thickening of the spleen in one male at 100 mg/kg/day (No. 512). These findings were considered not related to treatment as they did not show a clear dose-related trend or were observed in the control group only.

Organ weights – F1-Generation
Cohort 1A: At 100 mg/kg/day, a statistically significantly higher liver weight was recorded for females (absolute and relative to body weight, 1.13x and 1.07x, respectively). Means remained within the internal control data range. For males at 100 mg/kg/day, a statistically significant marginally higher liver to body weight ratio was recorded (1.06x), but a clear dose-related trend (also in absolute body weights) was absent. Therefore, liver weights in males were considered not affected by treatment. There were no other test item-related organ weight changes. The statistically significant higher absolute ovary weight at 30 mg/kg/day occurred in the absence of a dose-related trend and as such was considered to be unrelated to treatment.
Cohort 1B: Organ weights and organ to body weight ratios were considered not affected by treatment. The statistically significantly higher absolute and relative ovary weights at 100 mg/kg/day remained within the internal control data range and no clear dose-related response was apparent. Moreover, ovary weights did not show a similar change in Cohort 1A animals that were necropsied on a similar time point. Therefore, this change was considered not to be related to treatment.
Cohort Surplus: Brain and spleen weight were considered not affected by treatment.

Histopathology – F1-Generation:
Cohort 1A: Test item-related microscopic findings in Cohort 1A were noted in the stomach of both sexes at 100 mg/kg/day Microscopic alterations in the glandular stomach of the F1 Cohort 1A-animals were in general consistent with the findings in the F0-animals. However, these microscopic alterations in F1 - animals were recorded at a slightly lower incidence and severity and only regarded test item- related at 100 mg/kg/day. These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight). The inflammatory cell infiltrate recorded for the glandular stomach in the remaining dose groups including controls were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Spermatogenesis-staging (Cohort 1A): Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts (Cohort 1A): There were no test item-related effects on the ovarian follicle counts in the F1 females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Neuropathology and Morphometry– F1-Generation Cohorts 2A and 2B
Fixed brain weights of Cohort 2A animals and Cohort 2B animals were considered not affected by treatment. Brain dimensions (length and width of brain) of Cohort 2A animals and Cohort 2B animals were considered not affected by treatment. In the F1 animals (Cohort 2A), there were no test item-related effects on the H&E stained sections of brain or peripheral nervous system in the control or high-dose group males or females. In the F1 animals (Cohort 2B), there were no test item-related effects on the H&E stained sections of brain in the control or high-dose group males or females. Any variations observed during morphometric analysis of the brain of Cohort 2B animals and Cohort 2A animals were considered unrelated to treatment with the test item. Morphometric analysis of the brain of Cohort 2B animals revealed statistically significantly higher hippocampus thickness measurements in the 100 mg/kg/day group males when compared with the control group. Morphometric analysis of the brain of Cohort 2A animals revealed statistically significantly lower frontal cortex thickness, parietal cortex thickness, and hippocampus thickness measurements in the 100 mg/kg/day group males and higher frontal cortex thickness measurements in the 100 mg/kg/day group females. Trends between sexes were lacking, individual animal values were often within the range of the control group, and there were no differences that were observed at both time points or appeared worse with time. Additionally, there were no correlating changes in organ weights, histopathological findings, or clinical signs. Given these circumstances and the inconsistency of differences across brain regions, the differences were most likely unrelated to Hexahydro- 1,3,5-Trimethyl-1,3,5-Triazine.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive and developemntal parameters
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 12. Summary Test Item-Related Microscopic Finding F0 -Stomach

Dose level (mg/kg/day):

Males

Females

0

10

30

100

0

10

30

100

Stomacha

25

25

25

25

25

25

25

25

Lymphogranulocytic infiltrate glandular mucosa,

 

 

 

 

 

 

 

 

     Minimal

2

4

9

5

-

1

2

8

     Slight

-

-

-

16

-

1

-

11

     Moderate

-

-

-

4

-

-

-

1

Hemorrhage glandular mucosa 

 

 

 

 

 

 

 

 

     Minimal

-

1

-

7

1

-

3

7

     Slight

-

-

-

5

-

-

1

4

     Moderate

-

-

-

2

-

-

-

1

Erosion/ulceration glandular                                    mucosa

 

 

 

 

 

 

 

 

     Minimal

-

-

-

4

2

-

3

2

     Slight

-

-

-

7

-

1

1

1

     Moderate

-

-

-

1

-

-

-

-

Degeneration/regeneration

glandular mucosa

 

 

 

 

 

 

 

 

     Minimal

-

-

-

-

-

-

1

6

     Slight

-

-

-

3

-

-

-

2

Decreased number of parietal cells

 

 

 

 

 

 

 

 

     Minimal

-

-

-

2

-

-

-

-

     Slight

-

-

-

5

-

-

-

-

a = Number of tissues examined from each group.

Table 13. Summary Test Item-Related Microscopic Findings F0– male adrenal gland 

 

Males

Dose level (mg/kg/day):

0

10

30

100

 

 

 

 

 

Adrenal glanda

25

25

25

25

  Vacuolation zona glomerulosa

 

 

 

 

     Minimal

3

5

7

13

     Slight

1

-

-

2

a = Number of tissues examined from each group.

 

Table 14. Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.

Group

Dose level

mg/kg/day

Female/Male nos.

In-Life Reason

Histopathology

1

0

105/5

Not pregnant

Male 5[sb1] : massive bilateral tubular atrophy testes and reduced sperm eididymides

 

 

120/20

Not pregnant

No histopathological correlate

2

10

132/32

Not pregnant

No histopathological correlate

 

 

134/34

Not pregnant

No histopathological correlate

 

 

136/36

Not pregnant

No histopathological correlate

 

 

141/41

Implantation sites only

No histopathological correlate

 

 

144/44

Not pregnant

No histopathological correlate

 

 

146/46

Not pregnant

No histopathological correlate

3

30

156/56

Not pregnant

No histopathological correlate

 

 

168/68

Euthanized on Day 13 of lactation

Delivered pups, no findings suggesting lack of fertility

 

 

172/72

Not pregnant

No histopathological correlate

 

 

175/75

Not pregnant

Male 75: massive bilateral tubular atrophy testes and reduced sperm epididymides

4

100

176/76

Not pregnant

No histopathological correlate

 

 

179/79

Not pregnant

No histopathological correlate

 

 

182/82

Not pregnant

No histopathological correlate

 

 

199/99

Not pregnant

No histopathological correlate

Further results are included in background materials.

Conclusions:
Based on the criteria set in Regulation (EC) No 1272/2008, hexahydro-1,3,5 -trimethyl-1,3,5 -triazine is not classified for reproductive or developmental toxicity.

Executive summary:

OECD 443 (2019): The pre- and postnatal effects of the test item was evaluated in Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day, based on the results of aCombined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in the rat (Test Facility Study No. 491807). The following parameters and end points were evaluated included mortality/ moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm and splenic lymphocyte subpopulation analysis, organ weights and histopathological examinations. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood were conducted. The integrity and performance of the adult male and female reproductive systems  and developmental neurotoxicity  were also assessed.

F0-Generation:

Treatment resulted in adverse macroscopic and microscopic alterations in the stomach of both sexes at 30 and 100 mg/kg/day and non-adverse microscopic findings in the adrenal glands of males at 100 mg/kg/day. The stomach findings consisted of a combination of inflammatory and degenerative findings in the glandular stomach in the F0-animals at 30 and 100 mg/kg/day, consisting of an increased incidence and/or severity of lymphogranulocytic infiltrate, hemorrhage, erosion/ulceration, degeneration/regeneration mucosa, edema and/or decreased number of parietal cells. These findings were considered to be the result of irritating properties of the test item, and correlated to necropsy findings in these dose groups (foci in the glandular stomach; thickening of the limiting ridge observed at necropsy had no clear histopathological correlate).

