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

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

From April 16, 2019 to October 31, 2019

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 source

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

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:
Temperature range: 19.4 - 24.4 ºC
Relative humidity range: 46.0 - 104.4%
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
Water: Autoclaved primary-filtered water was supplied ad libitum

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 NOAEL at 100 mg/kg is determined on the results of previous studies (QPS Taiwan Study No.: T65316002-GN). The high dose at 300 mg/kg was chosen with the aim of inducing toxic effects but not death or severe suffering.

Duration of treatment / exposure:

There was a 15-day pre-exposure period from A5. 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 female of Group 2 (ID# 0022), two females of Group 3 (ID#s 0024 and 0032) and one female of Group 4 (ID# 0046) pairing was unsuccessful in a continued 6 or 7 days, re-mating of females with proven males of the same group was conducted.
The gestation period was 21 to 23 days. Males were sacrificed after a dosing period of four weeks (D29). Maternal females with pups were sacrificed on P13. The presumed pregnant but non-delivering females were sacrificed on G25, and one misjudged un-copulated female in each Group 2 and 4 was sacrificed on Day 24.

Frequency of treatment:

Once daily until the day before necropsy

Details on study schedule:

Study initiation date: 16 APR 2019
Experimental starting date: 25 APR 2019
Initiation of dosing: 14 MAY 2019
Completion of in-life: 11 JUL 2019
Experimental completion date: 31 OCT 2019
Study completion date: Report sign date

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

45 males & 52 females including spare animals

Control animals:

yes, concurrent vehicle

Details on study design:

Group Color Dose Level Dose Concentration Dose Volume Number of animals
No. code (mg/kg) (mg/mL) (mL/kg) (At least)
1 White 0a 0 10 10M/11F
2 Green 30 3 10 10M/12F
3 Yellow 100 10 10 10M/12F
4 Red 300 30 10 10M/12F
*Note: M = Males, F = Females

The NOAEL at 100 mg/kg is determined on the results of previous studies (QPS Taiwan Study No.: T65316002-GN). The high dose at 300 mg/kg was chosen with the aim of inducing toxic effects but not death or severe suffering.

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 A13 and during the study phase, once weekly by detailed clinical examinations and once daily by cage-side observation on other days except on G0 and P0 (Deviation 2). 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 weights
Body weight was performed as follow:
Animal/Frequency
Male/D1, D8, D15, D22 and D29
Female/D1, D8, D15 and D22
G0, G7, G14, and G20;
P0, P4 and P13; and at termination.
Body weight gain or relative body weight gain were calculated.
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 the study females with regular cyclicity and also daily from Day 1 until evidence of mating was recorded.

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.
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.

Postmortem examinations (offspring):

Surplus pups after litter size adjustment on P4 were euthanized by intraperitoneal injection of overdosed pentobarbital.
Dead pups except from litter ID#s 0003, 0014, 0016, 0020 and pups euthanized by 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 CJ309.

Offspring viability indices:

F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment with CJ309.

Clinical signs:

no effects observed

Description (incidence and severity):

Test article-related clinical sings were observed in animals at 100 mg/kg or above which included black feces in all animals at 100 and 300 mg/kg, grey urine in five males at 300 mg/kg (ID#s 1031, 1034, 1035, 1036, 1037) and one female at 300 mg/kg (ID# 0036), and grey skin in five males at 300 mg/kg (ID#s 1031, 1032, 1033, 1035, 1036).
Some clinical signs scattered in control and treated groups included hair loss in limbs/thorax/abdomen in one control, two Group 2 (30 mg/kg) and one Group 3 of males and in nine control, four Group 2, eight Group 3 and one Group 4 of females; red hair stain in forelimbs/nose in one Group 2 of male and in two control, one Group 2, one Group 3 and 2 Group 4 of females; and abrasion of skin in one female of Group 3. 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.
Overall, treatment with the test article resulted black feces in both genders of Groups 3 and 4, grey urine in both genders of Group 4 and grey skin in males of Group 4 during the study period.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the scheduled necropsy on D29, G25 or P13. No absorption or prematurely delivered was observed. One non-copulated female each of Group 2 and 4 (ID#s 0022 and 0046), as a result of incorrect mating determination, were sacrificed without necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in body weight and body weight gain between control and treatment animals during the study period in both genders.
Overall, treatment with the test article did not result in body weight changes in both genders.

