Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2020 to 11 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

EC Number:

300-504-3

EC Name:

Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Cas Number:

93941-05-0

Molecular formula:

C26H20ClN7O10S3.3Na

IUPAC Name:

trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Test material form:

solid: particulate/powder

Remarks:

Brown powder

Details on test material:

- Storage conditions: The test material was stored in the airtight container at room temperature (tolerance temperature: 10 - 30 ºC).

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

This strain is established as a laboratory animal and there is historical data of this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Obtained at 7 weeks old. The animals were 9 weeks old at the start of dosing.
- Weight at study initiation: Males: 379.1 g - 427.9 g; females: 227.2 g - 281.0 g
- Housing: All animals were housed in hanging stainless steel cages with wire-mesh floor (260 W × 380 D × 180 H mm, TOKIWA) for quarantine and acclimation at two or three in each cage according to the sex. After group allocation males and females were housed individually in hanging stainless cages with wire-mesh floor (TOKIWA) of 260 W × 380 D × 180 H mm and 165 W × 300 D × 150 H mm, respectively. Enrichment of irradiated hemp mats with gamma-ray.
In the mating period each female was housed in a male’s cage in each dose group. Copulated females were housed in polycarbonate cages (PC cage, 265 W × 426 D × 150 H mm, TOKIWA) with wooden bedding, enrichments of autoclaved gnawing wood and hemp mats from gestation day 14. Pups were housed with a dam after delivery.
Undertrays were changed at the end of quarantine and at group allocation, and twice a week after group allocation and abnormal urine was observed. Feeders, hanging cages and racks were changed at group allocation, once a month after group allocation and at the start of the recovery period. PC cages and wooden bedding were changed on gestation day 20, and postnatal days 4 and 8. All housing materials were autoclaved prior to use.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were observed once or more a day during the quarantine and acclimatisation periods until start of dosing. Oestrus cycle was observed in all females from the next day of receipt to the day before group allocation for 14 days.

DETAILS OF FOOD AND WATER QUALITY: The analytical data of contaminants in the diets, wooden bedding and enrichments were provided by the manufacturer or supplier. The tested parameters were confirmed to meet the requirements of CERI Hita according to the “Toxic Substances Control Act of US-EPA” (1979). Contaminants in drinking water were analysed twice a year according to the water regulations of the “Ordinance on drinking water quality standards” [No. 101 of MHLW]. The analytical data of contaminants in the water were confirmed to be in the stated ranges of CERI Hita.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target range 21 - 25 °C
- Humidity (%): Target range 40 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (7:00 - 19:00): 12 hours dark (19:00 - 7:00)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and formulations were administered by gavage once a day using a syringe (Terumo) connected to a nelaton catheter (Terumo) at the amount of 5 mL/kg based on the latest body weights.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was weighed and purified water was added to prepare the 20.0 w/v% formulation. A part of the 20.0 w/v% formulation was diluted with purified water to prepare the 2.00 and 6.00 w/v% formulations. The formulations and vehicle were subdivided into plastic containers, and stored at room temperature (target range: 10 to 30 ºC). On each dosing day formulations and vehicle were taken out from the storage place and dosed to the animals. The formulations were used within 15 days after preparation.


VEHICLE
- Justification for use and choice of vehicle: The test material dissolved in purified water at concentration of 20.0 w/v% (non-GLP). This vehicle is used for a general toxicity study and we have historical control data of this vehicle.
- Concentration in vehicle: 2.0 w/v % at 100 mg/kg/day, 6 w/v % at 300 mg/kg/day and 20 w/v % at 1 000 mg/kg/day.
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test material formulations at 20.0 and 0.200 w/v% were confirmed with high performance liquid chromatography (HPLC).
Actual concentrations measured after storage period were confirmed to be within the acceptable range compared to those measured immediately after preparation. These formulations were confirmed to be stable for 16 days at room temperature.
Concentrations of the test material formulations were confirmed with HPLC in the first preparation.
Actual concentrations of the 20.0, 6.00 and 2.00 w/v% formulations were 19.5, 5.87 and 1.90 w/v%, respectively. The formulations were within a permissible range of CERI Hita.

