CJ303

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The study does not need to be conducted because a developmental toxicity study is available.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The NOAEL of CJ303 in rat’s maternal toxicity, or teratogenic and embyo-/fetotoxicity performance was 1000 mg/kg/day (OECD TG414).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20, 2018 to March 26, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.
Number of animals: 140 females, and the remaining male rats were used for mating.
Animal age on arrival: 84~90 days old
Body weight: 348-600g for males and 256-377g for females.

Animal Husbandry
Husbandry:
All animals were housed in plastic cages (L46.0xW31.5xH20.0cm) on cage racks (L170.0xW50.0xH160.0cm). There were 5 cages per layer, and 4 layers per rack. There were two rats at most per cage, and mated females were housed individually in cages. During the gestation period, the cage location of females was determined by the mating date.
Environmental conditions: Temperature and humidity in animal room were controlled automatically and daily recorded. The temperature was controlled within 21.1-24.4℃ and the relative humidity was controlled within 44-74% in the animal room. Lighting was controlled with the sequence of 12 hours light and 12 hours dark.

Food, Water and Bedding:
The sterilized SPF rodent growth and breeding feed supplied by Beijing Keao Xieli Feed Co., Ltd were used in this test; the lot number of the feed was 18613123. All the nutrition components and contaminants were within the permitted limits described in the national standard. An analysis report of diet was supplied by the supplier.
Drinking water was purified with HT-R01000 purity system and routinely analyzed (at least annually), and all parameters were within the permitted limits described in the national standard.
The corn cob bedding supplied by Beijing Keao Xieli Feed Co., Ltd was used in this test, and the lot number was 18039813, 18069813. The contaminants of the bedding were within the permitted limits described in the national standard for animal feed. An analysis report of bedding was supplied by the supplier.
Diet and water were available to the animals ad libitum during test. The diet, water and bedding were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal Welfare:
Animal use in the whole study complied with national animal welfare laws and regulations (instructive notions with respect to caring for laboratory animals, PRC Ministry of Science, 2006). The animal care and use activities required for conduct of this study were supervised and approved by the testing facility Animal Care and Use Committee (IACUC).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route of administration: Gavage method (by oral).
Volume of administration: 5 ml/kg.bw. Animals in vehicle control group were given the same volume of drinking water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on the related requirements in OECD414 and according to the results of the preliminary test, three dose levels were used in this study including 100, 300 and 1000mg/kg.d. A concurrent vehicle control group was included at the same time.

The analytical check of test item concentrations was conducted on the first and final preparation. Formulations of different test-item concentrations and the vehicle control were sampled and analyzed. To be considered acceptable, the actual analysis results for at least 3/5 samples of each concentration are to be within ±10% of the nominal concentration and the relative standard deviation is not more than 10%.
The details about the methods used and the results obtained were shown in the analysis report attached in this final report. The results indicate that the concentrations of the prepared test item were within acceptable limits.

Details on mating procedure:

For each mating, two (or one) female rats and one male rat were put together at afternoon and separated in the next morning. The females were examined for presence of the sperms through vaginal smear method. The day of finding the sperms was defined as the day 0 of pregnancy (GD0). The female rats which failed to mate were put together with male rats and examined again until the number of pregnant rats met the requirements of the test. In the test, there were twenty-nine mated females in each group.
All treated animals survived until the end of the study were euthanized by CO2 inhalation. The spare animals were used for other studies.

Duration of treatment / exposure:

Experimental start date: July 11, 2018
(the beginning of animal mating)
Animal mating period: July 11~19, 2018
Animal dosage: July 17~August 07, 2018
Animal dissection: August 01~08, 2018
Soft tissue examination of fetus: Novembr 01~07, 2018
Skeleton examination of fetus: Octobor 31~Novembr 08, 2018
Preparation of test item: July 16~August 03, 2018

Frequency of treatment:

Schedule of administration: All animals were dosed daily in the morning during the day5~19 of pregnancy (GD5~GD19). The dosing volume for each animal was adjusted, based on the latest body weight.

