4-[(4-chloro-6-{[(3 or 4)-sulfophenyl]amino}-1,3,5-triazin-2-yl)amino]-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonic acid, lithium sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 5, 2019 to August 28, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[(4-chloro-6-{[(3 or 4)-sulfophenyl]amino}-1,3,5-triazin-2-yl)amino]-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonic acid, lithium sodium salts

EC Number:

942-795-5

Molecular formula:

Not applicable; this UVCB substance contains: C24H20ClN8O11S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 748.9 < MW < 797.0 g/mol (UVCB substance), C24H21N8O12S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 730.4 < MW < 778.6 g/mol (UVCB substance), C18H15Cl2N7O8S2.xLi.yNa, (x + y) = 2; 0 < (x,y) < 2 with 606.2 < MW < 638.3 g/mol (UVCB substance), C21H17N8O9S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 642.4 < MW < 690.5 g/mol (UVCB substance), C30H25N9O14S4.xLi.yNa, (x + y) = 4; 0 < (x,y) < 4 with 891.5 < MW < 955.7 g/mol (UVCB substnace), and traces of NaCl and Na2SO4.

IUPAC Name:

4-[(4-chloro-6-{[(3 or 4)-sulfophenyl]amino}-1,3,5-triazin-2-yl)amino]-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonic acid, lithium sodium salts

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

1) this species/strain used has been demonstrated to be sensitive to reproductive and developmental toxicants and has been widely used for reproductive and developmental toxicity evaluation;
2) historical data and experience exist at the Testing Facility

Sex:

male/female

Details on test animals or test system and environmental conditions:

Microbiologic status: Specific pathogen free (SPF)
Source: BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, ROC)
Ages: ~11 weeks old at estrous cycle evaluation
Body weights Males: 407-497 g at the first dosing
Females: 221-304 g (Deviation 2) at the first dosing
Number of animals: 45 males & 52 females including spare animals
Method of identification: Ear notch a and cage card

Housing style: Animals were individually housed with an enrichment toy and bedding. While mating, animals were pair-housed in stainless steel wire mesh cages. The presumed pregnant females were caged individually and provided with nesting materials. Lactating females were caged individually with their offspring.
Temperature range: 16.8 – 22.6 ºC
Relative humidity range: 38.6 – 94.8%
Illumination setting: 12 h light (lights on at 06:00) and 12 h dark (lights off at 18:00)
Fresh air change rate setting: 155 times per hour
Diet: Autoclavable Rodent Diet 5010 (PMI® Nutrition International, Inc., MO, USA, Appendix VIII) was supplied ad libitum (Deviation 3)
Water: Autoclaved primary-filtered water was supplied ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Male rats were weighed and euthanized by carbon dioxide exposure followed by exsanguinations. Vaginal washing was examined in the morning on the day of necropsy to determine the stage of the estrous cycle. Females were weighed and euthanized by carbon dioxide exposure followed by exsanguinations. A gross examination of the thoracic, abdominal and pelvic viscera were performed. The implantation sites were counted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The LD50 cut-off value at 5000 mg/kg is determined on the results of previous studies (QPS Taiwan Study No.: T65315001-GN). The NOAEL of repeated toxic of read-across substance is 400 mg/kg. The high dose at 1000 mg/kg was chosen with the aim of inducing toxic effects but not death or severe suffering.

Duration of treatment / exposure:

Calibration standard and check standard concentrations were determined based on the nominal concentrations.
Calibration standard samples at seven concentration levels (5, 10, 15, 30, 50, 80, and 100 µg/mL) and check standard samples at two concentration levels (10 and 80 µg/mL) were stored at -20C until analysis.
Data were acquired and processed (integrated) according to QPS SOP BA-T007 using the proprietary software application Shimadzu LCsolution (Version 1.25)/CLASS-Agent (Version 2.33).
Linear regression analysis calculations were performed with 1/x weighting using Watson LIMS (Version 7.4.1).
All statistics (e.g., Mean, S.D., %CV, and %RE) found in the data tables were calculated by Watson LIMS or based on the “precision as displayed” option of Microsoft® Excel.

Acceptance criteria for calibration standards and check standards were based upon the current version of SOP QPS-BA-004. For a run to be acceptable, a minimum of 75% of the total number of calibration standards in the calibration range should be within 10010.0% of their nominal values. The calibration curve must contain at least one calibration standard at both the LLOQ and ULOQ of the range. The regression coefficient of determination value (r2) must be >0.9900. In addition, at least 2/3 of the determined check standard concentrations of all check standard samples must be within 10010.0% of their nominal values, and at least half of the check standard samples at each concentration must be within 10010.0% of their nominal values.