Duration of treatment / exposure:

Males and females were administrated for 14 days before mating. Thereafter, males were treated for 29 days including the mating period. Females were administrated until day 13 of nursing period throughout the mating and gestation periods. Non-mating females were treated for 29 days.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females except for the non-mated high dose group and the non-mated vehicle control group which were both 10 females only.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day repeated-dose oral toxicity study was performed at the dose levels of 0, 50, 200, 500 and 1 000 mg/kg/day consisting of three males and three females for each group. Following changes were observed. Elevation of the limiting ridge of the forestomach was observed in the 500 and 1 000 mg/kg groups. This change was considered to be related to an irritation of the test material. Reddish changes of the kidney and reddish brown urine were observed in the same dose levels. No abnormal changes were noted in the body weights or organ weights. From these results, 1 000 mg/kg/day was set for the high dose, and 300 and 100 mg/kg/day were set for the middle and low doses for the present study.
- Rationale for animal assignment: The animals showed no abnormalities in clinical observations, body weights and oestrus cycle were allocated to groups using a body weight-stratified randomisation on the day before the first dosing. The body weights of each animal were confirmed to be in the range of ± 20 % of the mean body weights for each sex at group allocation. The animals not allocated were excluded from the study.
- Post-exposure recovery period in satellite groups: Five males of the mated vehicle control group and five females of the non-mated vehicle control group were allocated as the recovery group. Five mated males from the 1 000 mg/kg/day high dose group and five non-mated females from the 1 000 mg/kg/day high dose group were allocated to the recovery group.
- Section schedule rationale: Each five animals of the first half are the main group, later five animals are the recovery group.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs twice a day, i.e. before and after dosing including observation of mortality during the dosing period. Females were observed for delivery and nursing conditions. In the recovery period the animals were observed once a day.
- Cage side observations included: Clinical signs and mortality. Females were observed for delivery and nursing conditions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once before the dosing period and once a week thereafter. After the initiation of dosing, the animals were observed in a blind test basis with the random numbering and observation labels without identifying the dose levels.
Observations at removal from cage: Animal reactions for external stimuli (holding animals or bringing a hand close to animals to hold), such as easiness of removal and vocalisation were assessed by scoring method.
Handling observations: Muscle tone, subnormal temperature, hair appearance (piloerection, staining hair and unkempt hair), skin and mucous colour (paleness, reddening and cyanosis), eyes (lacrimation, exophthalmos and pupillary size), salivation and secretion were observed.
Observation in arena: Animals were placed on a standard arena (an observation platform of 90 cm × 60 cm) and observed for posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behavior and abnormal behaviour for one min or more (within five min). Frequencies of defecation (number of faeces) and urination (number of pools) were recorded for one min.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured using an electric balance on the following days:
Males on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery;
Non-mating females on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery;
Females on day 1, 3, 7, 14 of dosing, on gestation day 0, 7, 14, 20 and on postnatal day 0, 4, 8, 13, and day 21, 35, 42 and 49 for non-copulated females;
In addition, all animals were weighed on the necropsy days.

