1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

EC Number:

211-322-8

EC Name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Cas Number:

638-16-4

Molecular formula:

C3H3N3S3

IUPAC Name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: males 355.5-405.2 g, females 208.7-255.0 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 40-75%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26.05.2005 To: 20.07.2005 (not stated in the report, estimation based on 42 treatment and 14 recovery days)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % Carboxymethyl cellulose sodium [dissolved Japanese Pharmacopoeia Carmellose sodium with Japanese Pharmacopoeia injection solvent]

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 1.25 % (w/v), 2.5 % (w/v) and 5 % (w/v)
- Amount of vehicle (if gavage): 5 mL/kg
- Justification for choice of vehicle: test material was confirmed to be hardly soluble in the preliminary test

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg

PREPARATION OF DOSING SOLUTIONS:
A 5 w/v% liquid was prepared by weighing the test material on a balance, after grinding with a mortar, adding small amounts of the solvent medium. This was then serially diluted with the solvent medium.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For measurement of the test material concentration in prepared test specimen, 0.5 ml of the respective prepared test specimen was taken, and the test solution was prepared by diluting appropriately with methanol. Separately, the required amount of the test material was weighed and dissolved in methanol, and standard solution was prepared (1, 2, 5 μg/ml). Test solution and standard solution were measured by high performance liquid chromatography (HPLC), and the concentration was obtained by using the calibration curve created from standard solution.

Duration of treatment / exposure:

males: continuous 42 days from 2 weeks before mating through the longest 2-week mating period to the day before autopsy
females: 2 weeks before mating, mating period until copulation, gestation period, and lactation day 4 (42-48days)
females without delivery: until the day before autopsy date (pregnancy 25 days equivalent)
females of the satellite group: continuous to day 42

Frequency of treatment:

once a day, from 9-12 am

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 (main study)
5 females (satellite control and high-dose group)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: once daily during rearing and recovery period, 2 times daily before and after administration during administration period

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule for males: last day of medical inspection, administration days 7, 14, 21, 28, 35, and 42, for recovery group in addition days 7 and 14
- Time schedule for females: last day of medical inspection, administration days 7, 14, 21, 28, 35, and 42, for the case where observation day falls during delivery, observation was on lactation day 0; other delivery cases were observed between lactation day 0 to 4

BODY WEIGHT: yes
- Time schedule for males and females of the satellite group: on administration days 1, 7, 14, 21, 28, 35, and 42, recovery day 1, 7, and 14, and the day of autopsy (the day after the last administration and recovery day 15).
- Time schedule for females: before confirmation of mating on administration days 1, 7, 14, and 21, after confirmation of mating, pregnancy day 0 (confirmation date of mating), 7, 14, and 20, post partum, nursing day 0 (delivery date) and day 4, and the date of autopsy, measured for females undelivered on pregnancy day 0 (confirmation date of mating), 7, 14, 20, and 26 days equivalent

FOOD INTAKE: yes
- Food intake was calculated from measuring the difference between the feed and the residual feed.
- Food intake for males and females of the satellite group on administration days 1-2, 7-8, 14-15, 29-30, 35-36, and 41-42; days 6-7 and 13-14 of recovery
- Food intake for females before mating, administration days 1-2, 7-8, and 14-15, after confirmation of mating, pregnancy days 0-1, 7-8, 14-15, and 20-21, nursing days 3-4

Sacrifice and pathology:

SACRIFICE
- after 18-22 hours of abstinence from food, followed by lethal exsanguination under pentobarbital sodium anesthesia or blood collection, autopsy was conducted, and hematologic test, biochemical examination of blood, and pathology examination were performed

GROSS NECROPSY
- Macroscopic observation of organs and tissues were performed for all cases at autopsy
- Collection and storage of organs in case of lesions: brain, hypophysectomy, medulla, heart, airway tube, lung (includes bronchial tubes),liver, kidney, asymmetric thymus, spleen, adrenal gland, thyroid, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymidis, ventral prostate, seminal vesicles including coagulating glands, ovaries, uterus, vagina, bladder, mandible lymph nodes, mesenteric lymph nodes, sciatic nerve, femora, and bone marrow