Reproduction results – F0-generation:

No reproductive toxicity was observed up to the highest dose level tested (100 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. sperm motility, concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0 generation / F1 generation (pre-weaning):

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, live birth, viability and weaning indices, duration of gestation, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and brain and spleen weight

Developmental results – F1 generation (post-weaning):

No mortality occurred that was considered to be directly attributable to treatment with the test item. A total 13 F1 animals were found dead or were sacrificed in extremis during the first 4 days of treatment. The mortality incidence recorded over the dose groups did not show a dose-related trend. Based on the nature of the necropsy findings recorded for most of these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of the F1 animals that survived until their scheduled necropsy showed similar lesions, eg. in the respiratory tract.

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations, and body weight and food intake was considered not affected by treatment.

Treatment resulted in adverse macroscopic and microscopic alterations in the stomach of both sexes at 100 mg/kg/day (Cohort 1A-animals, PND 96 (nos. 456-459, 776-778), PND 100 (nos. 442-445) or PND 89-95 (other animals). These were in general consistent with the findings in the F0-animals, although these microscopic alterations in F1 -animals were recorded at a slightly lower incidence and severity and only occurred at 100 mg/kg/day.

These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight).

Macroscopically, these findings were supported by thickening of the limiting ridge and foci in the glandular mucosa, which were also observed at similar incidences in Cohort 1B animals (≥ PND 97; no histopathological examination was conducted for Cohort 1B animals). These findings were considered to be the result of irritating properties of the test item, and correlated to necropsy findings in these dose groups (foci and in the glandular stomach and thickening of the limiting ridge).

Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by treatment.

No treatment-related effects were recorded for developmental parameters including balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and morphology of reproductive organs.

No adverse changes in in-life or post-mortem developmental neurotoxicity endpoints were recorded for Cohort 2 animals. For Cohort 2A animals, these endpoints consisted of acoustic startle response, detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity. For both Cohort 2A and 2B animals, developmental neurotoxicity endpoints consisted of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain. Non-adverse changes at 100 mg/kg/day consisted of a lower mean rectal temperature for males and lower motor activity for females. Mean rectal temperature of males was marginally reduced (0.98x). Given the minor degree of this change that occurred in the absence of any other corroborative changes, this was considered not adverse.

The lower motor activity for females (both for total movements and ambulations) was not supported by clinical observations or other functional observation tests, remained wthin the normal range for rats of this age and strain, and had no supportive morphological correlates in examined neuronal tissues.

Also, all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Overall these changes were considered not to represent an adverse effect on neurobehaviour.

No treatment-related changes in developmental immunotoxicity endpoints were recorded, i.e. splenic lymphocyte subpopulations, lymphoid histopathology and organ weights.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following no-observed-adverse-effect levels (NOAEL) of Hexahydro-1,3,5-trimethyl-1,3,5-triazine were established:

General toxicity (F0 and F1): 30 mg/kg/day (based on lesions in the glandular stomach at 100 mg/kg/day in F0 animals, and F1 animals).

Developmental neurotoxicity: at least 100 mg/kg/day.

Reproduction:       at least 100 mg/kg/day.

Development:       at least 100 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The data-set for effects on fertility is consistent with data requirements under Annexes VII to X of REACH, and all studies are considered reliable and relevant for endpoint conclusions.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Based on the results of all reproductive and developmental studies conducted on the substance, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 30 mg/k bw//day

Reproductive NOAEL: ≥100 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

NOAEL (developmental, rat) = ≥ 100 mg/kg bw/d, OECD 422, van Tuyl (2010)

NOAEL (developmental, rat) = ≥ 120 mg/kg bw/d, OECD 414, de Raaf - Beekhuijzen (2016)