FOOD CONSUMPTION:
- Food weights were measured using an electric balance at the following days:
Males on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery;
Non-mating females on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery;
Females on day 1, 3, 7, 14 of dosing, on gestation day 0, 7, 14, 20 and on postnatal day 0, 4, 7 (animal no. 130), 8, 10, 13.
From dosing day 7, feeding weights were measured after remainder weights were measured and replenished with food. On dosing day 14, remainder weights were measured in both sexes because the mating period was started on the next day except for the non-mating group. On dosing day 21 feeding weights were measured for males which copulation was confirmed except for one male (animal no. 31). Feeding weights were measured on gestation and postnatal day 0. Remainder weights were measured on each final measurement day.
Mean food consumption per day was calculated from their feeding and remainder weights.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: Not specified
- How many animals: Blood was collected from the abdominal aorta under isoflurane anesthesia from the following animals:
Five males and five dams for the same animals of the sensorimotor function examinations;
Non-mating five females;
Males and females of the recovery group;
Two pups per litter on day 4 after birth;
Two pups per litter at termination on day 13 after birth.
- Parameters examined: Red blood cell count, haemoglobin conc., haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin conc., platelets count, reticulocyte count ratio, white blood cell count, differentiation of leukocytes (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Animals fasted: Not specified
- How many animals: Blood was collected from the abdominal aorta under isoflurane anesthesia from the following animals:
Five males and five dams for the same animals of the sensorimotor function examinations;
Non-mating five females;
Males and females of the recovery group;
Two pups per litter on day 4 after birth;
Two pups per litter at termination on day 13 after birth.
- Parameters examined: Serum samples were used to determine the following parameters. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, total cholesterol, triglyceride, blood urea nitrogen, creatinine, total protein, albumin, A/G ratio, glucose, total bilirubin, total bile acids, inorganic phosphorus, calcium, sodium, potassium, chloride and thyroid hormone (T4).
T4 levels were measured for parental males and pups on day 13 after birth.

URINALYSIS: Yes
- Time schedule for collection of urine: Five males of the main group (same animals of sensorimotor function examinations), non-mating females, and males and females of the recovery group were housed in a metabolic cages in the afternoon of the each last observation day for 15 - 17 h.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, free drinking and no feed until next morning.
- Parameters examined: Collected urines were examined for the following parameters. Urine sediments were examined in males and non-mating females in the control and 1 000 mg/kg groups.
Urine volume, colour, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The last dosing week
- Dose groups that were examined: The animals were examined for the parameters in five males of each dose group, five non-mating females of the control and high dose groups, and selected five parental females of each dose group. Reflex tests and measurements of grip strengths were performed on a blind test basis. Examinations in the recovery period were not performed since no abnormalities were noted in males and non-mating females of the high dose group in the examination of the dosing period.
- Battery of functions tested:
Optic response: Animal’s response when a tip of pen was brought approx. 3 cm from the nose for four seconds was assessed by scoring method.
Pinna response: Animal’s response when a sudden sound of a finger snap was produced was assessed by scoring method.
Pain response: Animal’s response when the animal’s tail was pinched with a clothespin between one-third and base of the tail was assessed.
Pupillary reflex: Following darkness adaptation of the animal’s eyes, pupil constriction in response to a bright beam of a light was observed.
Air righting reflex: Animal’s response when the animal was dropped from approx. 30 cm height with supine position was assessed.
Grip strength: Grip strengths of forelimbs and hindlimbs were measured with a grip strength meter (FGC-2, MATYS). Two trials were performed and the mean values were calculated for each animal.
Locomotor activity counts: Locomotor activity of each animal was counted with a rat activity monitoring system (ACTIMO-10NP, SHINFACTORY) by the number of crossing IR beam (17 mm interval in 119 mm × 255 mm) for one hour at 10 min intervals.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
- Gross Necropsy: All parental animals were subjected to a detailed gross necropsy including external surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents after bleeding from the ventral aorta under isoflurane anesthesia on the next day of last dosing and last recovery days. Non-delivered females, dam which all pups died and dead female were subjected as with a survived animal on each found day.
Vaginal smears were collected before gross necropsy in survived females, and stages of the oestrous cycle were determined with a light microscope after Giemsa staining. The number of implantation site of the uterus after incision were counted for the mating females.

