Reference type

Author

Year

Title

Bibliographic source

Testing laboratory

Report no.

Owner company

Company study no.

Report date

study report

MHW (Ministry of Health and Welfare), Japan

2007

A combined repeated-dose/reproductive-developmental toxicity study of trithiocyanuric acid by oral dosing in rats.

Japan Existing Chemical Data Base (JECDB)

Food and Drug Safety Center

R-04-007

data published

7.8.1 Toxicity to reproduction: Toxicity to reproduction.001

combined repeated dose and reproduction / developmental screening

no

Qualifier

Guideline

Deviations

according to

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

no

yes

yes

Identifier

Identity

CAS number

638-16-4

- Name of test material (as cited in study report): Trithiocyanuric acid
- CAS No.: 638-16-4
- Molecular formula: C3H3N3S3
- Molecular weight: 177.27
- Purity: 99.8% (HPLC)
- Impurities: 0.13% sulfur
- Physical state: Slightly pale yellow powder
- Melting point: ＞300°C
- Boiling point, specific gravity, partition coefficient, vapor pressure: No data
- Solubility: Sparingly soluble in water, slightly soluble in methanol, acetone, and dioxane, and soluble in cellosolve and THF
- Stability: The stable to normal handling
- Supplier: Kawaguchi Chemical Industry Co., Ltd.
- Lot No.: 407518
- Storage condition until use: Room temperature

rat

other: Crj: CD(SD), SPF

male/female

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks of age
- Weight at study initiation: 355.5 – 405.2 g for males and 208.7 – 255.0 g for females
- Housing: bracket-type metallic wire-mesh cages (W 220 × D 270 × H 190 mm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 to 26.0°C
- Humidity (%): 50.0 to 65.0%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light):12-hour lighting per day

oral: gavage

other: sodium carboxymethyl cellulose

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in corn oil for injection.
VEHICLE
- Justification for use and choice of vehicle: No data
- Amount of vehicle (if gavage): 5 ml/kg bw
- Supplier: Maruishi pharmaceutical industry Co., Ltd.
- Lot/batch no. (if required): 4720
- Dosing volume: 5 mL/kg
- Stability (test solutions): For 8 days
- Storage condition of test solution: Stored in a dark place with room temperature

yes

Test suspensions at each concentration (20 and 0.05 w/v%) of initial and final preparations were analyzed by the HPLC method at Hatano laboratory of Food and Drug Safety Center. Results showed that the concentration of the test article in each concentration was 94.3 to 101% of the nominal concentration.

(P) Males: 42 days including 14 days pre-mating (P)Females: Days including 14 days pre-mating, mating and gestation periods and the days until day 4 of lactation Female (no mating, satellite group): for 42 days

Daily: 7 times / week

0 (vehicle), 62.5, 125 and 250 mg/kg bw/day

Basis

actual ingested

12/sex/dose (main group), and 5 females/dose at 0 and 250 mg/kg bw/day (satellite group)

yes, concurrent vehicle

- Dose selection rationale: A preliminary study (study No. R-04-006) was conducted to determine the doses to be employed. Male and female rats were receiving 0, 250, 500, and 1000 mg/kg bw/day for 14 days. As a result, red colored urine was observed in more than half numbers of rats receiving 1000 mg/kg bw/day groups and all animals died in the 1000 mg/kg bw/day groups until Day 7. Black path zones of auricle and dark purple colored tail top were observed in the 500 mg/kg bw/day groups from Week 2, and moreover, one female sowing red colored urine, salivation, wasting and sedation were moribund . Body weight gains were depressed in male rats receiving 500 mg/kg bw/day at Week 2, and swelling of the spleen was observed at necropsy. No changes related to the test substance were observed in males receiving 250 mg/kg bw/day. From the results of the preliminary study, 500 mg/kg bw/day seemed to be the maximum tolerance dose for 14 days. Therefore, the high dose was set at 250 mg/kg bw/day for the main study, and the middle and low doses were set at 125 and 62.5 mg/kg bw/day using common ratio 2.