NOAEL (developmental, rabbbit) = ≥ 25 mg/kg/day, (OECD 414, Anon. 2019)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 September 2009 -10 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines in a GLP laboratory. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality).
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Age at study initiation: Approximately 9-10 weeks- Fasting period before study: no- Housing: Pre-matingAnimals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).MatingFemales were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).Post-matingMales were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).LactationPups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).GeneralSterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cageenrichment was provided during activity monitoring.- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). - Water (e.g. ad libitum): Free access to tap-water. - Acclimation period: At least 5 days prior to start of treatment.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 3°C (actual range: 20.2 – 21.9°C)- Humidity (%): 40 - 70% (actual range: 28 - 76%) - Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 22 September 2009 (allocation) To: 10 November 2009 (end of in-life phase)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for the density of the test substance (1.0202), the specific gravity of the vehicle (1.036) and the water content (66.44%).VEHICLE- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.- Concentration in vehicle: 2, 6 and 29 mg/mL (expressed as mas neat mg Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine/kg b.w.- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase (28 September and 08 October 2009), according to a validated method (NOTOX project 492054). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation: A maximum of 13 days was allowed for mating.- Proof of pregnancy: sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy- Further matings after two unsuccessful attempts: no- After successful mating each pregnant female was caged (how): individually- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Once daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the dose range finding study (NOTOX Project 491831)
Maternal examinations:
Parental animals: Observations and examinationsCAGE SIDE OBSERVATIONS: Yes - Time schedule: At least twice daily (early morning/late afternoon)DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.BODY WEIGHT: Yes- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4. FOOD CONSUMPTION: YesWeekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.WATER CONSUMPTION: No, Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.HAEMATOLOGY: Yes - Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. - Anaesthetic used for blood collection: Yes iso-flurane- Animals fasted: yes, but water was available - How many animals: 5 males/group (random) and all females with live pups- Parameters examined were: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated Partial thromboplastin time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. - Animals fasted: yes, but water available- How many animals: 5 males/group (random) and all females with live pups- Parameters examined were: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling). - Dose groups that were examined: all- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity Estrous cyclicity (Parental animals)not determined (not required)Sperm parameters (Parental animals)Parameters examined in all male parental animals:testis weight, epididymis weight.in addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis. Postmortem examinations (Parental animals)SACRIFICE- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).- Maternal animals: All surviving animals on lactation Days 5-6.- Females which failed to deliver: Post-coitum Day 27 (evidence of mating) or 22 days after the last day of the mating period (without evidence of mating).GROSS NECROPSYAll animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The number of former implantation sites and corpora lutea was recorded for all paired females.From all animals, samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):Identification marks (not processed), Cervix, Clitoral gland, Epididymides*, Ovaries, Preputial gland, Prostate gland, Seminal vesicles including coagulating gland, Testes*, Uterus, Vagina, All gross lesions.From 5 males/group (random) and all females with live pups, samples of the following tissues and organs were collected and fixed in addition to the abovementioned list:Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Colon, Duodenum, Eyes (including optic nerve and Harderian gland)*, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Lacrimal gland (exorbital), Larynx, Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), Nasopharynx, Oesophagus, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Thymus, Thyroid including parathyroid (if detectable), Tongue, Trachea, Urinary bladder.*Fixed in modified Davidson's solution and transferred to formalin after fixation for at least 24 hours.ORGAN WEIGHTS: YesThe following organ weights and terminal body weight were recorded from 5 males/group (random) and all females with live pups on the scheduled day of necropsy:Adrenal glands, Brain, Epididymides (all males), Heart, Kidneys, Liver, Ovaries Spleen, Testes (all males), Thymus, Uterus (including cervix), Prostate*, Seminal vesicles including coagulating glands*, Thyroid including parathyroid** weighed when fixed for at least 24 hours.HISTOPATHOLOGY: Yes The following slides were examined by a pathologist:- The preserved organs and tissues of 5 selected animals/sex of Groups 1 and 4.- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.- All gross lesions of all animals (all dose groups).- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy pups.
Fetal examinations:
Litter observationsPARAMETERS EXAMINEDThe following parameters were examined in F1 offspring:number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.GROSS EXAMINATION OF DEAD PUPS: YesIf possible, defects or cause of death were evaluated.Postmortem examinations (Offspring)SACRIFICEPups surviving to planned termination were killed by decapitation on lactation Days 5-6.GROSS NECROPSYAll pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.HISTOPATHOLOGY / ORGAN WEIGTHSno
Statistics:
Reproductive indicesFor each group, the following calculations were performed:Percentage mated females: Number of females mated/Number of females paired x 100Fertility index: Number of pregnant females/Number of females paired x 100Conception index: Number of pregnant females/Number of females mated x 100Gestation index: Number of females bearing live pups/Number of pregnant females x 100Duration of gestation: Number of days between confirmation of mating and the beginning of parturitionFor each group, the following calculations were performed:Percentage mated females: Number of females mated/Number of females paired x 100Fertility index: Number of pregnant females/Number of females paired x 100Conception index: Number of pregnant females/Number of females mated x 100Gestation index: Number of females bearing live pups/Number of pregnant females x 100Duration of gestation: Number of days between confirmation of mating and the beginning of parturitionOffspring viability indicesPercentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Checkx 100Number of live pups at First Litter Check Viability index (%) Number of live pups on Day 4 of lactation x 100Number of pups born alive
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg bw/d, salivation was noted for all animals from week 4 of treatment onwards. Rales (slight to moderate) were noted for two males and one female treated at 100 mg/kg bw/d for 2, 5 or 12 days. The male (no. 40) that showed rales for 12 days, also showed piloerection for 2 days.Rales (slight) were also noted for one male treated at 10 mg/kg bw/d for 2 days and one female treated at 30 mg/kg bw/d for 2 days. At this low incidence, these observations were not considered toxicologically significant.Alopecia was noted for three females. The finding occurred wthin the range of background findings to be expected for rats of this age and strain whcih are housed and treated under the conditions of the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Minor statistically significant decreases arising between controls and animals receiving 100 mg/kg bw/d (decreased haemoglobin and decreased APTT for males and increased APTT for females) were considered not to represent a change of biological significance as the changes were slight and the values remained within the range considered normal for rats of this age and strain.Individual increases of neutrophil counts with concurrently reduced lymphocyte counts was noted for one male (no. 3) of the control group. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg bw/d, total protein and albumin were slightly decreased for both sexes.Any statistically significant changes at 30 mg/kg bw/d (bile acids for males) and at 100 mg/kg bw/d (chloride for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution or remained within the range considered normal for rats of this age and strain.The increased values for bile acids (not statistically significant), noted for females of all dose groups were considered to have occured by chance and to be of no toxicological relevance as these changes were caused by individual animals only.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg body weight/day. The statistical significant decreased values for testes and epididymides weights (absolute and relative) at the high dose group were not considered toxicologically significant as the changes were very slight, no corroborative findings were noted at microscopic examination, and reproduction was unaffected.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg, five males and eight females showed a thickened limiting ridge of the stomach. This finding was also noted for one female treated at 30 mg/kg body weight/day. However, at this single occurrence and without microscopic correlate it was not considered toxicologically significant. For the male at 100 mg/kg/day that died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling, incomplete exsanguination was noted. One female of the control group showed one dead fetus in the right uterus horn. Other incidental findings included soft yellowish or greenish nodule at the epididymides, isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related morphologic alterations in Wistar (Han) rats subjected to a combined 28-Day study with Hexahydro-1,3,5-Trimethyl-S-Triazine via oral gavage at doses up to 100 mg/kg in stomach of both sexes and possibly eyes of females. There were no morphologic alterations at 30 mg/kg Hexahydro-1,3,5-Trimethyl-S-Triazine. In the stomach, minimal lymphogranulocytic inflammation of the glandular stomach was recorded in 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy. There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related. At 100 mg/kg body weight/day, salivation was noted for all animals from Week 4 of treatment onwards. Rales (slight to moderate) were noted for one female treated at 100 mg/kg for 2, 5 or 12 days. Rales (slight) were also noted for one female treated at 30 mg/kg for 2 days. At this low incidence, these observations were not considered toxicologically significant. Alopecia was noted for three females. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
BODY WEIGHT AND WEIGHT GAIN: Mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
FOOD CONSUMPTION: Food consumption before or after allowance for body weight was similar between treated and control animals.
NEUROBEHAVIOUR: No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The variation in motor activity did not indicate a relation with treatment.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No toxicologically significant effects on reproductive parameters were noted.
ORGAN WEIGHTS: No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg body weight/day.
GROSS PATHOLOGY: At 100 mg/kg, eight females showed a thickened limiting ridge of the stomach. This finding was also noted for one female treated at 30 mg/kg body weight/day. However, at this single occurrence and without microscopic correlate it was not considered toxicologically significant. One female of the control group showed one dead fetus in the right uterus horn. Other incidental findings included isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.
HISTOPATHOLOGY: NON-NEOPLASTIC: In the stomach, minimal lymphogranulocytic inflammation of the glandular stomach was recorded in 5/5 males and 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males and 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy. There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy) in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined. No abnormalities were seen in the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy offspring which could account for infertility
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
No toxicologically significant effects on developmental parameters were observed.No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant femalesGestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.One pup of Group 2 was found dead on Day 2 of lactation. This single occurrence was considered to have occurred by chance.Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level
Abnormalities:
not specified
Developmental effects observed:
not specified

Results of examinations: parental animals

Clinical signs (parental animals)

yes

Body weight and food consumption (parental animals)

no effects

Test substance intake (parental animals)

not examined

Reproductive function: estrous cycle (parental animals)

no effects

Reproductive function: sperm measures (parental animals)

no effects

Reproductive performance (parental animals)

no effects

Organ weights (parental animals)

no effects

Gross pathology (parental animals)

yes

Histopathology (parental animals)

yes

Details on results (parental animals)

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was however not considered treatment related. At 100 mg/kg body weight/day, salivation was noted for all animals from Week 4 of treatment onwards. Rales (slight to moderate) were noted for two males and one female treated at 100 mg/kg for 2, 5 or 12 days. The male that showed rales for 12 days, also showed piloerection for 2 days. Rales (slight) were also noted for one male treated at 10 mg/kg for 2 days and one female treated at 30 mg/kg for 2 days. At this low incidence, these observations were not considered toxicologically significant. Alopecia was noted for three females. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

BODY WEIGHT AND WEIGHT GAIN Mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION Food consumption before or after allowance for body weight was similar between treated and control animals.

NEUROBEHAVIOUR: No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The variation in motor activity did not indicate a relation with treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) No toxicologically significant changes.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) No toxicologically significant effects on reproductive parameters were noted.

ORGAN WEIGHTS No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg body weight/day. The statistical significant decreased values for testes and epididymides weights (absolute and relative) at the high dose group were not considered toxicologically significant as the changes were very slight, no corroborative findings were noted at microscopic examination, and reproduction was unaffected.

GROSS PATHOLOGY At 100 mg/kg, five males and eight females showed a thickened limiting ridge of the stomach. This finding was also noted for one female treated at 30 mg/kg body weight/day. However, at this single occurrence and without microscopic correlate it was not considered toxicologically significant. For the male at 100 mg/kg/day that died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling, incomplete exsanguination was noted. One female of the control group showed one dead fetus in the right uterus horn. Other incidental findings included soft yellowish or greenish nodule at the epididymides, isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related microscopic findings were present in stomach (males and females) and eyes (females) at 100 mg/kg:

- Stomach: Lymphogranulocytic inflammation of the glandular stomach (minimal) was recorded in 5/5 males and 4/8 females. Hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males (minimal) and 6/8 females (minimal-slight). This was in most cases the microscopic correlate to the thickening of the limiting ridge recorded at necropsy.

