CJ309

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 06, 2017 to March 16, 2018. 28 day administration

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,7-Naphthalenedisulfonic acid, 4-amino-6-[2-[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulfophenyl]diazenyl]-3-[2-(2,5-disulfophenyl)diazenyl]-5-hydroxy-, pentasodium salt and lithium chloride

Molecular formula:

Not applicable; this UVCB substance contains: C25H14ClN10O16S5.xLi.yNa, (x + y) = 5; 0 < (x,y) < 5 with 940.9 < MW < 1021.1 g/mol (UVCB substance), and traces of NaCl.

IUPAC Name:

Reaction products of 2,7-Naphthalenedisulfonic acid, 4-amino-6-[2-[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulfophenyl]diazenyl]-3-[2-(2,5-disulfophenyl)diazenyl]-5-hydroxy-, pentasodium salt and lithium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: BioLASCO Taiwan Co., Ltd.
- Age at study initiation: 58 Days
- Housing: One animal per cage in polycarbonate cages with Bedo'Cobs® 1/4″ (Andersons Lab Bedding) as bedding with enrichment toys given.
- Acclimation period: 13 days
- Temperature (°C): 20.6 to 22.6 °C
- Humidity (%): 34.3 to 66.5 %

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Duration of treatment / exposure:

28 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

For Group 1

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

For Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

For Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

For Group 4

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For male rats:
All animals in Group 2 through Group 4 (100, 300 and 1000 mg/kg/day) were observable that excretion of unusually blue-black-colored feces. All animals in Group 3 and Group 4 (300 and 1000 mg/kg/day) were observable that excretion of unusually blue-colored urine. For all animals in Group 4 (1000 mg/kg/day), the unusual blue-colored staining of the skin covering the entire body. One animal from Group 2 and two animals from Group 3 have small-to-medium amount of hair loss on the forelimb.

For female rats:
All animals in Group 2 through Group 4 (100, 300 and 1000 mg/kg/day) were observable that excretion of unusually blue-black-colored feces. All animals in Group 3 and Group 4 (300 and 1000 mg/kg/day) were observable that excretion of unusually blue-colored urine. For all animals in Group 4 (1000 mg/kg/day), the unusual blue-colored staining of the skin covering the entire body. One animal in Group 4 exhibited a mild increase in salivation on Day 21. Two animals in Group 4 also exhibited mild-to-moderated staining (blue) of the facial and forepaw hair on Day 1. Two animals from Group 1 have small-to-medium amount of hair loss on the forelimb.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of all study groups increased throughout the study period. A transient increase in the average relative body weight gain in the third week of the study (calculated on Day 22) was noted for female Group 4 animals (1000 mg/kg/day).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food consumption was seen to be increased at the highest dose level at 1,000 mg/kg/day, with statistically significant increases versus the control animals seen on Day 22 (end of Week 3) for both males and females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The observed statistical variations in hematology parameters remained within respective reference ranges, with no corresponding observable clinical changes.

The statistically significant decrease in red blood cell count (RBC) and hematocrit (HCT) were also seen in Group 3 (300 mg/kg/day) versus the control animals. A statistically significant decrease in the average percentage of lymphocytes (%LYMPH) concurrent with a statistically significant increase in the average percentage of neutrophils %NEUT (ID Nos. 0036 and 0038) were seen in Group 4 (1000 mg/kg/day).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For male rats:
Total bilirubin (TBil) increased was observable at the lowest dose level (100 mg/kg/day) and reached statistical significance at Group 3 and Group 4 (300 and 1000 mg/kg/day). The differences in mean TBil levels represent a 291- fold increase over the control (Group 1) animals. The average creatinine (Cre) levels increased, reaching statistical significance at the highest dose level (1000 mg/kg/day). The statistically significant changes included an increases of average total protein (TP) level among Group 4 (1000 mg/kg/day).