no

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Males and females: once before the start of dosing, two times/day during the dosing period, and once during the recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Male animals: the end of acclimation period, Day 7, 14, 21, 28, 35 and 42 during the dosing and Day 7 and 14 during the recovery period. Female animals: the end of acclimation period, Day 7, 14, 21, 28, 35 and 42 during the dosing, Day 0 at delivery period and Day 0 from 4 at delivery animals, and Day 7 and 14 of satellite groups during the recovery period. Test items are following: Body position, locomotor activity, vocalization, tremor, convulsion by cage side observation, ease of removing rat from cage, reactivity to being handled, heart rate, body temperature, fur, skin color, visible mucosa color, lacrimation, exophthalmos ophthalmocele, pupillary, and salivation by terminology for removing rat from cage, and body position, walking, grooming, phonation, straub tail, gait, stereotyped, abnormal behavior, tremor, convulsion, piloerection, and ophthalmorrhexis by observation of behavior on the working table.
FANCTIONAL OBSERVATION: Yes
- Time schedule:
End of the dosing period: male, female (dam), and female (satellite group) animals. End of the dosing period: male and female (satellite group) animals.
Test items are following: Prayer’s reaction, pupillary reflex, visual placing, startle reaction, withdrawal reflex, eyelid reflex, and righting reflex.

BODY WEIGHT: Yes

Males in the main and females in satellite groups were weighed on Day 1, 7, 14, 21, 28, 35 and 42 of dosing, and Day 1, 7, and 15 (necropsy day) at the end of the recovery period.
Females were weighed on Day 1, 7, 14, and 21 until successful copulation. Copulated females were weighed on Day 0, 7, 14 and 20 of gestation, days 0 and 4 of lactation, and the necropsy day.
FOOD CONSUMPTION : Yes
- Food consumption (g/day/rat) for each animal determined from the difference of the of the previous day's feeding amount: Yes
Measurement of food consumption was conducted on all animals at the following frequencies:
Males and females in the satellite groups: on Day 1-2, 7-8, 14-15, 29-30, 35-36, and 41-42 during the dosing period and Day 6-7 and 13-14 during the recovery period. Females in the main groups: on Day 1-2, 7-8, and 14-15 (before mating), on Day 0-1, 7-8, 14-15, and 20-21 (the gestation period). On Day 3-4 (the lactation period).
FOOD INTAKE: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 43 of the dosing period and Day 15 of the recovery period in males. On Day 5 of the lactation period (main group) and on Day 15 of the recovery period (satellite group) in females.
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes (for 18-22 hours)
- How many animals: 5 animals/sex/group
- Parameters examined red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, white blood cell count, differential white blood cell count, prothrombin time, activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as hematology examination.
- Animals fasted: Yes (for 18-22 hours)
- How many animals: 5 animals/sex/group
- Parameters checked: ASAT (GOT), ALAT (GPT), γ-GT, ALP, total bilirubin, blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chloride
URINALYSIS OF MALES: Yes
- Time schedule for collection of urine: Five males on Day 33 during the dosing period and all animals on Day 13 during the recovery period.
Five females on Day 34 (main group). All animals on Day 34 during the dosing period and Day 13 during the recovery period (satellite group).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: color, muddy, pH, occult blood, protein, glucose, ketones. bilirubin, urobilinogen, and sediments
BLOOD HORMONE: No
NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed clinical observation: on Day 7, 14, 21, 28, 35, and 42 of the dosing period, on Day 7 and 14 of the recovery period,
Functions test: on Day 42 of the dosing period and on Day 14 of the recovery period (males and satellite group females). On Day 4 of the lactation period (main group females).

GROSS PATHOLOGY AND ORGAN WEIGHTS：Yes Brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, and epididymis.
HISTOPATHOLOGY: Yes Brain, puitality, thymus, lymph nodes (including mesenteric and mandibular lymph nodes), trachea, lung (including bronchus), stomach, intestinal tract (duodenum, jejunum, ileum, cecum, colon, rectum), thyroids, heart, liver, spleen, kidneys, adrenals, urinary bladder, testes, epididymis, seminal vesicles (including the coagulating gland), prostate (ventral lobe), ovaries, uterus, vagina, bone marrow (one side femur), sciatic nerve (one side femur), spinal cord, and gross abnormalities site.

