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Administrative data

Description of key information

28-day NOAEL (parental, rat) = 30 mg/kg bw/d, OECD 422, van Tuyl (2010)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
OECD 422 Combined 28-day repeated dose toxicity study with the reproduction/developmental screening test
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 oral 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.- Dose volume: 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%.
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
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
Observations and examinations performed and frequency:
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.OPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: 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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes All 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.
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.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
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.HAEMATOLOGYMinor statistically significant differences arising between controls and animals receiving 100 mg/kg (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 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 CHEMISTRYAt 100 mg/kg, total protein and albumin were slightly decreased for both sexes.Any statistically significant changes at 30 mg/kg (bile acids for males) and at 100 mg/kg (chloride for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution or remained within the range considered normal for rats of this age and strain.The increased values for bile acids (not statistical significant) noted for females of all dose groups were considered to have occurred by chance and to be of no toxicological relevance as these changes were caused by individual animals only.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.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.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
other: actual ingested
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
eye
Organ:
retina
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Conclusions:
Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.At 100 mg/kg body weight/day, parental toxicity consisted of clinical signs (salivation, rales and/or piloerection),slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge and eyes (females only: degeneration of the retina). No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, body weight, food consumption, and organ weights) at 100 mg/kg.No parental toxicity was observedat 10 and 30 mg/kg/day. Based on these results, the following No Observed Adverse Effect Level (NOAEL) was derived:Parental NOAEL:            30 mg/kg/day
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database consists of one reliable study (Klimisch 1) which is suitable for completing this endpoint.It is considered that a “sub-chronic toxicity study (90 day)” as laid down in Annex IX, Section 8.6.2 of the REACH Regulation is not warranted in this case. This is because the available study is a reliable short term toxicity study that has already classified the substance as STOT-RE2 (Xn; R48/22), which meets the criteria for not conducting a 90 day study as Annex IX, 8.6.2 Column 2 – Specific rules for Adaptation from Column 1. For further details, refer to the 90 day study waiver endpoint record.
System:
eye
Organ:
retina

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Treatment with hexahydro-1,3,5-trimethyl-1,3,5-triazine by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/ bw/d resulted in sub-lethal adverse effects at 100 mg/kg bw/d. The only effect considered to be toxicologically significant was degeneration of the retinas observed in female subjects. Based on these results, the repeated dose NOAEL was determined as 30 mg/kg bw/d.

It is considered that a “sub-chronic toxicity study (90 day)” as laid down in Annex IX, Section 8.6.2 of the REACH Regulation is not warranted in this case. This is because the available study is a reliable short term toxicity study that has already classified the substance as STOT-RE2 (Xn; R48/22), which meets the criteria for not conducting a 90 day study as Annex IX, 8.6.2 Column 2 – Specific rules for Adaptation from Column 1. For further details, refer to the 90 day study waiver endpoint record.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This was the only study available for repeated dose via the oral route and meets the requirements of international guidelines and good laboratory practice.

Additional information

Justification for classification or non-classification

According to the criteria for classification and labelling of substances and mixtures (Regulation (EC) No. 1272/2008), hexahydro-1,3,5-trimethyl-1,3,5-triazine is classified as Specific Target Organ Toxicity Category 2 (H373), with eyes as the affected organs.