The concentration of each sample was determined using a validated HPLC-UV method (QPS Taiwan Study Number T653-1901). Accuracy, homogeneity and stability were assessed based on the determined sample concentration.

Frequency of treatment:

Once daily until the day before necropsy

Details on study schedule:

There was a 15-day pre-exposure period from A6. A 2-week per-mating period followed a 14-day mating period. In mating period, all animals were copulated at a ratio of 1:1. The female was placed with the same male until pregnancy occurred. One control female (ID# 0008) pairing was unsuccessful in a continued 8 days, re-mating of females with proven males of the same group was conducted.
The gestation period was 22 or 23 days. Males were sacrificed after a dosing period of four weeks (D29). Maternal females with pups were sacrificed on P13. Presumed pregnant but non-delivering females were sacrificed on G25, and one non-copulated female was sacrificed on D52.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/mL

Doses / concentrations 2

Dose / conc.:

10 other: mg/mL

Doses / concentrations 3

Dose / conc.:

40 other: mg/mL

Doses / concentrations 4

Dose / conc.:

100 other: mg/mL

No. of animals per sex per dose:

45 males & 52 females including spare animals

Control animals:

yes, concurrent vehicle

Details on study design:

Group No. Color code Dose Level(mg/kg) Dose Concentration(mg/mL) Dose Volume(mL/kg) Number of animals (At least)
1 White 0 0 10 10M/11F
2 Green 100 10 10 10M/12F
3 Yellow 400 40 10 10M/12F
4 Red 1000 100 10 10M/12F
*Note: M = Males, F = Females

The LD50 cut-off value at 5000 mg/kg is determined on the results of previous studies (QPS Taiwan Study No.: T65315001-GN). The NOAEL of repeated toxic of read-across substance is 400 mg/kg. The high dose at 1000 mg/kg was chosen with the aim of inducing toxic effects but not death or severe suffering.

Examinations

Parental animals: Observations and examinations:

MORTALITY ASSESSMENT
Mortality and moribundity of all animals were checked twice daily except for necropsy day, on which mortality and moribundity was checked only once before necropsy.

CLINICAL OBSERVATIONS
During acclimation, all animals were observed by detailed clinical examinations on A16. During study phase, once weekly by detailed clinical examinations and once daily by cage-side observation on other days except on G0 and P0 (Deviation 4). The maternal nursing behavior including pup clean and warm, pups grouped together, evidence of nursing activity or milk in the pup stomach were observed on P0.

BODY WEIGHT:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
The food consumption was measured on the same day as body weights were measured. During the mating period, food consumption was not measured.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before dosing to select for the study females with regular cyclicity and was also monitored daily from Day 1 until evidence of mating.

Litter observations:

Each litter was examined after delivery (P0) to measure the number and sex of pups, stillbirths, live births, and the presence of gross abnormalities. Live pups were counted and sexed and weighed together by litter on P0, P4 (before and after litter size adjustment) and P13. The dead pups were recorded during lactation period.

Postmortem examinations (parental animals):

Male rats were weighed and euthanized by carbon dioxide exposure followed by exsanguinations. A gross examination of the thoracic, abdominal and pelvic viscera were performed. The organs listed in following Table were sampled, weighed and fixed in appropriate fixative.
Vaginal washing was examined in the morning on the day of necropsy to determine the stage of the estrous cycle. Females were weighed and euthanized by carbon dioxide exposure followed by exsanguinations. A gross examination of the thoracic, abdominal and pelvic viscera were performed. The implantation sites were counted. The organs listed in following Table were sampled and fixed in appropriate fixative.

Postmortem examinations (offspring):

Surplus pups after litter size adjustment (see section 4.7.6.3) on P4 were euthanasia through intraperitoneal injection of overdosed pentobarbital.
Dead pups and pups sacrificed with euthanasia through intraperitoneal injection of overdosed pentobarbital at P13, were examined externally for gross abnormalities. At P13 the thyroid from 1 male and 1 female pup per litter were preserved in 10% Neutral buffered formaldehyde.

Statistics:

Body weight, including body weight change, food consumption and organ weights were analyzed for statistical analysis by utilizing Pristima® (see Section 4.4.1). The specific reproductive parameters were analyzed by utilizing SigmaStatTM Statistical Software for WindowsTM, Release 3.0 (Jandel Scientific Inc., USA). A p value of < 0.05 was used for the determination of statistical significance. Statistical analysis was performed separately for each sex of study animals. Summary statistics (mean, standard deviation and number) were calculated.
The homogeneity of data were assessed first by Bartlett’s test or Equal Variance Test depending on Pristima® or SigmaStatTM was used. When the data was homogeneous, a one-way analysis of variance (ANOVA) was applied, then Dunnett’s LSD test was performed to compare each test article-treated group (Groups 2 to 4) against control group (Group 1) if the result of one-way ANOVA was significant. Otherwise, Kruskal-Wallis one way ANOVA on ranks was used when data were heterogeneous, then Dunn Rank Sum test was performed to compare each test article-treated group (Groups 2 to 4) against control group (Group 1) if the result of Kruskal-Wallis one way ANOVA was significant.

