2-{[4-({4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfo-1-naphthyl}diazenyl)-(6 or 7)-sulfo-1-naphthyl]diazenyl}benzene-1,4-disulfonic acid, lithium sodium salts

Endpoint summary

Description of key information

The NOAEL of CJ309was 100 mg/kg/day (OECD TG407).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

28 Days

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From February 06, 2017 to March 16, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read-across target has structure similarity to CJ313. Therefore, the data is applied.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 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 %

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

28 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

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):

For male rats:
Statistically significant changes in hematology parameters featuring increased mean corpuscular hemoglobin concentration (MCHC) were observed in Group 4 (1000 mg/kg/day) versus the control animals (Group 1).
For female rats:
The increases in MCHC levels were seen in Group2, Group 3 and Group 4 (100, 300 and1000 mg/kg/day). 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).

Behaviour (functional findings):

not specified

Immunological findings:

not specified

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

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

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

neuropathology

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Conclusions:

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

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 included increases in mean corpuscular hemoglobin concentration and increased 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: via oral route-systemic effects

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 included increases in mean corpuscular hemoglobin concentration and increased 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.