- Tissue Collecting and Organ Weight Measurements: The following organs/tissues were taken from all parental animals. Trachea, lungs and urinary bladder were inflated with 10 % neutralised buffered formalin before removal. Stomach and intestines were filled and fixed with 10 % neutralised buffered formalin and the contents were washed away with water. All macroscopic lesions were taken.
Liver, heart, kidneys, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, brain, spleen, thymus, thyroid and adrenals were weighed using an electric balance and their relative weights were calculated based on the body weight at the time of necropsy. Bilateral organs were weighed right and left together. Prostate was measured with a part of the urethra. Seminal vesicles were taken with coagulation gland after binding at the region of the origin. Thyroid adhered to the trachea including the parathyroid was fixed with 10 % neutralised buffered formalin, and the right and left lobes were removed from the trachea and weighed on the next day of the necropsy. Uterus was measured containing enteral solution in non-mating females of the control and high dose groups and females of the recovery group.

- Organs and Tissues: Trachea, lungs, submandibular glands, stomach, intestines (duodenum to rectum, including Peyer’s patches), pancreas, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate (ventral and dorsolateral lobes), seminal vesicles (including coagulating gland), ovaries, uterus (horn and cervix), vagina, brain (including cerebrum, cerebellum and pons), spinal cord (thoracic), sciatic nerve, bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid (including parathyroid), adrenals, eyes, skeletal muscle (femoral region), bone (femur), and mammary gland.


HISTOPATHOLOGY: Yes. All organs/tissues were preserved in 10 % neutralised buffered formalin. Testes and epididymides were fixed in modified Davidson’s fixative. Light microscopic examinations were performed after embedding in paraffin, sectioning and haematoxylin and eosin (HE) staining in each five animals of the control and high dose groups. When the lesions which considered to be attributable to the test substance were observed in the high dose group, histopathological examinations were done for the organs/tissues in each five animals of the intermediate and low dose groups and the recovery group. All macroscopic lesions were examined.
In the stomach, histopathological examinations were performed for all treatment groups since treatment-related changes were suspected in the necropsy.
In the kidney, histopathological examinations were performed for all treatment groups since treatment-related changes were suspected in the organ weights or histopathological examinations.
Immunohistochemistry of α2u-globulin was performed in males of the control and high dose groups (animal no. 1 and 34) since hyaline droplets were observed in the kidney of the high dose group.
Positive substance was observed in the proximal tubules in the high dose group, while a little reaction was confirmed in those of the control group.
Light green staining was performed in males and females of the control and high dose groups (animal no. 1, 34, 111 and 154) since deposition of brown pigments was suspected in the proximal tubules of the kidney. Brown pigments were observed in the high dose group while no brown pigments were observed in those of the control group.
A peer review on the histopathological data of the stomach and kidney was performed by a second pathologist since the test material-related changes were observed in theses organs.

Other examinations:

OESTROUS CYCLE EXAMINATIONS
Vaginal smears of all females in the mating group were collected from day 1 to 14 of dosing. The stages of oestrous cycle were determined with a light microscope after giemsa staining. The days from oestrous to the next oestrous were regarded as an oestrous cycle length and the mean oestrous cycle length was calculated. When the oestrous was successive the first day was regarded as an oestrous.

REPRODUCTIVE CAPABILITY EXAMINATIONS
Each female was cohabited in the cage of a male in the same dose level, as a pair of male and female at night on the dosing day 15, and the females were returned to the original cage next morning. Cohabitation was continued until showing evidence of copulation or maximum of 14 days. When a vaginal plug or sperms in vaginal smear were confirmed it was considered to be an evidence of mating (day 0 of pregnancy).
When the dams delivered spontaneously, built a nest or milked their pups by 10:00, those were regarded as an evidence of delivery and the day was designated as delivery day (postnatal day 0). If the delivery finished after 10:00, the delivery day was regarded as a next day.
Delivered dams with implantation site of the uterus was regarded as a pregnant female.
Number of cohabited animals (number of mated pairs), number of animals confirmed for copulation (number of copulated pairs), number of animals confirmed for gestation (number of pregnant females) and number of animals which had surviving pups on postnatal day 0 and 4 (number of dams with live newborns) were calculated from the results of copulation, delivery and gestation. In addition, days from the first mating to the copulation (pairing days until copulation), days from copulation to the day before delivery (gestation length) and number of implantation sites were calculated.
In the following items, the value of each animal, and the mean values and standard deviations were
calculated for each dosing group:
- Copulation index (Number of copulated pairs / Number of mated pairs) × 100
- Conception index (Number of pregnant females / Number of copulated pairs) × 100
- Delivery dam index (Number of pregnant females with live pups / Number of pregnant females) × 100
- Delivery index (Number of pups born / Number of implantation sites) × 100.