Changes in estrous cyclicity and conception rate were analyzed by Fisher’s test. Graded pathological data was analyzed by Mann-Whitney’s U test (significance level = 0.05) and pathological data with number of positive and negative animals was analyzed by one-sided Fisher’s test.
Other data, obtained values in each animal or mean of a litter was one data, and these data were compared among the satellite groups and other among the groups. These data were analyzed using F-test for homogeneity of distribution. The Student’s t-test and the Aspin-Welch’s t-test were conducted for homogenous and non-homogenous distribution, respectively to compare the control and individual treatment groups. Three or more groups setting, these data were analyzed using Bartlett’s test for homogeneity of distribution. The Dunnett’s multiple comparison test after the ANOVA and the Dunnett’s-type mean rank sum test after Kruskal-Wallis’s H test were conducted for homogenous and non-homogenous distribution, respectively to compare the control and individual treatment groups. Significance level was set at 0.05 compared with the control group and among the groups.

Endpoint

Effect level

Based on

Sex

Basis for effect level / Remarks

LOAEL

62.5 mg/kg bw/day (actual dose received)

test mat.

male/female

Effects of lesions in the deposit of pigment in proximal tubule in kidneys and diffuse hypertrophy in adrenals in males and females receiving 62.5 mg/kg bw/day or more groups.

yes

yes

yes

yes

yes

yes

yes

yes

yes

CLINICAL SIGNS AND MORTALITY: Mortality: One male and one female died in the 250 mg/kg bw/day group on Day 8 and Day 42, respectively. Clinical signs: Incomplete eyelid opening and emaciation were observed in one dead female receiving 250 mg/kg bw/day, but no clinical sign was observed in one dead male receiving 250 mg/kg bw/day. The followings were observed in the 250 mg/kg bw/day groups: black area of the pinna in one male and three females, dark purple distally of the tail in two males, nodule of the tail in four males and one female, nodule of the pinna and emaciation in one male and one female, anemic in one male, induction of the scrotum in one male, nodule of the scrotum in three males, swelling of the left forelimb in one male, and crust formation of the snout in one female. Reddish urine was observed in one male receiving 125 mg/kg bw/day and three males and one female receiving 250 mg/kg bw/day groups. Sporadically salivation just after dosing was observed in six males receiving 125 mg/kg bw/day and eight males and 10 females receiving 250 mg/kg bw/day groups. Loss of fur was observed in two males in the control group, one male and one female receiving 125 mg/kg bw/day, and one female receiving 250 mg/kg bw/day. Crust formation of the dorsal neck was observed in one female receiving 62.5 mg/kg bw/day and mass of the perineal region was observed in one female receiving 125 mg/kg bw/day. No adverse changes were observed in both sexes during the recovery period.

DETAILED CLINICAL OBSERVATIONS AND FANCTIONAL OBSERVATION: Detailed clinical observations, no changes were observed in both sexes during the recovery period. When removing rats from the cage or handling rats, vocalization and flinches were observed in both sexes sporadically, and running around in the cage was observed in one female rat receiving 250 mg/kg bw/day. When handling rats, tenseness or rigidity was observed in one female rat receiving 125 mg/kg bw/day and 250 mg/kg bw/day. Rearing of stereotypy was observed in two females receiving 250 mg/kg bw/day.
Functional observation, no changes were observed in both sexes during the dosing and recovery periods.


BODY WEIGHT: Significant decreases in body weights, weight gains, and cumulative weight gains were observed in males receiving 250 mg/kg bw/day from Day 21 of the dosing period to Day 14 of the recovery period, from Day 7 to Day 35 of the dosing period, and from Day 14 to Day 42 of the dosing period. Significant decreases in body weights, weight gains, and cumulative weight gains were observed in females receiving 250 mg/kg bw/day from Day 42 of the dosing period to Day 14 of the recovery period, from Day 21 to Day 35 of the dosing period, and from Day 35 to Day 42 of the dosing period. No changes in body weights were observed in both sexes receiving 125 mg/kg bw/day and 62.5 mg/kg bw/day groups compared with the control groups during the dosing and recovery periods.
FOOD CONSUMPTION: Significant decreases in food consumption were observed in males receiving 250 mg/kg bw/day from Day 7 to Day 8 and Day 29 to Day 30 during the dosing period. Significant decreases in food consumption were observed in females receiving 250 mg/kg bw/day of the satellite group from Day 1 to Day 2 and Day 29 to Day 30 during the dosing period. No changes in food consumption were observed in both sexes receiving 125 mg/kg bw/day and 62.5 mg/kg bw/day groups compared with the control groups during the dosing and recovery periods.

URINALYSIS: Slight turbidity was observed in one male receiving 250 mg/kg bw/day during the dosing period. Occult blood was observed in each one male in the 250 mg/kg bw/day group and the control group during the dosing period. Red blood cells in sediments were observed in six males receiving 250 mg/kg bw/day and remained in one male during the recovery period.