Reproductive indices:

Mating and fertility indices and estrous cycle were un-effected by treatment with CJ301.

Offspring viability indices:

F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment with CJ301.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs during the pre-mating, gestation and lactation period, body weights and feed consumptions were comparable across all groups. Mating and fertility indices and estrous cycle were un-effected by treatment with CJ301. F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment with CJ301. Reproductive evaluation in gross or histopathology in adult males and females and external gross examination in pups were unaffected by test article treatment.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the scheduled necropsy on D29, D52, G25 or P13. No absorption or prematurely delivered was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males, there were no statistically significant differences in body weight between control and treatment animals during study period. In Week 2 (Day 15), increased body weight gains was observed in Group 4 (3.36 g/day) compared to control (2.10 g/day). The increase was transient and was not reflected in the body weight on Day 15 or following days and was not considered to be an adverse effect.

In females, there were no statistically significant differences in body weight between control and treatment animals during study period including gestation and lactation periods. In Week 2 (Day 15), increased body weight gains was observed in Group 4 (2.23 g/day) compared to control (0.07 g/day). The increase was transient and was not reflected in the body weight on Day 15 or following days and was not considered to be an adverse effect.

Overall, treatment with the test article did not result in body weight changes in both genders.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males, statistically significantly increased food consumption was observed in Group 4 (32.47 g/day) compared to control (27.46g/day) on Week 2 (Day 15). There was concurrent change in body weight gain but was not reflected in the body weight on Day 15 and was not considered to be an adverse effect.
In females, statistically significantly increased food consumption was observed in Group 4 (21.40 g/day) compared to control (17.90g/day) on Week 2 (Day 15). There was concurrent change in body weight gain but was not reflected in the body weight on Day 15 and was not considered to be an adverse effect. There was no statistically significantly difference in food consumption between control and treated groups during gestation and lactation period.
Overall, treatment with the test article did not result in food consumption changes in both genders.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

In males, a positive indication of fertility was obtained for 8 of 10, 10 of 10, 9 of 10 and 10 of 10 animals in Groups 1 to 4, resulting in a fertility index of 90%, 100%, 90% and 100%, respectively.
In female, a positive indication of mating was obtained for 10 of 11, 12 of 12, 12 of 12, 12 of 12 animals in Groups 1 to 4. The mating index was 91%, 100%, 100% and 100% for Group 1 to 4, respectively. Females were mated usually in a 4-day period of cohabitation. There was one animal (ID# 0035) in Group 3 that showed a precoital interval of 6 days and one animals (ID# 0046) of Group 4 showed 5 days. The average Pre-coital period was 2.0, 2.2, 2.9 and 2.2 days for Group 1 to 4, respectively. Two Group 1 and one Group 3 animals (IDs# 0002, 0003, and 0025) with positive indications of copulation was later found not to be pregnant. The fecundity index was 80%, 100%, 92% and 100% for Group 1 to 4, respectively. The fertility index was 73%, 100%, 92% and 100% for Group 1 to 4, respectively. One control female (ID# 0008) was not copulated even re-mating with proven males of the same group was conducted.
Overall, no test article-related effects on fertility as assessed by time to mating, mating index, fecundity index, or fertility index (In-Text Table 2).

Details on results (P0)

Mortality
No mortality occurred during the whole duration of the study.

Clinical Observations
Treatment with the test article did not result clinical adverse effects in both genders during the study period.

Body Weight Development
Treatment with the test article did not result in body weight changes in both genders.
 
Food Consumption
Treatment with the test article did not result in food consumption changes in both genders.

Estrous Cycle
The test article did not have an effect of the estrus cycle.

Reproductive Performance
No test article-related effects on fertility as assessed by time to mating, mating index, fecundity index, or fertility index.

Litter Observation
There were no test article-related effects in implantation, no. of live pups at birth and on P4, pups gender difference, litter/pup weight on P0, P4 and P13, AGD measurement and no. of nipple retention. The lower birth index in Group 4 was noteworthy.

T4 Determination
Statistically significantly Increased T4 was observed in adult males and pups age of 13 days in Group 4. The increased value may be related to test article but were not considered to be adverse effect because still within the historical data range (male: 28-56 ng/mL; pups on P13: .36-64 ng/mL) and no concurrent change in histopathologic examination of thyroid/parathyroid (section 5.9).