Duration of test:

Experimental start date: July 11, 2018
(the beginning of animal mating)
Experimental completion date: November 08, 2018

Dose / conc.:

0 mg/kg bw/day

Remarks:

Solvent control

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose level

Dose / conc.:

300 mg/kg bw/day

Remarks:

Middle dose level

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose level

No. of animals per sex per dose:

For each mating day, positive females were allocated to four groups based on body weight. All animals were identifiable by the individual cage card and be marked on fur at the same time. Details are shown as following:
Group Dose level (mg/kg.d) Sex Number of animals Animal Code

Solvent control 0 female 29 2000-2028
Low-dose level 100 female 29 2100-2128
Mid-dose level 300 female 29 2200-2228
High-dose level 1000 female 29 2300-2328

Control animals:

yes, concurrent vehicle

Details on study design:

Prior to the start of this study, a preliminary test was performed in this lab. In the test, one dose group of 1000mg/kg.d and solvent control group were included. There were five female rats in each group. All animals were treated by gavage for two weeks. The animals were observed daily and weighed once per three days.
No dead and any toxic symptom were observed in the all dosed animals. At the same time, the body weights of the animals in the dosed group had no significant decrease compared to the control.

In this test, animal drinking water was used as solvent. As the preparation, calculated the theoretical weight and range of the test item according to the prepared volume and concentration shown below, weighed the test item and placed into a calibrated grinding-mouth jar, poured some vehicle into the jar and stirred with a glass rod to dissolve test item completely. After that, washed the glass rod with vehicle and poured the washing water into the jar. Finally, added vehicle to the calibrated scale line on the jar, shook well and labeled it for use.
Group Dose (mg/kg.d) Volume of administration (ml/kg.bw) Theoretical concentration (mg/ml)
Solvent control 0 5 0
Low dose level 100 5 20.0
Middle dose level 300 5 60.0
High dose level 1000 5 200.0
Note: Theoretical Concentration = dose/volume of administration
Theoretical weight of test item = prepared volume (ml) x concentration (mg/ml)
Weight range (the lowest value) = theoretical weight (g) x 98%
Weight range (the highest value) = theoretical weight (g) x 102%
Based on the stability analysis result in the validation test, the test item formulations were prepared and stored at RT no longer than four days based on the stability analysis result in the validation test, and were subpackaged as use. After administration, the remainder will be kept temporarily in special cabinet in building A and was transported to external professional environmental protection company for disposal.

Maternal examinations:

Maternal Data:
-Number of animals at test start, number of animals surviving, number of pregnant animals, number of animals with total intrauterine mortality
-Clinical signs (by gestation day)
-Mortality (by gestation day), if any
-Body weight and body weight gain: mean ± SD.
-Corrected body weight on GD20 (body weight - gravid uterine weight) and corrected body weight gain: mean ± SD.
-Net body weight change (body weight on GD20 – body weight on GD5 – gravid uterine weight): mean ± SD.
-Food consumption: mean ± SD.
-Gravid uterine weight

Fetal examinations:

Caesarean Section and Necropsy Data:
-Number of corpora lutea: mean ± SD.
-Number of implantations: mean ± SD.
-Number of viable fetuses: mean ± SD.
-Percent of viable fetuses
-Number and percent of intrauterine mortality: Sub-divided into pre-implantation loss, post-implantation loss, early and late embryonic as well as fetal death.
-Fetal body weight (accuracy 0.001g): mean ± SD.

Statistics:

Calculate the average and standard deviation of the data for each group including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses and body weight of fetuses, etc. All of these data were statistic analyzed by decision tree. The data about abnormal clinical sign, ratio of absorptive fetus, dead fetus and viable fetus, and incidence of individual anomalies were evaluated by the chi-square test. All statistical analysis was done in SPSS 17.0.