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histological examination was conducted for ovaries, testis, and epididymidis of all cases, iin cases where changes were observed macroscopically, of appropriate test animals, and for organs and tissues other than these of the respective 5 cases, for which hematologic test and biochemical examination of blood were performed, from the control group and 250 mg/kg group of males for autopsy at the completion of administration and of female delivery cases
- Organ weights (actual weight): brain, heart, asymmetric thymus, liver, kidney, spleen, adrenal gland, ovary, and epididymidis

GROSS NECROPSY (offspring)
- among newborn pups, necropsies on dead pups were immediately performed and preserved
- necropsies were performed on all cases of newborn pups after lethal sacrifice by inhalation of ether day 4 of nursing

Other examinations:

- continueous observation of the estrous cycle was conducted until the day before dividing groups and after the start of administration, vaginal smear specimens were prepared every day and the estrous cycle was observed until cohabitation, and mating was confirmed
- estrous cycle was divided into estrus, proestrum, and anestrus, and after the start of administration, the frequency of test animals that changed to other than 4-day interval of estrous cycle was calculated for each group
- histological examination of testis and epididymidis in all cases
- On nursing day 0, the number of live and dead pups were counted by females and males separately, gender and existence or nonexistence of external malformation were observed.

Statistics:

Concerning frequency and fertility index of test animals with change of estrous cycle, Fisher's exact probability test was conducted (significance level: 5%) from histological examination findings of the test material administered groups, a significance test was conducted between the control group with the data sorted out by grade by Mann-Whitney U test (significance level: 5%) and with the total values of positive grades by Fisher’s exact probability one-sided test (significance level: 5%).
For other data, values obtained for each individual or average-value by every litter were made into a specimen and compared within the satellite and other groups. At this point, when the object of analysis was 2 groups, F test was first conducted, and if significant difference was not identified, Student’s t test was conducted. When a significant difference was identified by F test, the Aspin-Welch test was then performed. When the object of analysis was more than 3 groups, a test was conducted on uniformity of dispersion of the respective group by the Bartlett method (significance level: 5%). When the dispersion was uniform, one-way analysis of variance (significance level 5%) was performed, and when significance was identified among groups, multiple comparisons were made by the Dunnett method (significance level 5%).On the other hand, when dispersion in any of the groups was 0 and dispersion was not uniform, the Kruskal-Wall rank order test (significance level: 5%) was performed, and when significance was recognized, multiple comparisons were made by the Dunnett test method (significance level: 5%).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Auricular black spot, purple-black tail tip, tail node, auricular tubercle, scrotum hardening, scrotum nodule were observed in females and males of the 250 mg/kg dose group and seen also in continuous withdrawal.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male (dead on 8th day of administration) and one female (dead on 1st day of nursing) animal of the 250 mg/kg bw/day group died throughout the study. Change in clinical symptoms was not observed before the death of the male case, but since congestion and edema in lung were identified in histopathology, the cause of death seems to be respiratory depression. In female death, incomplete eyelid opening and tabefaction the day after delivery were followed and confirmed by death, and though the stress of delivery could have induced incidence of death, a wide range of necrosis in proximal tubule of cortex of kidney was identified by histopathology.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In regard to body weight trend, remarkable weight gain was identified with males of the 250 mg/kg dose group, the same changes, could be seen with the same group females of the satellite group, but the changes vanished along with withdrawal. Lower feed intake quantity was identified with males of the 250 mg/kg dose group, the same changes, could be seen with the same group females of the satellite group, but the changes vanished along with withdrawal.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

With males of the 250 mg/kg dose group, the hematocrit value with hematologic test was lowered significantly in serum albumin concentration with the biochemical examination of blood.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the completion of the administration period in the test, albumin concentration in the blood in males of the 62.5 and 250 mg/kg dosage groups lowered significantly, compared with the control group; moreover, creatinine concentration in males of the 250 mg/kg dosage group was lowered significantly. At the completion of the recovery period in the test, creatinine concentration in males and females of the 250 mg/kg dosage group, compared with the control group, was lowered significantly. Moreover, triglyceride level in females of the 250 mg/kg dosage group increased significantly compared with the control the group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