Gross Examination, Organ Weight and Histopathology
There were no treatment-related microscopic changes in study animals including pups on P13 examined. Microscopic findings observed were considered spontaneous background changes common in this species, incidental, toxicologically irrelevant or biologically insignificant due to low incidence/severity grade or lack of dose response.

Overall, no test article-related gross, organ weight changes, or microscopic findings in the study animals examined were found.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

All animals survived until the scheduled necropsy on D29, D52, G25 or P13. No absorption or prematurely delivered was observed.

Bright yellow urine was observed in two females at Group 3 (IDs# 0026 and 0029), two females and one male of Group 4 (ID#s 0045, 0047 and 1032). The clinical signs may be test article-related. The findings were incidental and not considered to be adverse effects.

In males, some clinical signs scattered in treated groups including small to medium amount of hair loss in forelimb/head in two of Group 1, one of Group 2 and one of Group 4; and red or yellow hair stain in forelimbs in one Group 2 and one Group 4. These clinical signs were not considered to be test article-related due to absence of a dose-dependency, or also presence in the control group (Group 1).

In females, some clinical signs scattered in treated groups including small to large hair loss in abdomen/forelimb/thorax in five Groups 1 and 2, seven Group 3 and Group 4; red mild hair stain in forelimb/mouth/muzzle in two Groups 1 and 2, three Group 3 and one Group 4; and skin scars in one Group 1. These clinical signs were not considered to be test article-related due to absence of a dose-dependency, or also presence in the control group (Group 1).

One Group 3 female (ID# 0030) showed a mass in thorax then abrasion since Day 8 to P12 and was confirmed to be as hyperplasia/hypersecretion  by histopathologic examination and were regarded as incidental background changes.

Overall, treatment with the test article did not result clinical adverse effects in both genders during the study period.

Applicant's summary and conclusion

Conclusions:

CJ301 (100, 400 or 1000 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 56 days, depending on the time of copulation and gestation. CJ301 did not cause adverse effects in clinical observations, body weight, food consumption, male and female rats’ reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition and offspring parameters including litter weight, AGD and nipple retention. The no observed adverse effect level of CJ301 in rat’s reproductive performance was 1000 mg/kg.

Executive summary:

ABSTRACT

The purpose of this study was togeneratelimited information on the effects ofCJ301on male and female rats’ reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Methods

Animals were assigned to four groups and received vehicle (water for injection, WFI) orCJ301(Lot 8507) at dose levels of 100, 400 and 1000 mg/kg by oral gavage at 10 mL/kg for 28 consecutive days in males and 40–56 days in females. The parent males were sacrificed after the mating period (Day 29). One un-copulated control female was sacrificed on Day 52, one in each control and Group 3 females were copulated without delivery and sacrificed on Gestation Day 25; and others reared their offspring (F1) and then sacrificed on Postnatal Day 13. The pups were sacrificed on Postnatal Day 4 or 13. The study design is depicted below:

Group		Color	Dose Level	Dose Concentration	Dose Volume	Number of animals (At least)
No.		code	(mg/kg)	(mg/mL)	(mL/kg)
1		White	0^a	0	10	10M/11F
2		Green	100	10	10	10M/12F
3		Yellow	400	40	10	10M/12F
4		Red	1000	100	10	10M/12F
^a	WFI
Note	M = Males, F = Females

Measurements

The following parameters were evaluated: adult mortality and clinical observations, adult estrus cycle, adult and litter body weights, adult food consumptions, offspring parameters including number, sex, gross abnormalities, androgen-dependent developmental markers (e.g., anogenital distance (AGD) and number of nipple retention), adults and pups thyroid hormones (T4) determination, adults gross necropsy (including implantation site examination), organ weight and microscopic examinations of collected tissues.

Results

The stability, homogeneity and target concentrations ofdoseformulations were confirmed. The test article was not detectable in the control group.Treatment of animals withCJ301did not induce death, abortion or premature delivery. Increased T4 concentration was observed in adult males and pups of P13 but was not considered to be adverse effects because there were no concurrent changes in the histopathological examination of the thyroid gland. Clinical signs during the pre-mating, gestation and lactation period, body weights and feed consumptions were comparable across all groups. Mating and fertility indices and estrous cycle were un-effected by treatment with CJ301. F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment withCJ301. Reproductive evaluation in gross or histopathology in adult males and females and external gross examination in pups were unaffected by test article treatment.

Conclusion

CJ301(100, 400 or 1000 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 56 days, depending on the time of copulationand gestation.CJ301did not cause adverse effects in clinical observations, body weight, food consumption, male and female rats’ reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition and offspring parameters including litter weight, AGD and nipple retention.The no observed adverse effect level ofCJ301in rat’s reproductive performance was 1000 mg/kg.