- Eyes (females): Degeneration of the retina (atrophy) was recorded in 2/5 females (minimal-slight). All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar Han rats of this age and strain and therfore not considered to be treatment related. No abnormalities were seen in the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy offspring which could account for infertility. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Results of examinations: offspring

Viability (offspring)

no effects

Clinical signs (offspring)

no effects

Body weight (offspring)

no effects

Sexual maturation (offspring)

not examined

Organ weights (offspring)

not examined

Gross pathology (offspring)

no effects

Histopathology (offspring)

not examined

Details on results (offspring) No toxicologically significant effects on developmental parameters were observed. No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females Gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment. One pup of Group 2 was found dead on Day 2 of lactation. This single occurrence was considered to have occurred by chance. Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

Conclusions:
Formulation analysis showed that the formulations were prepared accurately and homogenously and were stable for at least 6 hours at room temperature. At 100 mg/kg body weight/day, parental toxicity consisted of clinical signs (salivation, rales and/or piloerection),slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge. No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, body weight, food consumption, and organ weights) at 100 mg/kg. No parental toxicity was observed at 10 and 30 mg/kg/day. No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg body weight/day. Based on these results, the following No Observed Adverse Effect Level (NOAEL) was derived: Parental NOAEL: 30 mg/kg/day and reproductive and developmental toxicity of NOAEL: ≥100 mg/kg/day.
Executive summary:

OECD 422 (2010) - In a combined repeat dose toxicity study with reproductive/developmental toxicity screening (OECD 422), Hexahydro-1,3,5-Trimethyl-S-Triazine in rats by oral gavage at dosage of 10, 30 and 100 mg/kg bw/day. Male were exposed for 28 days i.e. 2 weeks prior to mating, during mating and up to termination. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, post coitum and during at least 4 days of lactation. A summary of adult responses to the test item are described below;

No mortality occurred during the study period that was considered to be related to treatment with the test.  At the highest dose group, clinical signs included salivation, rales and/or piloerection, slightly decreased total protein and albumin. A thickening limiting ridge of the stomach at macroscopic examination and microscopic finding such as lymphogranulocytic inflammation of the glandular stomach was recorded in 5/5 males and 4/8 females. Minimal hyperplasia of the epithelium of the limiting ridge was recorded in 2/5 males and 6/8 females (minimal-slight).

There were microscopic findings which were considered possibly treatment-related: bilateral degeneration (atrophy)  in the outer nuclear layer of the retina recorded in Group 4 in 2/5 females (1: minimal, 1: slight; the minimal degree of unilateral degeneration of the retina of animal No. 71 was regarded to be within background pathology). This was characterized by reduced cellularity, mainly in the central area (close to the optic nerve) and mid-peripheral area of the retina. Severity was scored as “minimal” where multifocal loss of cells of the outer nuclear layer was seen, and “slight” when the distribution was more diffuse and more pronounced. Based on a peer-review, bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined.

Food consumption was unaffected and the mean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.  

No toxicologically significant effects on reproductive parameters were observed; percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites were unaffected by the treatment.

No difference in maternal care were noted. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among pregnant females.

Gestation index duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index and early postnatal pup development (mortality, clinical signs, bodyweight and external macroscopy) were unaffected by the treatment.

On pup of group 2 (pup 8 of litter 55) was found dead on day 2 of lactation. This single occurrence was considered to have occurred by chance.

Incidental clinical symptoms consisted of small size, blue spot on the neck, pale appearance and broken tail. Incidental macroscopic findings included small size and scab on the apex of the tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.  

The No Observed Adverse Effect Level (NOAEL) was derived as:

Parental NOAEL = 30 mg/kg bw/day

Reproductive and developmental toxicity of NOAEL ≥100 mg/kg/day

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines in a GLP laboratory. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl: WI (HAN) (outbred, SPF-Quality)
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Age at study initiation: Approximately 10-14 weeks- Fasting period before study: no- Housing: Females were individually housed in Macrolon cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage enrichment were supplied.General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. - Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). - Water (e.g. ad libitum): Free access to tap-water. - Acclimation period: At least 5 days prior to start of treatment.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 3°C (actual range: - Humidity (%): 40 - 70% (actual range: - Air changes (per hr): 10- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 10 January 2016 (start treatment) To: 28 January 2016 (end of in-life phase)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for the density of the test substance (1.0236), the specific gravity of the vehicle (1.036) and the water content (58.4249%).VEHICLE- Based on trial formulations performed at WIL Research Europe B.V.- Dose volume: 5 mL/kg bodyweight, experimental dose levels of neat Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine was 0, 10, 40 and 120 mg hexahydro-1,3,5-trimethyl-1,3,5-triazine/kg b.w.- Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase (11 January (analysed the same day) and 26 January (refrigerated and analysed the day after) 2016), according to a validated method (ABL study no. 15342; WIL project no. 511297). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature was also determined (highest and lowest concentration) on one occasion.The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
Untreated females were mated at the supplier and were at Day 0 or 1 post coitum on arrival at the test facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
Rats were treated for 14 days between days 6 to 20 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose over the complete treatment period
Duration of test:
23 days including mating of females at supplier and necrospy.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection was based on a 28-day repeated dose toxicity study, (Wil project 491807), a dose range finding study (WIL project 491831) and an acute oral toxicity study (NOTOX project 491805).
Maternal examinations:
Parental animals: Observations and examinationsMORTALITY/VIABILITY OBSERVATIONS: Yes - Time schedule: At least twice daily (early morning/late afternoon)DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily 1 hour after dosing (+/- 30 minutes) from Day 2 post-coitum onwards up to the day prior to necropsy. At least once daily 1 hour after dosing (± 30 minutes) from Day 2 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. BODY WEIGHT: Yes- Time schedule for examinations: Females were weighed on Days 2, 6, 9, 12, 15, 18 and 21 post-coitumFOOD CONSUMPTION: YesFood consumption was measured on Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitumWATER CONSUMPTION: No, subjective appraisal will be maintained during the study, but no quantitative assessment was introduced as no treatment related effect was suspected.NECROPSY EXAMINATIONS: All animals were sacrificed on Day 21 post-coitum using an oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).The stomach and the eyes of all animals were dissected and examined. The stomach was fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). The eyes were fixed in modified Davidson’s solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial; all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:-The number of corpora lutea.-The weight of the (gravid) uterus.-The number and distribution of live and dead fetuses.-The number and distribution of embryo-fetal deaths-The weight of each fetus.-The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).-Externally visible macroscopic fetal abnormalities. In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).EXTERNAL: All viable fetuses were examined, weighed and sexed. All live fetuses were euthanized by administration of 0.5 mL of sodium pentobarbital (Euthasol(R) 20 %, AST Farma B.V., Oudewater) into the oral cavity using a small metal or plastic feeding tube. For late resoprtions a gross external examination was performed. Late resorptions were discarded. VISCERAL (INTERNAL): Approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was confirmed by internal examination.The heads were removed from these fetuses and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination. After examination, the tissues without variations or malformations were discarded. Tissues with variations or malformations were stored in 10% buffered formalin.Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded. The carcasses were processed and stained with Alizarin Red S (as described below (SKELETAL)), but not examined in first instance.SKELETAL:From the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96 % aqueous ethanol, macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands). Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.-The Fisher Exact-test was applied to frequency data.-The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.-Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection was noted during treatment for eight females at 40 mg/kg and six females at 120 mg/kg. As it was seen for 1-2 days only and also occurred during pretest for some animals, it was not considered toxicologically relevant. Other signs of toxicity seen in the treatment groups (i.e. uncoordinated movements, hunched posture and rales) were observed for single animals for up to 3 days only, and therefore not considered to be related to treatment.Incidental findings that were noted included alopecia, scabs, scales, erythema and salivation. As these findings occurred within the range of background findings observed in rats of this age and strain under the conditions in this study and were not consistent over time, they were considered not to be toxicologically significant.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related microscopic findings:Stomach (Glandular):- Secretory depletion was present in 6/10 females treated at 120 mg/kg/day up to slight degree.- Inflammatory cell infiltrate, granulocytic was present at increased incidence and severity in 7/10 females treated at 120 mg/kg/day up to slight degree, compared to a single female at a minimal or slight degree in the remaining dose groups.The remainder of the recorded microscopic findings, including those in the eyes, were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Relatively high mean value for post-implantation loss (7.4% per litter) was noted at 10 mg/kg, compared to the control group and the other treatment groups. This was primarily the result of the high values in two individual females. The post-implantation values for the other females in the 10 mg/kg group were in the same range at noted for the other groups. As no dose-dependent relationship was observed, this was considered as incidental and not treatment-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female in the 40 mg/kg group was non-pregnant. All pregnant females had litters with viable fetuses.
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects: yes. Remark: some microscopic, none toxicologically relevant effects were observed. Details on maternal toxic effects: One female was non-pregnant (at 40 mg/kg bw) All females had litters with viable fetuses. In comparison to the control group the 10 mg/kg bw group had a relatively high mean value for post-implantation loss (7.4 % per litter). This was due to high values in two individual females. No dose-dependent relationship was observed, this was considered as incidental. MACRO- AND MICRO-SCOPIC EXAMINATION: Macroscopic examination at necropsy revealed no treatment-related findings. One female at 120 mg/kg showed an interrupted left horn of the uterus. This was considered as incidental and not toxicologically significant. One control female showed alopecia. There were test item-related microscopic findings: Stomach (Glandular):-Secretory depletion was present in 6/10 females treated at 120 mg/kg/day up to slight degree.-Inflammatory cell infiltrate, granulocytic was present at increased incidence and severity in 7/10 females treated at 120 mg/kg/day up to slight degree, compared to a single female at a minimal or slight degree in the remaining dose groups. The remainder of the recorded microscopic findings, including those in the eyes, were within the range of background pathology encountered in rats of this age and strain. There was no test item‑related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to 120 mg/kg.Mean combined (male and female) fetal body weights were 5.0, 5.1, 5.2 and 5.2 for the control, 10, 40 and 120 mg/kg groups, respectively.The statistically significant changes noted for female fetal body weights at 40 and 120 mg/kg were not considered toxicologically relevant as the increase was very slight, did not show a dose response relationship, and all values were within normal limits.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 120 mg/kg.Skeletal malformations noted in this study were bent limb bones (Group 2 fetus A035-01 and control fetuses A013-01 and -03 of the same litter) and a rib anomaly (Group 2 fetus A043-01). Due to the single occurrence at the low dose level and/or occurrence in control fetuses, these malformations were not considered to be treatment related.The variation of 14th rudimentary ribs was noted at an incidence of 71.8%, 54.1%, 50.4% and 48.0% per litter in Groups 1, 2, 3 and 4, respectively. Compared with the current control incidence, the incidences in Groups 3 and 4 were statistically significantly reduced. From historical control data it appears that the incidence of 14th rudimentary ribs ranged from 19.0% to 72.0% per litter. In comparison with this, the current control value lay at the maximum historical control value, while thevalues of all treated groups lay closer the historical mean value of 44.1% per litter. Taken this into account, it was considered that the striking group distribution of 14th rudimentary ribs in this study occurred by chance and was not related to treatment.Other skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend and/or occurred infrequently.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from a fetus at 40 mg/kg (A061-02), which had situs inversus whereby all organs were laterally transposed, there were no other viscerally malformed fetuses observed.The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, discolored adrenal and liver, partially undescended thymus horn(s), convoluted ureter and retroesophageal right subclavian artery. These variations occurred at low incidences and/or in the absence of a dose-related incidence trend and therefore were not considered to be treatment related.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
There were no treatment related effects on any litter size for any test group as compared to the control. All litter sizes were 11 +/- 0.5. Sex ratio was unaffected by treatment and were ~50:50 for all treatment and control groups. There were no effects on foetal bodyweight and these were 5.0 +/- 0.2 g. MORPHOLOGICAL EXAMINATIONS: EXTERNAL: There were no treatment related effects on external morphology following treatment up to 120 mg/kg. The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group. There were no treatment related effects on external morphology following treatment up to 120 mg/kg. The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group. No dose-dependent relationship was observed, this was considered as incidental. VISCERAL: There were no treatment related effects on visceral morphology following treatment up to 120 mg/kg. Apart from a fetus at 40 mg/kg (A061-02), which had situs inversus whereby all organs were laterally transposed, there were no other viscerally malformed fetuses observed. The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, discolored adrenal and liver, partially undescended thymus horn(s), convoluted ureter and retroesophageal right subclavian artery. These variations occurred at low incidences and/or in the absence of a dose-related incidence trend and therefore were not considered to be treatment related. SKELETAL: There were no treatment related effects on skeletal morphology following treatment up to 120 mg/kg. Skeletal malformations noted in this study were bent limb bones (Group 2 fetus A035-01 and control fetuses A013-01 and -03 of the same litter) and a rib anomaly (Group 2 fetus A043-01). Due to the single occurrence at the low dose level and/or occurrence in control fetuses, these malformations were not considered to be treatment related. The variation of 14th rudimentary ribs was noted at an incidence of 71.8%, 54.1%, 50.4% and 48.0% per litter in Groups 1, 2, 3 and 4, respectively. Compared with the current control incidence, the incidences in Groups 3 and 4 were statistically significantly reduced. From historical control data it appears that the incidence of 14th rudimentary ribs ranged from 19.0% to 72.0% per litter. In comparison with this, the current control value lay at the maximum historical control value, while the values of all treated groups lay closer the historical mean value of 44.1% per litter. Taken this into account, it was considered that the striking group distribution of 14th rudimentary ribs in this study occurred by chance and was not related to treatment. Other skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend and/or occurred infrequently. CONCLUSIVE REMARK No toxicologicallly relevant effects were observe.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No developmental toxicity observed
Abnormalities:
not specified
Developmental effects observed:
no
Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for hexahydro-1,3,5-trimethyl-1,3,5-triazine was established as being 40 mg/kg for local toxicity based on microscopic findings in the stomach. The maternal NOAEL for systemic toxicity was at least 120 mg/kg.The developmental NOAEL was established as being at least 120 mg/kg.
Executive summary:

Study outline

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 10, 40 and 120 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on two days during treatment were analyzed for accuracy, homogeneity and/or stability.

 

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortemand external, thoracic and abdominal macroscopic findings were recorded. Stomach and eyes were collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach and eyes from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

 


RESULTS

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

 

Maternal findings

No maternal toxicity was observedin the 10, 40 and 120 mg/kg groups.

 

There were test item-related microscopic findings in the stomach (glandular) at 120 mg/kg. Findings consisted of secretory depletion and inflammatory cell infiltrate, both up to slight degree.

Developmental findings

No developmental toxicity was observed in the 10, 40 and 120 mg/kg groups.

 

CONCLUSION

 

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Effect Level (NOEL) for Hexahydro-1,3,5-trimethyl-1,3,5-triazine was establishedas being 40 mg/kg for local toxicity based on microscopic findings in the stomach. The maternal NOAEL for systemic toxicity was at least 120 mg/kg.