EXAMINATION OF PUPS
After finishing of delivery (postnatal day 0), number of males and females including stillborns, number of live pups and their total number were counted. Clinical observations including the external surface were carried out once a day until postnatal day 13. Males and females were counted for the number of live pups and the total number on postnatal day 4 and 13, and their body weights were measured on postnatal day 0, 4 and 13.
Anogenital distance (AGD) of each pup was measured on postnatal day 4. Each AGD was collected with the cube root of the body weight on the same day. The number of nipples/areolae in male pups was counted on postnatal day 13.
Thyroid was collected from pups on postnatal day 13 and fixed with 10% neutralized buffered formalin.

From these results, the value of each litter, the mean values and standard deviations of each dosing group were calculated as follows:
- Birth index (Number of live pups at birth / Number of implantation sites) × 100
- Viability index at birth (Number of live pups at birth / Number of pups at birth) × 100
- Sex ratio of pups at birth Number of live males at birth / Number of live pups at birth
- Viability index on day 4 after birth (Number of live pups on day 4 / Number of live pups at birth) × 100
- Sex ratio of pups on day 4 after birth Number of live males on day 4 / Number of live pups on day 4.

Statistics:

See below.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dosage Period
In males, reddish urine was observed in the 100, 300 and 1 000 mg/kg groups. This change was considered related to the colour of the test material.
In females, reddish urine was observed in all treatment groups. In the postpartum period, one animal showed no retrieving in the 300 mg/kg group.
In males, defecation and urination were increased in the 1 000 mg/kg group in week 3. Defecation was increased in the 1 000 mg/kg group in week 4. There were no treatment-related changes in the 100 or 300 mg/kg groups during the dosing period.
In females, no abnormal changes were noted at any dose levels.

Recovery Period
Reddish urine was observed in males and females of the 1 000 mg/kg group.
There were no treatment-related observations in either sex of the 1 000 mg/kg group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (animal no. 144) in the 1 000 mg/kg group died during delivery (gestation day 23) and this was considered to be not treatment-related since there were no pathological changes related to death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Dosage Period
No significant changes were noted in either sex of any treatment groups during the dosing period.

Recovery Period
Body weights were not affected during the recovery period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dosage period
In males, PT and APTT were elongated in the 1 000 mg/kg group. Haematocrit value was increased in the 100 and 1 000 mg/kg groups without dose-relationship and the changes were slight. Reticulocytes were decreased in the 100 mg/kg group without dose-relationship. Monocytes were increased in the 1 000 mg/kg group and the changes were slight. PT was elongated in the 100 mg/kg group and the change was slight. No abnormal changes were noted in the 300 mg/kg group.
In females, MCV was decreased in the 300 mg/kg group without dose-relationship. Reticulocytes were decreased in the 1 000 mg/kg group. No abnormal changes were noted in the 100 mg/kg group.
In non-mating females, haemoglobin conc. and haematocrit value were decreased in the 1 000 mg/kg group. Platelet count was increased in the 1 000 mg/kg group.
All of these changes were slight within our historical control ranges and considered to be not treatment-related.

Recovery period
In males, there were no abnormal changes in the 1 000 mg/kg group.
In females, reticulocytes were decreased in the 1 000 mg/kg group, and this change was slight within our historical control range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dosage period
In males, chloride level was increased in the 300 mg/kg group without dose-relationship. There were no abnormal changes in the 100 or 1 000 mg/kg groups.
In females, total protein, albumin and calcium levels were decreased in the 1 000 mg/kg group. There were no abnormal changes in the 100 or 300 mg/kg groups.
In non-mating females, BUN and total bilirubin were decreased in the 1 000 mg/kg group, and these changes were slight within our historical control ranges and considered to be not treatment-related.
T4 level were not affected in males and pups on postnatal day 13.