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:

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: 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 10 of 10, 10 of 10, 8 of 10 and 10 of 10 animals in control and Groups 2 to 4, resulting in a fertility index of 100%, 100%, 80% and 100%, respectively.
In females, a positive indication of mating was obtained for 11 of 11, 11 of 11, 12 of 12, 11 of 11 animals in control and Groups 2 to 4. The mating index was 100% for all groups. Females were mated usually in a 5-day period of cohabitation. There was one animal each control and Group 3 (ID#s 0003 and 0026) that showed a precoital interval of 6 or 7 days, and two Group 3 (ID#s 0024 and 0032) that showed a 8-day precoital interval. The average pre-coital period was 3.7, 3.0, 3.7 and 2.3 days for control and Groups 2 to 4, respectively. One control (ID# 0011) with positive indications of copulation was later found not to be pregnant. The fecundity index was 91% for control and 100% for Groups 2 to 4. The fertility index was 91%, 100%, 100% and 100% for control and Groups 2 to 4, respectively.
Overall, no test article-related effects on fertility as assessed by time to mating, mating index, fecundity index, or fertility index (In-Text Table 3).

Estrous Cycle
The individual animal data of estrus cycle examination showed the estrus cycles of all animals during 14 consecutive days of pre-mating period. The sequence of the stages was: proestrus, estrus, metestrus and diestrus. It was normal to observe extra diestrus and estrus stages. The estrus was cycling when the proestrus was followed by the estrus. A 3–4 day cycle was observed in almost all animals. Some animals showed 1 or 2 cycles during pre-mating, which included one control animals (ID# 0011), three animals in Group 2 (ID#s 0012, 0015 and 0022), two Group 3 (ID#s 0024 and 0035), and one Group 4 (ID# 0037). Except for ID# 0011, which was not pregnant and ID# 0022, which was misjudged as non-copulated (see 4.7.8.5), other animals mentioned above were mated in 1 8 days (see section 5.6) and confirmed pregnant. There were no apparent or dose-dependent differences in the average number of cycles during the pre-mating period, which was 3.2, 2.9, 3.3 and 3.3 in the control and Groups 2 to 4, respectively.
Overall, the test article did not have an effect of the estrus cycle.

Litter Observation
Except one Group 3 (ID# 0025), all pregnant females were parturition. The average gestation length was 22 days. The litter size was 10, 11, 11 and 11 in control and Groups 2 to 4, respectively, delivered live pups. The live litter size index was 100% in control, Groups 2 and 4 and 92% in Group 3.
The mean number of implantation sites per dam were 17.5, 16.6, 15.1 and 15.7 in control and Groups 2 to 4, respectively. The mean number of live pups per dam at birth was 15.8, 15.5, 14.0 and 14.5 in control and Groups 2 to 4, respectively, as well as 15.8, 15.5, 13.8 and 14.4 on P4. There were no statistically significant differences in the above three parameters between control and treatment groups. In one dam each control and Group 4 and four dams of Group 3 (ID#s 0003, 0024, 0027, 0028, 0030 and 0041), the number of post-implantations was equal or greater than 3, and one of Group 3 (ID# 0025) had 2 implantations but no parturition. The viability at birth was 96.3%, 98.8%, 100% and 99.4% in control and Groups 2 to 4, respectively, as well as 100%, 99.4%, 98.7% and 99.4% at P4. No externally abnormal pups were observed at birth in the control and treated groups.
At birth, 42.8% to 56.2% of pups were male and on P4, 44.3% to 51.3% pups were male. There was no apparent dose-dependency in gender distribution. The gender difference at birth and on P4 was a consequence of difficult sex determination at birth.
There were no statistically significant differences in litter weight at birth (P0), on P4 and P13 between control and treated groups. The mean pup weight was 6.7 g, 6.6 g, 6.9 g and 6.9 g at birth in control and Groups 2 to 4, respectively, as well as 10.6 g, 10.6 g, 11.3 g and 11.2 g on P4 before culling; and 35.2 g, 34.5 g, 35.5 g and 34.9 g after culling on P13. There were no apparent changes in pup weight between control and treatment groups at birth, on P4 or P13.
There were no statistically significant differences in average AGD, normalized AGD and pup weight at the time of AGD measurement between control and treatment groups in male and female pups. No nipple retention was observed in male pups on P13 in all groups.
Overall, there were no test article-related effects in implantation, no. of live pups at birth and on P4, pup gender difference, litter/pup weight on P0, P4 and P13, AGD measurement and no. of nipple retention.