 

The developmental toxicity was established as being over or equal to 120 mg/kg (the highest dose tested).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2018 - 20 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidlines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
Temporary Deviations from the maximum level of target humidity occure but it did not impact the integrity of the study
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
yes
Remarks:
Temporary Deviations from the maximum level of target humidity occure but it did not impact the integrity of the study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
yes
Remarks:
Temporary Deviations from the maximum level of target humidity occure but it did not impact the integrity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
 TEST ANIMALS
- Source: On 28 Sep 2018, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France).
- Females (if applicable) nulliparous and non-pregnant: Time mated female were used, Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: Females: 3.035 - 4.307 kg.
- Fasting period before study: No
- Housing: Individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles .
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: 2 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21 ºC
- Humidity (%): 56 to 69 %
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 28th Sept 2018 To Day 29 post-coitum
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Propylene Glycol 400 was selected as suitable vehicle based on trial preparation performed at the test facility according to SOP-TSO-B-018.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared prior to treatment and used within 6 hours after preparation. 


VEHICLE
- Justification for use and choice of vehicle (if other than water): Polyethylene glycol 400
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): Concentration range 100 to 600 mg/mL,
- Lot/batch no. (if required): No specified
- Purity: not specified but specific gravity was reported as 1.125.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20146537).
Details on mating procedure:
Mating procedure was not disclosed but the females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
Day 6 to Day 28 post-coitum
Frequency of treatment:
Once daily
Duration of test:
23 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
8 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with the test item by oral gavage at a dose volume of 0.5 mL/kg body weight at 0, 5, 15 and 50 mg/kg bw/day along with the concurrent vehicle control.
Based on the findings, 0, 2, 8 and 25 mg/kg/day dose levels at a dose volume of 0.5 mL/kg body weight were selected for the definitive study.

- Rationale for animal assignment (if not random): N/A
 - Other:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.
The dosing formulations were stirred continuously during dose administration.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule: during clinical observations

- Cage side observations checked in table [No.?] Not stated
 


DETAILED CLINICAL OBSERVATIONS: Yes 

- Time schedule: All rabbits were observed for morbidity and mortality twice daily, i.e. once in the morning and once in the afternoon. Rabbits were observed for clinical signs during the treatment period of the study: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy.


 BODY WEIGHT: Yes 
 - Time schedule for examinations: Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes 
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: On days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum. .

 WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: monitored on regular basis throughout the study by visual inspection of the water bottles/containers.
 
 POST-MORTEM EXAMINATIONS: Yes
 - Sacrifice on gestation day # GD 29
- Organs examined: Yes, external and visceral organs

 OTHER: The following data were recorded i.e.
* The number of corpora lutea.
* The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
* The number of implantation sites.
* The number and distribution of live and dead fetuses.
* The number and distribution of embryo-fetal deaths.
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

 - Other: Yes, gross evaluation of placenta, pregnancy status
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:
- Other: The following litter data were recorded:
* Total number of fetuses
* Number of live fetuses
* Number of dead fetuses
* Individual fetal body weight
* Fetus sex
Statistics:
STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. This included Dunnett-test (many-to-one-t-test), Steel-test (many-to-one rank test), Kruskal-Wallis nonparametric ANOVA test and Mann Whitney test.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Indices including; pre and post-implantation loss (%) and fetuses viability index (%).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Across all test item treated groups, severity/incidence of reduced faeces production appeared increased compared to the control group, but without a clear dose-related trend. This finding was considered to be related to the temporarily lower food intake recorded across the dose groups and as such was considered to be related to treatment with the test item.
At 25 mg/kg, piloerection was recorded for 3/22 animals (nos. 77, 82 and 84) between post-coitum Days 7-26, along with lean appearance for one of these animals (no. 77) between post-coitum Days 16-26.
Other clinical signs observed during the study among animals surviving until scheduled necropsy were considered to be unrelated to treatment as their incidence occurred in the absence of a clear dose-related trend, were recorded at low incidence in a few animals only and/or occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study.
Female no. 33 (2 mg/kg) that delivered her offspring on the scheduled day of necropsy showed blood in the dosing tube on post-coitum Day 25, followed by laboured respiration, gasping, pale and lean appearance between post-coitum Days 25-28. This animal also showed weight loss from post-coitum Day 24 onwards (13% compared to post-coitum Day 6). It was therefore considered likely that a gavage-related incident had resulted indirectly to an early delivery.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three cases of mortality occurred that were considered not related to treatment. One Control female (no. 5) was sacrificed for ethical reasons on post-coitum Day 18. This animal appeared inactive/lethargic, had a pale appearance and had significant blood loss (red fluid on manure tray). During abdominal palpation, the abdomen appeared soft and a significant amount of blood was expelled through the vagina. At necropsy, this animal showed reddish/watery-cloudy contents in the left horn of the uterus. These findings were most likely related to abortion. Two females at 2 mg/kg (nos. 26 and 41) were sacrificed for ethical reasons on post-coitum Day 22 or 18, respectively. Female no. 41 appeared in a moribund state and showed rales and gasping after dosing, during which a minor amount of blood was noted on the outside of the dosing tube and on the nose. Necropsy showed that the lungs were not collapsed and had many dark red foci. These observations indicate that this was to be considered a gavage-related death. Female no. 26 did not consume food since post-coitum Day 6, showed weight loss (9% compared to post-coitum Day 6) and showed piloerection between post-coitum Day 15-21 and lean appearance on post-coitum Day 21. This animal was subsequently sacrificed for ethical reasons. No findings were noted at necropsy for this animal. As none of the other animals at this dose group nor at higher dose levels showed similar findings, this was considered to be an incidental occurrence unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg, a slight statistically significant mean body weight loss was recorded over post-coitum Days 6-9 (on average -1% compared to post-coitum Day 6 mean). On an individual animal basis, the weight loss varied from -1 to -6% in 13/22 animals. Mean body weight gain remained statistically significantly lower on Day 12 post-coitum (1% weight gain vs. 5% in the control group over Days 6-12), but recovered to levels similar to control means from approximately post-coitum Day 18 onwards.
One female at 25 mg/kg (no. 73) lost 9% weight between post-coitum Days 27 and 29. This was considered to be related to this animal having 11 dead fetuses. This animal had lost 24% weight when corrected for gravid uterus weight (i.e. exceeding the historical control data range ). One other female at this dose (no. 84) had a body weight being lower than Day 6 body weight throughout the treatment period (ranging from -3 to-11% of the body weight on Day 6). This animal had a normal number of fetuses. This animal had lost 16% weight when corrected for gravid uterus weight (i.e. exceeding the historical control data range1).
Mean body weight gain corrected for gravid uterus remained within the normal range at all dose levels. Body weight and body weight gain at 2 and 8 mg/kg was considered not affected by treatment with the test item
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg, food consumption before and after correction for body weight was statistically significantly lower than control means between post-coitum Day 6-12. Mean (relative) food consumption recovered to control mean levels around post-coitum Day 15-18.
At 2 and 8 mg/kg, food consumption after correction for body weight was also slightly lower than controls, achieving statistical significance on several occasions between post-coitum Days 9-18, but without a clear dose-related trend. Mean food consumption recovered to control mean levels around post-coitum Day 18-21.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No adverse maternal findings were recorded up to a dose of 25 mg/kg.
At 25 mg/kg, mean body weight and food intake were temporarily reduced during the first week of treatment, but recovered as the treatment period progressed. At 2 and 8 mg/kg, food consumption was also slightly reduced during the first two weeks of treatment, without a clear dose-related trend, but also recovered as the treatment period progressed. This was accompanied by an increased severity/incidence of reduced faeces production across all test-item treated group, but without a clear dose-related trend. Piloerection and/or lean appearance was recorded for a few animals at 25 mg/kg during the treatment period. Given the temporary/incidental nature of these findings, these were considered not adverse.
Mean body weight gain corrected for gravid uterus remained within the normal range at all dose levels and no treatment-related changes were noted at macroscopic examination. The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
The number of gravid females in the control group (17/22, i.e. 77.3%) was slightly outside the historical control range (P5-P95: 78.9-100%, n=26 studies). The number of gravid rabbits was however considered sufficient to adequately interpret the study results, and is within guideline requirements (par 11, OECD414: “Each test and control group should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate”).
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
No adverse maternal findings were recorded up to a dose of 25 mg/kg.
At 25 mg/kg, mean body weight and food intake were temporarily reduced during the first week of treatment, but recovered as the treatment period progressed. At 2 and 8 mg/kg, food consumption was also slightly reduced during the first two weeks of treatment, without a clear dose-related trend, but also recovered as the treatment period progressed. This was accompanied by an increased severity/incidence of reduced faeces production across all test-item treated group, but without a clear dose-related trend. Piloerection and/or lean appearance was recorded for a few animals at 25 mg/kg during the treatment period. Given the temporary/incidental nature of these findings, these were considered not adverse.
Mean body weight gain corrected for gravid uterus remained within the normal range at all dose levels and no treatment-related changes were noted at macroscopic examination in all organs including thyroid gland. The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
The number of gravid females in the control group (17/22, i.e. 77.3%) was slightly outside the historical control range (P5-P95: 78.9-100%, n=26 studies). The number of gravid rabbits was however considered sufficient to adequately interpret the study results, and is within guideline requirements (par 11, OECD 414: “Each test and control group should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate”).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment related effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two external malformations were observed ithe study at scheduled necropsy. Fetus A012-02 and A013-09 both had an omphalocele and as both these fetuses were control fetuses this was considered to be spontaneous in origin. In addition, Group 2 fetus A033-02 that was delivered on Day 29 had hyperextension of a hindlimb, which was considered to be a chance finding. External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 25 mg/kg, an increased incidence of unossified metacarpals and tarsals (skeletal variations) was recorded. For metacarpals, incidences were 2.9% and 9.5% per litter in the control and 25 mg/kg group, respectively. Incidences were within the historical control range (0.0% - 10.1% per litter). However, the high dose incidence was slightly above the p95 value of 9.4% per litter. For tarsals, incidences were 0.9% and 2.5% per litter in the control and 25 mg/kg group, respectively. The incidence at the high dose was not significantly different from the control group. The incidence at 25 mg/kg exceeded the historical control range (0.0% - 2.1% per litter; p95 value of 1.9% per litter).
The incidence of these skeletal variations at the high dose level was mainly caused by two litters (A082 and A084). Half or more fetuses of these two litters had unossified metacarpals, and four fetuses of litter A084 had unossified tarsals. Most fetuses in both these litters also had a body weight that was below the historical control range (minimum value: 36.4 or 36.0 for male and female fetuses, respectively) and most of these fetuses also had unossified metacarpals and/or tarsals.
All other variations noted were considered not treatment-related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in control fetuses only.
There were no treatment-related skeletal malformations following treatment up to 25 mg/kg/day. Only one fetus in Group 4 (A068-06) had a skeletal malformation consisting of an anomaly of the vertebral column. As in two control fetuses (A012-5 and A018-9) a vertebral anomaly was observed as well, this was not considered to be treatment-related. Besides a control fetus with costal cartilage anomaly (no. A003-12), there were no other skeletal malformations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 25 mg/kg/day. Three visceral malformations were observed in this study. Group 4 fetus A084-09 had a small right eye, Group 3 fetus A055-03 had a diverticulum of the intestine and Group 2 fetus A034-02 had a cardiovascular malformation consisting of an atrial septum defect and dilated aortic arch. The single occurrence and group distribution of these malformations does not indicate a treatment relationship and therefore all were considered to be spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Fetal Examinations
At 25 mg/kg, a non-adverse increased incidence of metacarpals/metatarsals and tarsals (skeletal variations) occurred (9.5% vs. 2.9% per litter in the control group for metacarpals/metatarsals and 2.5% vs. 0.9% in the control group for tarsals). The incidences at 25 mg/kg slightly exceeded the 95% confidence interval of the historical control data range. These slightly higher incidences were mainly caused by two litters of which half or more fetuses had unossified metacarpals and/or several fetuses has unossified tarsals. It was considered that the higher incidence of unossified metacarpals and tarsals (main ossification parameters) in the 25 mg/kg group was a result of the low fetal body weights of these two litters. This higher incidence of unossified metacarpals/tarsals along with lower body weights was confined to these two litters and this was not recorded for other litters of this group. As these variations may be considered to represent a transient delay in development (Ref. 5), these were considered not adverse.
There were no treatment-related effects in external or visceral morphology.
No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio and fetal body weights).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed under the condition of the study
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
Treatment related:
no