Recovery period
Any test parameters were not affected in the 1 000 mg/kg group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Dosage Period
In males, red urine was observed in one of the 1 000 mg/kg group. pH 8.0 of urine was noted in the 300 mg/kg group without dose-relationship. Occult blood (1+) was observed in the 1 000 mg/kg group, and this change was considered to be a single occurrence since there was no related changes or the change was slight. In the 100 mg/kg group urinalyses did not show significant changes.
In females, red urine was observed in one of the 1 000 mg/kg group. In the control group cloudy urine and pH 7.0 were noted. In the 1 000 mg/kg group cloudy urine, pH 7.0 and occult blood (1+) were noted. These changes were considered to be single occurrences since the same changes were noted in the control group or the change was slight.

Recovery Period
In males, slight red urine was observed in one of the 1 000 mg/kg group. In the control group pH 7.5 and occult blood were noted.
In females, cloudy urine and occult blood were noted in the control or 1 000 mg/kg groups without test material related.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Dosage Period
In males, motor activity in 20 - 30 min was decreased in the 1 000 mg/kg group. There were no abnormalities in the 100 or 300 mg/kg groups.
In females, any test parameters were not affected by the test material.

Recovery Period
Examinations were not performed in either sex during the recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dosage period
In males, no significant changes were noted at any dose levels.
In females, absolute and relative weights of the kidneys were increased in mating animals of the 1 000 mg/kg group. Organ weights were not affected in the 100 or 300 mg/kg groups.
In non-mating females, absolute and relative weights of the kidneys were increased in the 1 000 mg/kg group. Absolute weight of the adrenals was increased in the 1 000 mg/kg group, and there were no related microscopic changes related to this.

Recovery period
Relative weight of the kidneys was increased in males and females of the 1 000 mg/kg group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dosage period
In males, treatment-related changes were as follows: Elevation of the limiting ridge of the forestomach in 3/10 of the 300 mg/kg group and 5/5 of the 1 000 mg/kg group; bilateral discolouration of the kidneys in 10/10 of the 300 mg/kg group and 5/5 of the 1 000 mg/kg group.
The remainder of macroscopic regions in males were spontaneous changes: Unilateral enlargement of the testis in the 100 mg/kg group; cyst in the pituitary gland in the 300 mg/kg group.
In females, treatment-related changes were as follows: Elevation of the limiting ridge of the forestomach in 6/9 of the 1 000 mg/kg group; bilateral discolouration of the kidneys in 6/9 of the 300 mg/kg group and 9/9 of the 1 000 mg/kg group. Bilateral discolouration of the kidneys and small thymus were observed in a dam which all her pups died in the 300 mg/kg group. In non-mating females, elevation of the limiting ridge of the forestomach and bilateral discolouration of the kidneys in 5/5 of the 1 000 mg/kg group as treatment-related changes.
The dead female in the 1 000 mg/kg group had elevation of the limiting ridge of the forestomach, bilateral discolouration of the kidneys and bilateral enlargement of the adrenals.
The remainder of macroscopic regions in females were spontaneous changes: Defect of the left lobe of the thyroid in the control group; blackish region of the mucosa of the glandular stomach in the control, 100, 300 and 1 000 mg/kg group; whitish region in the liver in the 1 000 mg/kg group.