Calculatioins:
Pre-implantation loss: %, group mean
(Number of corpora lutea - Number of implantation)x100/Number of corpora lutea

Post-implantation loss: %, group mean
(Number of implantations - Number of viable fetuses)x100/Number of implantations

Sex distribution: %, group mean
Number of male (female) fetuses x 100/Number of fetuses

External abnormalities/litter: %, group mean
Number of fetuses with abnormality x 100/Number of fetuses

Visceral abnormalities/litter: %, group mean
Number of fetuses with abnormality x 100/Number of fetuses

Skeletal abnormalities/litter: %, group mean
Number of fetuses with abnormality x 100/Number of fetuses

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All of rats in the dose groups had blue faeces after being administrated, but which was considered to be caused with the color of the test item and without adverse effect. All rats in the vehicle control group exhibited no symptoms.

Mortality:

no mortality observed

Description (incidence):

No animal in all groups was dead in the test.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the administration period, the body weight of pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05). The body-weight gain of pregnant rats in the low- and high- dose groups had a significantly increased from GD8 to GD11 (P≤0.05 or P≥0.01), but that was considered without toxicological significance. At the same time, the mean corrected body-weight gain during the gestation period and the mean maternal net body-weight gain from the day of treatment (GD5) to the termination (GD20) in the pregnant rats in all dose groups had no significant difference compared with the control group (P≥0.05).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant difference in food consumption of the pregnant rat during the treated period was observed in all dose groups as compared with the vehicle control group (P>0.05).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant difference in the gravid-uterine weight and total placenta weight was observed in all dosed groups, compared with the vehicle control group (P>0.05).

Number of abortions:

no effects observed

Description (incidence and severity):

Within all dose-groups, no statistically significant difference was observed in the percent of absorbed fetuses compared with the vehicle control group (P>0.05).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

no statistically significant increase was observed in the pre-implantation loss and the post-implantation loss compared with the control group (P>0.05).

Dead fetuses:

no effects observed

Description (incidence and severity):

Within all dose-groups, no statistically significant difference was observed in the percent of dead fetuses compared with the vehicle control group (P>0.05).

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

food consumption and compound intake

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference in body weight of live fetuses was observed in all dosed groups, compared with the control group (P>0.05).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant difference in sex distribution of live fetuses was observed in all dosed groups, compared with the control group (P>0.05).

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus respectively in the low- and high-dose groups showed signs of anury, 2, 4, 2 and 3 fetuses in the control group, low-, mid- and high- dose groups showed signs of hydronephrosis in one or double kidneys, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05) and which were considered as incidental findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus respectively in the low- and high- dose groups showed signs of anury, 2, 4, 2, and 3 fetuses in the control group, low-, mid- and high- dose groups showed signs of hydronephrosis in one or double kidneys, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05) and which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations except that some examined fetuses had less than six sternal ossification points, but there was no difference in the mean number of sternal ossification points in all dosed groups (P>0.05).

Developmental effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

no

Table 1-1. Observation results of animal symptoms (symptoms/animal number)

Gestation day	Vehicle control	Low-dose	Mid-dose	Hgih-dose
	0 mg/kg.d.	100 mg/kg.d.	300 mg/kg.d.	1000 mg/kg.d.
Number#	29	29	29	29
GD0-4	-	-	-	-
GD5	-	-	-	-
GD6	-	B/29**	B/29**	B/29**
GD7	-	B/29**	B/29**	B/29**
GD8	-	B/29**	B/29**	B/29**
GD9	-	B/29**	B/29**	B/29**
GD10	-	B/29**	B/29**	B/29**
GD11	-	B/29**	B/29**	B/29**
GD12	-	B/29**	B/29**	B/29**
GD13	-	B/29**	B/29**	B/29**
GD14	-	B/29**	B/29**	B/29**
GD15	-	B/29**	B/29**	B/29**
GD16	-	B/29**	B/29**	B/29**
GD17	-	B/29**	B/29**	B/29**
GD18	-	B/29**	B/29**	B/29**
GD19	-	B/29**	B/29**	B/29**
GD20	-	B/29**	B/29**	B/29**

Note: #Number of all tested rats including non-pregnant females

GD5~19 are dosing period; GD20 is the day of scheduled kill;

---: No symptom; B: Blue faeces

Statistic analysis by Chi-square test in SPSS17.0;

Compared with the control group, **-p≤0.01.