With males of the 250 mg/kg dose group, a high frequency of erythrocytes in urine were identified.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At completion of the administration period in the test, body weight on autopsy of males in the 250 mg/kg dosage group, compared with the control group, decreased significantly; relative weights of brain, heart, kidney, and adrenal gland increased significantly, compared with the control group. No significant difference was identified between females in the control group and the TTC administered group.
In the test at the recovery period completion, the actual weight of body weight, liver, and kidney on autopsy in males of the 250 mg/kg dosage group, compared with the control group, lowered significantly, while relative weights of brain, heart, kidney, spleen, adrenal gland, ovary, and epididymidis increased significantly compared with the control group. Actual weight, on autopsy, of body weight and asymmetric thymus in females of the 250 mg/kg dosage group, compared with the control group, decreased significantly, while relative weights of heart and liver, compared with the control group, increased significantly.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the one male of the high dose group found dead, lung showed suppressed involution and edema, and swelling of dark reddish kidney was observed. In the one female of the high dose group found dead, lung and asymmetric thymus showed edema, and swelling and pale color of kidney were observed, pancreas turning to pale color was smaller in size, and bone marrow also turned pale in color.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Multinucleated giant cell in subcutaneous tissue was observed together with granuloma and inflammatory cell infiltration. With histological examination of kidney, very slight edema of the papilla was identified in males of the 62.5 mg/kg dose group and females of the 250 mg/kg dose group (only at the end of recovery period), and lipofuscin of the proximal tubule was identified for both females (very slight to slight) and males (slight to moderate) of the 62.5 mg/kg dose group. With histological examination of adrenal glands in males, diffuse enlargement of cortical cells adhesion was identified with the administration groups above 62.5 mg/kg. Changes described above in the kidney and adrenal gland was also identified in animals after withdrawal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No influence of the test material administration could be identified in the estrous cycle, copulation rate, birth rate, pregnancy period, and delivering rate. In histological examination of testes and epididymidis, seminiferous tubule atrophy and intraductal cell debris in males other than the infertile cases are seen, but they are localized and the change is mild and does not suggest toxicity of the test material. Three cases of infertility evidenced in the 250 mg/kg dose group and the number of corpora lutea for the same group was reduced significantly compared to the control group, but significant change in the rate of implantation was not identified. However, no clear influence was seen in oestrous cycle, and abnormality was also not observed in ovaries; therefore, whether a decrease of number of corpora lutea was induced by administration of the test material or not could not be determined. All cases of pregnant females gave live births and no significant difference was observed between the control group and the TTC respective dosage group during gestation period.

With regard to survival of pups no change suggesting the influence of administration of the test material was seen. Furthermore, concerning morphology of pups, abnormality was not observed in morphological observation of deaths and 0-day nursing, and further, during autopsy of 4-day nursing; thus, this test material does not influence development growth and differentiation of pups. In the 250 mg/kg dosage group, whitish nodule was observed on the body surface of nursing pups observed after the death of dam animal, but the change was only in 1 litter, and no abnormality was observed in 0-day nursing; thus, findings did not originate from administration of the test material.

Effect levels

1

Dose descriptor:

LOEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pigmentation in the proximal tubule of kidney and diffuse enlargement of the adrenal cortex in dose groups above 62.5 mg/kg bw/day

2

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: infertility was identified in the 250 mg/kg bw/day dose group

3

Dose descriptor:

NOAEL

Remarks:

F1 generation

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

4

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: auricular black spot, purple-black tail tip, tail node, auricular tubercle, scrotum hardening, scrotum nodule

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

integumentary

Organ:

skin

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The LOEL for the toxicity of repeated trithiocyanuric acid administration under the conditions of this study was determined to be 62.5mg/kg/day for both females and males based on edema in papilla combined with pigmentation of cortex in proximal tubule of the kidenys. Based on the presence of auricular black spot, purple-black tail tip, tail node, auricular tubercle, scrotum hardening, scrotum nodule in females and males of the 250 mg/kg dose group and seen also in continuous withdrawal the NOAEL of 125 mg/kg bw/day was derived. The NOAEL for reproductive toxicity was determined to be > 250 mg/kg/day due to no advers effects on reproduction. No adverse toxicological effects in pups were reported, therfore the NOAEL for F1 generation is determined to be > 250 mg/kg/day.