HAEMATOLOGY: At the end of the dosing period, significant decreases in hematocrit value and Increasing tendency in reticulocyte ratio were observed in males receiving 250 mg/kg bw/day. No changes were observed in female receiving 250 mg/kg bw/day. At the end of the recovery period, significant decreases in hemoglobin concentration, hematocrit value, MCV, and MCH were observed in females receiving 250 mg/kg bw/day. No significant differences in males receiving 250 mg/kg bw/day compared with the control group.

CLINICAL CHEMISTRY: At the end of the dosing period, significant decreases in albumin value were observed in males receiving 62.5 mg/kg bw/day and 250 mg/kg bw/day. A significant decrease in creatinine value was observed in males receiving 250 mg/kg bw/day and remained after the recovery period. No changes were observed in female receiving 250 mg/kg bw/day compare with the control group after the dosing period. At the end of the recovery period, a significant decrease in a creatinine value and a significant increase in triglyceride value were observed in females receiving 250 mg/kg bw/day.

ORGAN WEIGHTS: At the end of the dosing, A significant decrease in body weight was observed in male receiving 250 mg/kg bw/day; therefore, significant increases in relative brain, heart, and adrenals weights were observed in this group. No changes in organ weight were observed in females.
At the end of the recovery period, significant decreases in absolute liver and kidneys weights and significant increases in relative brain, heart, kidneys, spleen, adrenals, testes, and epididymis were observed in males receiving 250 mg/kg bw/day. Significant decreases in absolute thymus weight and significant increases in relative brain, heart, and liver weights were observed in females receiving 250 mg/kg bw/day compared with the control group.

GROSS PATHOLOGY: Edematous and subinvolution in the lungs and enlargement and dark red colored in the kidneys were observed in the dead male. Edematous in the lungs and thymus, enlarges and pale colored kidneys, pale colored and small sized spleen, and pale colored bone marrow of femur were observed in the dead female. At the end of the dosing period, abscess, yellow zone, and adhesion with scrotum in the epididymis were observed in one or two males receiving 250 mg/kg bw/day. Enlargement and rough surface in the kidneys, small sized spleen, nodule and dark red zone in tail were observed in males receiving 250 mg/kg bw/day. Enlargement of the kidneys and white spot in the liver were observed in males receiving 62.5 mg/kg bw/day. Small sized spleen and thymus, black spot or recessed area in mucosa of the glandular stomach, and nodule and black spot of skin in the auricle were observed in females receiving 250 mg/kg bw/day. Cyst in the kidney, mass of around the vulva in the subcutis, black spot of mucosa in the glandular stomach, and white spot in the liver were observed in females receiving 125 mg/kg bw/day. At the end of the recovery period, abscess and adhesion with scrotum, nodule of scrotum in skin, and black spot of auricle in skin were observed in one male receiving 250 mg/kg bw/day. Alopecia of skin in clavicular area were observed in one female receiving 250 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC: Granuloma with multinucleated giant cells and inflammatory cell infiltration was observed in subcutaneous tissues in lesion of the auricle, tail and scrotum. Necrosis or edemas of the papilla of the kidneys were observed in males receiving 62.5 mg/kg bw/day and more and females receiving 250 mg/kg bw/day groups. Deposit of pigment of proximal tubule in the kidneys was observed in both sexes receiving 62.5 mg/kg bw/day or more. Diffuse hypertrophy of fascicular cells in the adrenal glands was observed in both sexes receiving 62.5 mg/kg bw/day or more. Lesions in the kidneys or adrenal glands were observed in both sexes receiving 250 mg/kg bw/day at the end of the recovery period.

Based on the effects of dosing on the kidney and adrenal gland, the LOAEL for the male and female rat repeated dose toxicity of 1,3,5-triazine-2,4,6(1H,3H,5H)-trithione was determined to be 62.5 mg/kg bw/day.

UUID

IUC5-6e3a7ad5-8227-4baa-bd43-d4991de751c6

Dossier UUID

0

Author

dra / National Institute of Health Sciences / Tokyo / Japan

Date

2017-01-04 16:29:42 JST

Remarks

Purpose flag

key study

Study result type

experimental result

Reliability

1 (reliable without restriction)

Rationale for reliability incl. deficiencies

OECD Test Guideline study under GLP condition