Table 1-2. Body weight and body-weight gain of pregnant rats (g)

Gestation day	Vehicle control	Low-dose	Mid-dose	Hgih-dose
	0 mg/kg.d.	100 mg/kg.d.	300 mg/kg.d.	1000 mg/kg.d.
Number of pregnant rats	26	27	27	27
GD0	292.1±17.7	294.9±23.4	289.4±24.6	292.2±23.3
GD5	318.0±20.7	322.7±26.5	321.0±22.4	316.1±25.7
GD8	328.7±19.0	333.8±25.6	334.2±22.6	330.1±26.3
GD11	342.6±19.5	352.9±27.2	351.9±25.0	350.0±27.8
GD14	360.0±22.2	368.4±27.4	367.7±26.5	364.0±31.5
GD17	393.5±27.8	400.9±30.1	401.5±30.4	394.8±35.8
GD20	442.9±33.5	452.8±33.0	448.0±35.6	443.4±43.2
GD5-8	10.8±5.4	11.1±6.7	13.3±6.0	14.0±6.0
GD8-11	13.9±8.4	19.1±6.1	17.6±8.3	19.8±7.4**
GD11-14	17.3±8.3	15.5±5.5	15.9±5.7	14.1±8.2
GD14-17	33.5±8.6	32.5±8.0	33.7±7.5	30.7±8.6
GD17-20	49.5±10.3	51.9±8.0	46.5±9.8	48.6±12.3
GD5-20	125.0±20.5	130.1±15.8	127.0±22.8	127.3±30.0
Corrected body-weight on GD20	355.4±22.7	363.8±27.0	363.6±26.8	358.0±35.0
Corrected body-weight gain	63.3±13.0	68.9±11.8	74.2±25.0	65.8±23.3
Net body-weight change	37.4±8.9	41.1±11.8	42.7±12.3	41.9±21.2

Note: Data expressed as mean ± SD

Statisctic analysis by ANOVA, followed by Duncan test as p<0.05 in SPSS17.0;

Compared woth the control group, *-p≤0.05, **-p≤0.01

Corrected body-weight on GD20 = Body weight on GD20 - Gravid uterine weight

Corrected body-weight gain = Correted Body weight on GD20 - Body weight on GD0

Net body-weight change = Body weight on GD20 - Body weight on GD5 - Gravid uterine weight

Table 1-3. Food consumption of animals (g/day)

Gestation day	Vehicle control	Low-dose	Mid-dose	Hgih-dose
	0 mg/kg.d.	100 mg/kg.d.	300 mg/kg.d.	1000 mg/kg.d.
Number of pregnant rats	26	27	27	27
GD7-8	23.6±3.0	25.8±4.2	26.0±4.1	24.3±4.7
GD10-11	24.5±3.8	25.6±3.9	26.2±4.9	25.6±5.2
GD13-14	23.7±3.1	25.4±2.8	26.1±4.4	26.0±4.2
GD16-17	27.6±3.3	27.0±4.2	28.7±5.3	28.4±4.3
GD19-20	26.9±3.7	27.3±3.3	27.4±4.0	27.5±4.5

Note: Data expressed as mean ± SD

Statisctic analysis by ANOVA, followed by Duncan test as p<0.05 in SPSS17.0.