Table 3. Clinical Signs Summary

Dose group

Sign (max. grade) Location

Treatment

Week: 1………………………………………4.

Day:    12345671234567123456712

 

 

 

 

 

 

 

 

 

Group 1 (control)

Behaviour

Lethargy (3)

G:         …………………….2…………………

%:         …………………….0…………………

Skin/fur

Pale (3)

G:         …………………….1…………………

%:         …………………….0…………………

Alopecia (3)

G:         11111111111111111111111

%:         00000000000000000000000

Scabs (3) (Mouth)

 

G:         …………..11111111………………

%:         …………..00000000………………

Scabs (3)

(Nose)

 

G:         11111111…………………………..

%:         00000000………………………….

Scabs (3) (Flews)

 

G:         11111111111111111111111

%:         00000000000000000000000

Secretion / excretion
Faeces production reduced (3)

 

G:         ……………11111111111111111

%:         ……………11155544433322233

Red fluid on manure tray (3)

 

G:         …………………….3…………………….

%:         …………………….0…………………….

Various Overgrown (3)

(Upper incisors)

 

G:         11111111…………………………….

%:         00000000…………………………….

Missing (1)

(Upper incisors)

G:         11111111…………………………….

%:         00000000…………………………….

 

 

 

 

 

 

 

 

 

 

 

Group 2 (2 mg/kg)

Breathing
Laboured respiration (3)

G:         ……………………..………………111

%:         ……………………..………………111

Rales (3)

G:         …………………….3……………………

%:         …………………….0…………………..

Gasping (1)

 

G:         …………………….1………………1…

%:         …………………….0………………1…

Skin / fur Swelling (4)

(Flews)

 

G:         11……………….111…………………

%:         00……………….000...………………

Pale (3)

G:         ……………………..…………………1..1

%:         ……………………..…………………1..1

Piloerection (1)

 

G:         …………1111111111111………….

%:         …………0000011111110………….

Alopecia (3)

 

G:         …………………….………..111111111

%:         …………………….………..001111111

Scars (3) (Flews)

 

G:         …………………….………………….1111

%:         …………………….………………….1111

Scabs (3)

(Nose)

 

G:         11122111111111111……………….

%:         00000000000000000……………….

Scabs (3) (Flews)

G:         …1111111111111111111111111

%:         …0000001111111111111111111

Wound (3) (Flews)

G:         …………..1…………………………………

%:         …………..0………………………………….

Secretion / excretion
Faeces production reduced (3)

G:         ….22211133333311111111

%:         ….11133366656644444433

Various Moribund (1)

G:          …………………..1………………….

%:          …………………..0………………….

Lean (1)

G:          ………………………..1…………….1

%:          ………………………..0…………….1

Broken (1) (Lower incisors)

G:          11111111111111……………….

%:          00000000000000……………….

 

 

 

 

Group 3 (8 mg/kg)

Skin / fur
General erythema (4) (Throat region)

G:          ………………………………………….1

%:          ………………………………………….0

Piloerection (1)

 

G:          ……111………………………………..

%:          ……000………………………………..

Scabs (3) (Nose)

 

G:         1111111111111111111……….

%:         0000000000000000000……….