T4 Determination
Statistically significantly increased T4 was observed in Group 4 in adult males. The increased T4 may have been related to test article treatment but was not considered to be an adverse effect because it was still within the historical data range (male: 28-56 ng/mL) and no concurrent change in histopathologic examination of thyroid/parathyroid at 1000 mg/kg in previous repeat dose study.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see "Remarks"

Remarks on result:

other: Generation: P + F1 (migrated information)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Litter Data
There were no effects of toxicological relevance on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4 of the dose groups, when compared to the respective control groups. There were no statistically significant changes noted for these litter data.

Litter Weight Data
There were no statistically or biologically significant effects on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4 of the dose groups, when compared to the respective control groups. Values were within the normal range of variation for animals of this strain.

 
Precoital Interval and Duration of Gestation
There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the respective control groups. Values were within the normal range of variation of historical control data.

Pre- and Post-Natal Data
There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups, when compared to the respective control groups.
A slight tendency towards a higher number of corpora lutea in the HDsup group (14.40) compared to the respective Csup group (12.75) is considered incidental, as no dose dependency was observed.
The HDsup group was noted to have a slightly higher percentage of pre-implantation loss compared to the other groups (HDsup: 20 %, C: 7 %, Csup: 10 %, LD: 12 %, MD: 4 %). Values were within the range of historical control data and lacked statistical significance and dose-dependency. Thus, this effect cannot be attributed to the treatment with the test item.


Reproductive Indices
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control groups.
A slightly reduced copulation index (number of copulated females / number of pairs x 100) was only observed in the Csup group (90 % compared to 100 % in all other groups) and is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals.
A slightly reduced delivery index in the C group (90 %) compared to 100 % in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally.
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control groups.
A slightly reduced copulation index (number of copulated females / number of pairs x 100) was only observed in the Csup group (90 % compared to 100% in all other groups) and is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals.
A slightly reduced delivery index in the C group (90 %) compared to 100 % in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally.
The viability index (no. of live offspring at day 4 / no. of live offspring at birth) X 100) was marginally lower in the Csup group (98.96 %), the LD group (99.09 %) and the HDsup group (99.17 %) when compared to 100 % in each the C and MD group. As this was attributed to the death of one pup in one single dam per group, respectively, and without dose-dependency, this finding is considered incidental.



Pup Survival Data
There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group.
A marginally higher mean mortality of pups between PND 1 and PND 4 was observed in the Csup group (1.04 %), the LD group (0.91 %) and the HDsup group (0.83 %) compared to 0.0 % in the C and MD group. As this outcome did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 103 of the Csup group and female no. 58 of the LD group, one pup was missing on PND 1, respectively. The loss of pups was attributed to cannibalism by the dam. One pup of female no. 132 of the HDsup group was found dead on PND 1.



Pup External Findings
No gross external abnormalities of toxicological relevance were observed in any of the groups.
A dark snout observed in one single pup of the C group, one single pup of the Csup group and two pups of the LD group is considered incidental.
There were few incidental further external findings such as a wound in two pups of the C group and one pup of the MD group as well as a swollen eye in one single pup of the LD group and a cold pup with pale skin in the MD group. These findings are considered to be spontaneous and not related to the test item.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

Conclusions:

CJ309 (30, 100 or 300 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 41 to 58 days, depending on the time of copulation and gestation. CJ309 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 NOAEL (no observed adverse effect level) of CJ309 in rat’s reproductive performance was 300 mg/kg.