Table 1-4. The devlopmental endpoint results

Parameter	Vehicle control	Low-dose	Mid-dose	Hgih-dose
	0 mg/kg.d.	100 mg/kg.d.	300 mg/kg.d.	1000 mg/kg.d.
Mated female (n)	29	29	29	29
Pregnant female (n)	26	27	27	27
Corpara lutea
Total number	438	468	440	444
Mean±SD	16.8±3.3	17.3±2.8	16.3±3.6	16.4±3.1
Implantation
Total number	405	431	406	410
Mean±SD	15.6±3.2	16.0±2.6	15.0±3.4	15.2±3.1
Gravid uterine weight (g)
Mean±SD	87.5±18.3^A	89.0±14.1	84.4±20.6	85.4±18.0
Total placental weight (g)
Mean±SD	11.6±2.7^A	11.5±2.4	10.7±2.8	11.1±2.5
Live fetus
Number of litters	26	27	27	27
Total number	396	418	393	397
Mean ± SD	15.2±3.3	15.5±2.6	14.6±3.5	14.7±3.1
Percent (%)	97.8(396/405)^C	97.0(418/431)	96.8(393/406)	96.8(397/410)
Absorbed fetus
Total number^1	7	9	10	12
Mean ± SD	8	13	13	13
Percent (%)	2.0(8/405)^C	3.0(13/431)	3.2(13/406)	3.2(13/410)
Dead fetus
Total number^2	1	0	0	0
Mean ± SD	1	0	0	0
Percent (%)	0.2(1/405)^C	0(0/431)	0(0/406)	0(0/410)
Pre-implantation loss (%)	0.2(1/405)^C	7.9(37/468)	7.7.(34/440)	7.7.(34/444)
Post-implantation loss(%)	2.2(9/405)^C	3.0(13/431)	3.2(13/406)	3.2(13/410)

Note: ¹Including early absorbed fetuses and late absorbed fetuses

²Including early dead fetuses and late dead fetuses

^CStatistic analysis by Chi-square test in SPSS17.0;

^AStatisctic analysis by ANOVA, followed by Duncan test as p<0.05 in SPSS17.0.

Compared with the control group, *-p≤0.05

Table 1-5. Fetal examination results

Parameter	Vehicle control	Low-dose	Mid-dose	Hgih-dose
	0 mg/kg.d.	100 mg/kg.d.	300 mg/kg.d.	1000 mg/kg.d.
Body weight of fetus (g)
Mean ± SD	3.59±0.27	3.68±0.26	3.65±0.25	3.68±0.27
Sex
Number of male fetuses	199	195	198	212
Sex distribution(%) (males/total)	50.3(199/396)^C	46.7(195/418)	50.4(198/393)	53.4(212/397)
Number of female fetuses	197	223	195	185
Sex distribution(%) (females/total)	49.7(197/3396)	53.3(223/418)	49.6(195/393)	46.6(185/397)
External exam
Number of examined fetuses/litter	396/26	418/27	393/27	393/27
Number of external normal fetuses	396	417	393	396
Number of external abnormal fetuses/litter	2/1	4/2	2/1	3/2
Anury(%)	0(0/396)^C	0.2(1/418)	0(0/393)	0.3(1/397)
External abnormalities (%)	0(0/396)^C	0.2(1/418)	0(0/393)	0.3(1/397)
External abnormalities litter (%)	0(0/26)^C	3.7(1/27)	0(0/27)	3.7(1/27)
Soft tissue exam
Number of examined fetuses/litter	205/26	216/27	203/27	202/27
Number of visceral normal fetuses	203	212	201	199
Number of visceral abnormal fetuses/litter	2/1	4/2	2/1	3/2
left side pyelectasis (%)	1.0(2/205)^C	0(0/216)	1.0(2/203)	0.5(1/202)
right side pyelectasis (%)	0(0/205)^C	0.5(1/216)	0(0/203)	0.5(1/202)
bilateral pyelectasis (%)	0(0/205)^C	1.4(3/216)	0(0/203)	0.5(1/202)
Visceral abnormalities (%)	1.0(2/205)^C	1.9(4/216)	1.0(2/203)	1.5(3/202)
Visceral abnormalities litter(%)	3.8(1/26)^C	7.4(2/27)	3.7(1/27)	7.4(2/27)
Skeleton exam
Number of examined fetuses/litter	191/26	202/27	190/26	195/27
Number of skeletal normal fetuses	191	202	190	195
Skeletal abnormalities (%)	0	0	0	0
Sternal ossification points
Mean ± SD	5.62±0.39^A	5.57±0.42	5.62±0.32	5.63±0.49

Note: ^CStatistic analysis by Chi-square test in SPSS17.0;

^AStatisctic analysis by ANOVA, followed by Duncan test as p<0.05 in SPSS17.0.