Recovery period
In males, elevation of the limiting ridge of the forestomach was observed in 3/5 of the 1 000 mg/kg group. Bilateral discolouration of the kidneys was observed in 5/5 of the 1 000 mg/kg group. In the control group unilateral whitish region in the epididymis was observed.
In females, elevation of the limiting ridge of the forestomach was observed in 1/5 of the 1 000 mg/kg group. Bilateral discolouration of the kidneys was observed in 5/5 of the 1 000 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dosage period
In males, treatment-related changes were observed in the stomach and kidney as follows: Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in 3/5 of the 100 mg/kg group, 7/7 of the 300 mg/kg group and 5/5 of the 1 000 mg/kg group; hyperplasia of the surface epithelial cells in the fundic mucosa of the glandular stomach in 2/5 of the 300 mg/kg group and 5/5 of the 1 000 mg/kg group; accumulation of the hyaline droplets in the proximal tubules of the kidney in 1/5 of the 100 mg/kg group, 6/10 of the 300 mg/kg group and 5/5 of the 1 000 mg/kg group; vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney in 5/5 of the 1 000 mg/kg group.
The remainder of microscopic regions in males were spontaneous changes: Focal myocarditis of the heart, lymphocyte infiltration in the ventral prostate, focal myositis of the skeletal muscle in the control group; basophilic tubules of the kidney, unilateral diffuse atrophy and dilatation of the seminiferous tubules of the testis in the 100 mg/kg group; solitary cyst in the cortex and medulla of the kidney, cyst formation in the pars intermedia of the pituitary gland in the 300 mg/kg group; mineralisation in Peyer’s patches of the jejunum, microgranuloma in the liver, focal myocarditis in the heart, lymphocyte infiltration in the ventral prostate in the 1 000 mg/kg group.

In females, treatment-related changes were observed in the stomach and kidney as follows: Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in 1/5 of the 100 mg/kg group, 4/5 of the 300 mg/kg group and 8/8 of the 1 000 mg/kg group, hyperplasia of the surface epithelial cells in the fundic mucosa of the glandular stomach in 1/5 of the 1 000 mg/kg group; vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney in 9/9 of the 1 000 mg/kg group.
In non-mating females, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in 5/5 of the 1 000 mg/kg group; hyperplasia of the surface epithelial cells in the fundic mucosa of the glandular stomach in 3/5 of the 1 000 mg/kg group; vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney in 5/5 of the 1 000 mg/kg group as a treatment-related change.
The dead female in the 1 000 mg/kg had atrophy of the axillar lymph node and hypertrophy of the cortical cells of the adrenal.
The remainder of microscopic regions in females were spontaneous changes: Erosion in the glandular stomach, solitary cyst in the medulla of the kidney, cyst formation in the pars distalis of the pituitary gland, mineralisation in the Peyer’s patches of the jejunum, aplasia of the left lobe, ectopic thymic tissue and ultimobranchial rest in the thymus in the control group; erosion in the glandular stomach, basophilic tubules of the kidney of the 100 mg/kg group; erosion in the glandular stomach, unilateral pyelitis and solitary cyst in the medulla of the kidney, atrophy of the thymus in the 300 mg/kg group; erosion in the glandular stomach, focal necrosis of the hepatocytes, Rathke’s pouch remnant in the pituitary gland, vacuolar change of the limiting ridge of the forestomach, mineralisation in the Peyer’s patches of the jejunem, scar and solitary cyst in the medulla of the kidney, focal myositis in the skeletal muscle in the 1 000 mg/kg group.

Recovery period
In males, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in 4/5, regeneration of the tubules of the kidney in 4/5, vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney in 5/5 of the 1 000 mg/kg group as a treatment-related change. In the control group unilateral spermatic granuloma in the epididymis was observed as a spontaneous change.
In females, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in 4/5, regeneration of the tubules of the kidney in 2/5, vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney in 5/5 in the 1 000 mg/kg group.
The remainder of microscopic regions in females were spontaneous changes: Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach, vacuolar change of the limiting ridge of the forestomach, solitary cyst in the medulla of the kidney in the control group; lymphocyte infiltration in the interstitium of the kidney, solitary cyst in the medullar of the kidney in the 1 000 mg/kg group.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Food Consumption:
Dosage Period
Food consumption did not show significant changes in any treatment groups during the dosing period.

Recovery Period
There was no abnormal food consumption in either sex.