Executive summary:

ABSTRACT

The purpose of this study was to generate limited information on the effects of CJ309 on 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) or CJ309 (Lot 8505) at dose levels of 30, 100 and 300 mg/kg by oral gavage at 10 mL/kg for 28 consecutive days in males and 41 to 58 days in females. The parent males were sacrificed after the mating period (Day 29). One misjudged un-copulated female in each Groups 2 and 4 was sacrificed on Day 24, one in each control and Group 3 females was copulated without delivery and sacrificed on Gestation Day 25 (G25); and others reared their offspring (F1) and then sacrificed on Postnatal Day 13 (P13). The pups were sacrificed on Postnatal Day 4 or 13. The study design is depicted below:

Group No.	Color code	Dose Level (mg/kg)	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of animal
1	White	0^a	0	10	10M/11F
2	Green	30	3	10	10M/12F
3	Yellow	100	10	10	10M/12F
4	Red	300	30	10	10M/12F

^aWFI

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 thyroxine (T4) determination, adults gross necropsy (including implantation site examination), organ weight and microscopic examinations of collected tissues.

 

Results

The stability, homogeneity and target concentrations of dose formulations were confirmed. The test article was not detected in the control group.

Treatment of animals with CJ309 did not induce death, abortion or premature delivery.

Treatment-related findings were clinically black feces in both sexes at 100 mg/kg and above, grey urine in both sexes at 300 mg/kg, clinically grey skin in males at 300 mg/kg, grossly blue discoloration of the kidney in both sexes at 100 mg/kg and above and blue discoloration of the testis in males at 100 mg/kg and above. The above findings were not considered to be adverse effects due to no changes in body weight, food consumption and histopathology examination.

Increased T4 concentration was observed in adult males but was not considered to be adverse effects because there were no histopathological thyroid gland changes at 1000 mg/kg/day examined in the previous repeat dose study (T65316002-GN).

Mating and fertility indices and estrous cycle were un-effected by treatment with CJ309. F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment with CJ309. Organ weight or histopathology in adult males and females and external gross examination in pups did not reveal test article-related effects on reproductive parameters.

Conclusion

CJ309 (30, 100 or 300 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 41 to 58 days, depending on the time of copulation and gestation. CJ309 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 NOAEL (no observed adverse effect level) of CJ309 in rat’s reproductive performance was 300 mg/kg.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

96

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this study was togeneratelimited information on the effects ofCJ309on 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) orCJ309(Lot 8505) at dose levels of 30, 100 and 300 mg/kg by oral gavage at 10 mL/kg for 28 consecutive days in males and 41 to 58 days in females. The parent males were sacrificed after the mating period (Day 29). One misjudged un-copulated female in each Groups 2 and 4 was sacrificed on Day 24, one in each control and Group 3 females was copulated without delivery and sacrificed on Gestation Day 25 (G25); and others reared their offspring (F1) and then sacrificed on Postnatal Day 13 (P13). The pups were sacrificed on Postnatal Day 4 or 13.

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 thyroxine (T4) determination, adults gross necropsy (including implantation site examination), organ weight and microscopic examinations of collected tissues.

 

The stability, homogeneity and target concentrations ofdoseformulations were confirmed. The test article was not detected in the control group.

Treatment of animals withCJ309did not induce death, abortion or premature delivery.

Treatment-related findings were clinically black feces in both sexes at 100 mg/kg and above, grey urine in both sexes at 300 mg/kg, clinically grey skin in males at 300 mg/kg, grosslyblue discoloration of the kidney in both sexes at 100 mg/kg and above and blue discoloration of the testis in males at 100 mg/kg and above. The above findings were not considered to be adverse effects due to no changes in body weight, food consumption and histopathology examination.

Increased T4 concentration was observed in adult males but was not considered to be adverse effects because there were no histopathological thyroid gland changes at 1000 mg/kg/day examined in the previous repeat dose study (T65316002-GN).

Mating and fertility indices and estrous cycle were un-effected by treatment withCJ309. F1 pup viability, litter body weight, sex, AGD and nipple retention were not affected by treatment withCJ309. Organ weight or histopathology in adult males and females and external gross examination in pups did not reveal test article-related effects on reproductive parameters.

CJ309(30, 100 or 300 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 41 to 58 days, depending on the time of copulationand gestation.CJ309did 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 NOAEL (no observed adverse effect level) ofCJ309in rat’s reproductive performance was 300 mg/kg.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

CJ309 (30, 100 or 300 mg/kg) was given orally by gavage once daily to male rats for 28 days and female rats for 41 to 58 days, depending on the time of copulation and gestation. CJ309 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 NOAEL (no observed adverse effect level) of CJ309 in rat’s reproductive performance was 300 mg/kg.