For female rats:
The statistical significance of the increase of Total bilirubin (TBil) was achieved in Group 4 (1000 mg/kg/day). The differences in mean TBil levels represent a 208-fold increase over the control (Group 1) animals. The statistically significant changes included a decrease of average aspartate aminotransferase (AST) level in Group 3 (300 mg/kg/day) and an increase of average phosphate (P) level in Group 4 (1000 mg/kg/day).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

For male rats:
An increase of the urine specific gravity (SG) was noted for male Group 4 (1,000 mg/kg/day). Increased presence of sperm cells and triple phosphate crystals in the urine sediment of male dosed animals was also observed. The color of urine changed from yellow to green (300 mg/kg/day) and then blue (1,000 mg/kg/day) with progressively increasing dose levels. Marked increases of urine bilirubin (BIL), ketone (KET) and urine protein (PRO) were observed in Gourp 3 and Group 4 (300 and 1,000 mg/kg/day). Increased presence of urine glucose (GLUU), pH level, urobilinogen (URO), occult blood (BLO), and leukocyte esterase (Leu) was also seen among Group 4 (1,000 mg/kg/day).

For female rats:
The color of urine changed from yellow to green (300 mg/kg/day) and then blue (1,000 mg/kg/day) with progressively increasing dose levels. Marked increases of urine bilirubin (BIL), ketone (KET), urine glucose (GLUU; male only), pH level, urine protein (PRO), urobilinogen (URO), occult blood (BLO), and leukocyte esterase (Leu) were observed in Group 4 (1,000 mg/kg/day). Increased URO was also observed in Group 3 (300 mg/kg/day).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For male rats,
A very slight trend of splenic weight increase was noted but the increase was similar at all dose levels.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Many organs/tissues were discolored blue in the study animals examined as following.
For male rats:
The adrenal, epididymis, pituitary gland, prostate/seminal vesicles, Harderian gland, cecum, duodenum, mandibular and mesenteric lymph nodes, salivary gland, thymus and eye in Group 4 (1000 mg/kg/day). The colon, rectum, dorsal neck skin, urinary bladder, stomach, ileum, jejunum and testis in Group 3 and Group 4 (300 and 1,000 mg/kg/day). The kidney in Group2, Group 3 and Group 4 (100, 300 and 1,000 mg/kg/day).

For female rats:
The Harderian gland, cecum, duodenum, mandibular and mesenteric lymph nodes, salivary gland, thymus, eye, ileum, jejunum, vagina, ovary and oviduct in Group 4 (1,000 mg/kg/day). The colon, rectum, dorsal neck skin, urinary bladder, stomach and uterus in Group 3 and Group 4 (300 and 1,000 mg/kg/day). The kidney in Group2, Group 3 and Group 4 (100, 300 and 1,000 mg/kg/day).

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

According to OECD 407 test method, the NOAEL of CJ309 was 100 mg/kg/day.

The 28-day repeat-dose toxicity findings revealed no specific, target organ toxicity arising from a repeated exposure to CJ309. The observed statistical variations in hematology parameters remained within respective reference ranges, with no corresponding observable clinical changes. Changes in total bilirubin (TBil) were not correlated to any corresponding observable clinical, gross, or microscopic changes or adverse effects to the hepatic or urinary functions; it is possible that they reflect an adaptive response rather than target toxicity. Overall, the findings in this study support a non-classification of STOT-RE.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316002-GN and OECD 407 (OECD, 2008). The test article was administered to the three treatment groups by oral gavage in a dose of 100, 300 and 1000 mg/kg/day for 28 consecutive days. There were five male and five female Sprague-Dawley rat in each group.

All study animals survived to study completion. Test article-related changes included blue feces discoloration, blue urine discoloration, and blue dermal staining of the entire body for both sexes. Transient increase in body weight relative gain and elevated food consumption were also seen in the third week of the study and attributable to the test article effect. Changes in hematology parameters inciuded increases neutrophil percentage concurrent with a decrease in lymphocyte percentage. Changes in serum chemistry parameters included increased total bilirubin and creatinine level. Changes in urinalysis parameters included urine discoloration and SG, and marked presence of sperm cells and triple phosphate crystals. Other urine changes in urinalysis parameters including urine bilirubin, urobilinogen, ketone, protein, urine glucose, pH level, occult blood, and leukocyte esterase were not considered due to possible interference of test outcomes by test article presence in urine.

There were no treatment-related gross, organ weight or microscopic changes in the study animals examined. No test article-related changes were identified in coagulation parameters or the assessment of sensory reactivity to stimuli, grip strength, and motor activity. Although there were no clinical or gross findings other than those attributable to the physical presence of the test article, the altered hematology and serum chemistry are suggestive of possible hemolytic event that could be considered adverse. The noobserved- adverse-effect level (NOAEL) of the study is therefore set at 100 mg/kg/day (94.79 mg/kg/day when adjusted for test article purity), at which dose level the above-mentioned findings were not yet apparent.