Oestrous Cycle Stages at Necropsy:
Dosage period
Oestrous stage of each dose group was as follows: Metoestrus in 7/10, dioestrus in 3/10 of the control group; metoestrus in 5/8, dioestrus in 3/8 of the 100 mg/kg group; metoestrus in 5/9, dioestrus in 4/9 of the 300 mg/kg group; metoestrus in 5/9, dioestrus in 4/9 of the 1 000 mg/kg group.
Non-mating females had following oestrous stage: Oestrus in 3/5, dioestrus in 2/5 of the control group; proestrus in 1/5, oestrus in 2/5, metoestrus in 1/5, dioestrus in 1/5 of the 1 000 mg/kg group.

Recovery period
Oestrous stage of each dose group was as follows: Proestrus in 1/5, oestrus in 3/5, metoestrus in 1/5 of the control group; oestrus in 1/5, metoestrus in 2/5, dioestrus in 2/5 of the 1 000 mg/kg group.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL of the test material was considered to be less than 100 mg/kg/day for repeated-dose toxicity.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed to assess the repeated-dose toxicity of the test material in accordance with OECD Guideline 422 and in compliance with GLP.

Male and female Crl:CD(SD) rats at 9 weeks of age were treated with the test material for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively. The dosage levels were set at 100, 300 and 1 000 mg/kg/day. Control animals were similarly dosed with purified water. Recovery groups were set for the vehicle control and high dose groups.

Reddish urine was observed in males and females of all test material groups. This change was related to colour of the test material. One female died in the 1 000 mg/kg group during delivery and this was considered to be not treatment-related.

In urinalyses, red urine was observed in male and female of the 1 000 mg/kg group. In haematology, PT and APTT were elongated in males of the 1 000 mg/kg group. There were no abnormal changes in detailed clinical observations, sensorimotor function examinations, body weights, food consumption or blood chemistry.

In organ weights, absolute and relative weights of the kidneys were increased in females of the 1 000 mg/kg group. At the end of the recovery period relative weights of the kidneys were increased in males and females of the 1 000 mg/kg group.

In macroscopic examinations, elevation of the limiting ridge of the forestomach was observed in males of the 300 or 1 000 mg/kg groups and females of the 1 000 mg/kg group. Bilateral discolouration of the kidneys was observed in males and females of the 300 and 1 000 mg/kg groups. At the end of the recovery period, these changes were also observed in males and females of the 1 000 mg/kg group.

In histopathological examinations, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach was observed in males and females of the 100, 300 and 1 000 mg/kg groups. Hyperplasia of the surface epithelial cells in the fundic mucosa of the glandular stomach was observed in males of the 300 and 1 000 mg/kg groups and females of the 1 000 mg/kg group.

Accumulation of hyaline droplets in the proximal tubules of the kidney was observed in males of the 100, 300 and 1 000 mg/kg groups. This change was considered to be male rats specific and not toxicologically significant since relationship to α2u-globulin was confirmed. Vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney was observed in males and females of the 1 000 mg/kg group. This change was considered to be related to colour of the test material. At the end of the recovery period, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach, regeneration of the tubules of the kidney, vacuolisation of the proximal tubules with brown pigment deposition in the cortex of the kidney were observed and the effects of the test material was considered to be reversible.

The test material did not affect parameters of gonadal function, mating behavior and implantation of the parental animals. No adverse effects of the test material were noted in the body weights, viability index, AGD or number of nipples/areolae of pups.

 

The NOAEL was not determined due to the localised histopathological changes in male and female rats observed in the forestomach at all dose groups (100, 300, 1000 mg/kg bw/day), which is not present in humans. Hyperplasia of the surface epithelial cells in the fundic mucosa of the glandular stomach was observed in both sexes at 300 or 1000 mg/kg bw/day, with no other associated functional changes. Based on the currently available data set for the registered substance, human relevance of the localised irritation effects observed in the rat forestomach is considered limited due to anatomical differences in between humans and rodents.

LOAEL = 100 mg/kg/day.