Scabs (3)

(Flews)

 

G:          ………………111111111111111

%:          ………………000000000000000

Scabs (3) (Throat region)

G:          ……………………………………..111

%:          ……………………………………..000

Wound (3) (Flews)

G:          ……………1…………………………….

%:          ……………0…………………………….

Secretion / excretion Faeces production reduced (3)

G:          ……….2222222222221111111

%:          ……….2222266666444433322

Various Overgrown (3) (Upper incisors)

G:          …………………………………….1111

%:          …………………………………….0000

 

 

 

 

 

 

 

 

Group 4 (25 mg/kg)

Skin / fur Swelling (4) (Flews)

G:          ……111…………………….11……….

%:          ……000…………………….00……….

Piloerection (1)

G:          . 1111111111111111111111..

%:          . 0000000001111111111100..

Alopecia (3)

G:          ……………………………………11111

%:          ……………………………………00000

Scars (3) (Flews)

G:          …………………………………111111

%:          …………………………………000000

Scabs (3) (Flews)

G:          . . . . 111111111111. . . . . . . ..

%:          . . . . 011000000000. . . . . . . . .

Wound (3) (Flews)

G:          . . . . 11. . . . . . . . . . 1. . . . . . . .

%:          . . . . 00. . . . . . . . . . 0. . . . . . . .

Red staining (1) (Vagina)

Secretion / excretion

G:          . . . . . . . . . . . . . . . . 1. . . . . . . .

%:          . . . . . . . . . . . . . . . 0. . . . . . . .

Faeces production reduced (3)

G:          . 113321112222221111111111

%:          . 027775555555555533322244

Red fluid on manure tray (3)

G:          . . . . . . . . . . . . . . . . . .. 2111 . . . .

%:          . . . . . . . . . . . . . . . . . . 0000 . . . .

Various Lean (1)

G:          . . . . . . . . 111111111111111 . . .

%:          . . . . . . . . .00000000000000 . . .

Broken (1)

Upper incisors

G:          . . . 11111111111111111111111

%:          . . . 00000000000000000000000

G: Median value of the highest individual daily grades
%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,..., A=more than 95%) .: Observation performed, sign not present

Table 4. Mean Body Weight (g) and % Weight Gain

Post Coitum Day

Group 1 Control

 

Group 2

2 mg/kg

Group 3

8 mg/kg

Group 4

25mg/kg

Weight gain

% Gain

Weight gain

% Gain

Weight gain

% Gain

Weight gain

% Gain

Day 6

3557

0

3528

0

3542

0

3567

0

Day 9

3665

3

3606

2

3602

2

3512

-1**

Day 12

3727

5

3652

4

3654

3

3612

1**

Day 15

3774

6

3724

6

3696

4

3682

3

Day 18

3822

7

3766

7

37-9

5

3719

5

Day 21

3826

8

3762

7

3757

6

3758

6

Day 24

3885

10

3750

10

3817

8

3814

7

Day 27

3913

11

3881

11

3866

9

3866

9

Day 39

3944

12

3941

12

3903

10

3881

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

Table 5. Mean Food Consumption(g/animal/day)and Relative Food Consumption (g/animal/day)

Post Coitum Day

Group 1 Control

 

Group 2

2 mg/kg

Group 3

8 mg/kg

Group 4

25mg/kg

Food consumption

Relative food consumption

Food consumption

Relative food consumption

Food consumption

Relative food consumption

Food consumption

Relative food consumption

6 -9

148

40

122

33

121

33

60**

17**

9 - 12

139

37

106*

29*

109*

30

102**

28**

12 - 15

101

27

68*

18*

65*

17*

84

23

15 - 18

103

27

88

23

72*

19*

93

25

18 - 21

107

30

96

27

102

28

123

44

21 - 24

111

29

100

28

118

31

121

31

24- 27

79

20

90

23

88

23

90

23

27 - 29

77

20

90

24

90

23

86

22

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

Table 6. Summary of Macroscopic Findings

Post Coitum

Group 1 Control

Group 2

2 mg/kg

Group 3

8 mg/kg

Group 4

25 mg/kg

Animals examined

22

22

22

22

Animals without findings

20

18

20

19

Animals affected

2

4

2

3

General observation

Early delivery

0

1

0

0

Lungs Not collapsed

Focus/foci

0

0

1

3

0

0

0

0

Uterus Content

1

0

0

0

Skin

Alopecia

Scab formation

Scar(s)

 

0

0

1

 

0

0

0

 

0

1

0

 

1

0

1

Bone

teeth, upper incisor: right side, overgrown

Broken

 

0

 

0

 

0

 

0

 

1

 

0

 

0

 

1

Broken

1

0

0

0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

NOTE: Further details on study results ia attached in the background data

Conclusions:
The substance is not considered a developmental toxicant under the conditions of the study and does not meet the the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).
Executive summary:

OECD 414 - (2019): The potential of Hexahydro-1,3,5-trimethyl-1,3,5-triazine to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, at dosages of 0, 2, 8, 25 mg/kg/day in polyethylene glycol 400.  In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

In maternal animals, no adverse maternal findings were recorded up to a dose of 25 mg/kg. At 25 mg/kg, mean body weight and food intake were temporarily reduced during the first week of treatment, but recovered as the treatment period progressed. At 2 and 8 mg/kg, food consumption was also slightly reduced during the first two weeks of treatment, without a clear dose-related trend, but also recovered as the treatment period progressed. This was accompanied by an increased severity/incidence of reduced faeces production across all test-item treated group, but without a clear dose-related trend. Piloerection and/or lean appearance was recorded for a few animals at 25 mg/kg during the treatment period. Given the temporary/incidental nature of these findings, these were considered not adverse.

Mean body weight gain corrected for gravid uterus remained within the normal range at all dose levels and no treatment-related changes were noted at macroscopic examination. The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.  The number of gravid females in the control group (17/22, i.e. 77.3%) was slightly outside the historical control range (P5-P95: 78.9-100%, n=26 studies). The number of gravid rabbits was however considered sufficient to adequately interpret the study results, and is within guideline requirements (par 11, OECD 414: “Each test and control group should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate”).

For the foetus, at 25 mg/kg, a non-adverse increased incidence of metacarpals/metatarsals and tarsals (skeletal variations) occurred (9.5% vs. 2.9% per litter in the control group for metacarpals/metatarsals and 2.5% vs. 0.9% in the control group for tarsals).  The incidences at 25 mg/kg slightly exceeded the 95% confidence interval of the historical control data range. These slightly higher incidences were mainly caused by two litters of which half or more fetuses had unossified metacarpals and/or several fetuses has unossified tarsals. It was considered that the higher incidence of unossified metacarpals and tarsals (main ossification parameters) in the 25 mg/kg group was a result of the low fetal body weights of these two litters.  This higher incidence of unossified metacarpals/tarsals along with lower body weights was confined to these two litters and this was not recorded for other litters of this group. As these variations may be considered to represent a transient delay in development (Ref. 5), these were considered not adverse.

There were no treatment-related effects in external or visceral morphology.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio and fetal body weights).

In conclusion, based on the above findings, under the test conditions employed in the study, it was concluded that No - Observed - Effect Level (NOEL) for: Maternal toxicity and fetal developmental toxicity is 25 mg/kg/day. Therefore, the  substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The data-set for effects on fertility is consistent with data requirements under Annexes VII to X of REACH, and all studies are considered reliable and relevant for endpoint conclusions.
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

Based on the results of all reproductive and developmental studies conducted on the substance, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 30 mg/k bw//day

Developmental NOAEL: ≥100 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

There was no evidence of reproductive or developmental toxicity that requires analysis of the mode of action of the substance, nor any relevance to humans.

Justification for classification or non-classification

Based on the criteria set in Regulation (EC) No 1272/2008, hexahydro-1,3,5 -trimethyl-1,3,5 -triazine is not classified for reproductive or developmental toxicity.