Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key 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)
Deviations:
no
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: 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
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
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
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 GAINMean body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.FOOD CONSUMPTIONFood 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 WEIGHTSNo 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 PATHOLOGYAt 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-NEOPLASTICTreatment-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 3/5 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.
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
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
Conclusions:
Treatment with Hexahydro-1,3,5-Trimethyl-S-Triazine by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity at 100 mg/kg body weight/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 Levels (NOAEL) were derived:Parental NOAEL: 30 mg/kg/dayReproductive NOAEL: ≥100 mg/kg/dayDevelopmental NOAEL: ≥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:
subacute
Species:
rat
Quality of whole database:
The database consists of one reliable study (Klimisch 1) study which is suitable for completing this endpoint. Whilst it is acknowledged that the available OECD 422 “Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test” (van Tuyl, 2010) does not address all aspects of reproduction that can be derived from an OECD 416 study, the available study is sufficient in order to conclude that no reproductive toxicity was observed at any dose level tested. A waiving argument has been proposed for further reproductive toxicity endpoints as the registrant believes that instead of recommending that further animal testing be conducted, consideration should be given to the hazardous properties of MMA triazine, namely it is a corrosive and sensitising substance and this is the leading health effect.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

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)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key 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):
One male at 100 mg/kg bw/d (no.33) 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):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Eyes (females): Degeneration of the retina (atrophy) was recorded in 3/5 females (minimal-sight)
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 examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg bw/d, 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 bw/d. However, at this singlr occurrence and without microscopic correlate it was not considered to be toxicologically significant.For the male at 100 mg/kg bw/d (no.33) that dies on the day of scheduled necropsy, incomplete exsanguination was noted. One female (no. 42) of the control group showed one dead fetus in the right uterus horn. Other incidental findings are not summarised. The incidenced of all gross pathological findings were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence. All necropsy findings were considered to be of no toxicological significance.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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
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 3/5 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:
Treatment with Hexahydro-1,3,5-Trimethyl-S-Triazine by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity at 100 mg/kg body weight/day. No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg body weight/day. Based on the results, the following No Observed Adverse Effect Levels (NOAEL) were derived:Parental NOAEL: 30 mg/kg/dayReproductive NOAEL: ≥100 mg/kg/dayDevelopmental 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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): 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 observedDetails 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):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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:
Embryotoxic / teratogenic effects:no effectsDetails 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 fetl 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 REMARKNo 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 examinationpost-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 observedin 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).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database consists of two reliable (Klimisch 1) studies, one for developmental screening in rats (OECD 422) and the prenatal developmental toxicity in rats (OECD 414). The data presented meet the requirements of REACH Annexes VII to X for this endpoint and the OECD 414 was conducted following a final decision on compliance check by ECHA.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

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.