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Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexahydro-1,3,5-Trimethyl-S-Triazine- Substance type: Clear pale yellow liquid- Physical state: liquid- Stability under test conditions: Stable- Storage condition of test material: At room temperature in the dark

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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