Table 2-1. Individual body weight (g)

Animal codes	GD0	GD5	GD8	GD11	GD14	GD17	GD20
2000	256	283	303	323	336	360	407
2001	276	290	301	320	338	366	426
2002	277	292	310	323	331	361	419
2003	285	310	321	331	365	402	460
2004	286	305	310	323	344	369	419
2005	292	302	310	318	328	339	357
2006	294	308	328	340	360	390	439
2007	308	335	353	372	391	423	481
2008	323	349	360	376	384	420	468
2009	271	295	304	310	313	310	310
2010	272	296	315	324	340	375	420
2011	291	326	332	340	320	336	336
2012	291	317	324	331	349	378	422
2013	301	323	329	350	374	416	470
2014	306	351	353	380	400	432	488
2015	306	323	331	337	352	397	447
2016	338	373	381	372	416	464	527
2017	274	310	320	344	350	393	452
2018	278	313	320	342	360	396	446
2019	287	301	308	320	337	360	398
2020	288	332	342	362	380	422	456
2021	296	320	337	360	362	392	443
2022	299	325	330	352	371	420	466
2023	305	335	341	360	373	408	468
2024	310	340	347	357	364	361	362
2025	275	310	328	324	342	378	409
2026	276	310	318	328	345	373	432
2027	293	314	328	342	356	396	444
2028	313	340	346	354	375	400	452
2100	260	278	300	304	324	352	395
2101	276	307	328	338	353	386	442
2102	277	291	316	328	340	383	432
2103	283	305	316	338	358	391	450
2104	286	315	323	354	370	401	453
2105	291	305	324	345	367	396	457
2106	295	320	331	353	371	400	457
2107	306	340	350	380	391	430	480
2108	323	360	372	388	409	436	481
2109	267	297	309	330	341	381	435
2110	274	308	320	330	352	387	438
2111	289	320	331	348	378	387	454
2112	293	310	326	334	336	340	358
2113	299	313	327	351	363	403	455
2114	309	327	345	344	345	350	334
2115	295	318	330	350	371	400	446
2116	373	403	421	438	457	496	554
2117	270	300	305	320	330	360	406
2118	283	313	322	339	350	377	412
2119	286	315	320	342	358	382	440
2120	289	325	325	346	364	389	446
2121	296	316	320	334	350	376	424
2122	301	329	340	360	370	410	466
2123	305	336	340	362	376	406	442
2124	311	340	340	364	371	402	446
2125	339	378	378	400	410	452	506
2126	280	310	322	351	356	395	438
2127	293	320	333	350	367	396	456
2128	315	350	369	386	400	450	514
2200	264	289	298	315	326	365	410
2201	274	297	320	336	358	385	432
2202	278	296	321	338	354	390	431
2203	283	305	325	346	360	395	451
2204	286	300	310	334	344	382	428
2205	290	315	330	360	377	404	446
2206	296	328	350	368	356	383	381
2207	302	320	340	375	389	426	478
2208	328	356	378	399	416	446	500
2209	263	296	305	320	342	370	418
2210	277	305	310	322	350	390	444
2211	287	315	324	326	331	350	362
2212	296	310	326	342	356	383	413
2213	298	320	342	362	377	416	462
2214	332	370	388	387	416	455	503
2215	285	323	330	349	366	392	449
2216	208	327	345	356	383	417	468
2217	270	305	321	326	343	367	415
2218	284	337	347	370	384	413	466
2219	285	303	312	322	332	356	392
2220	291	320	329	347	362	401	456
2221	294	314	320	340	351	378	421
2222	301	346	360	382	400	446	492
2223	305	337	347	370	384	416	452
2224	320	360	366	396	412	463	518
2225	332	366	371	393	406	442	486
2226	290	316	328	335	350	390	439
2227	292	318	331	352	360	402	461
2228	326	363	370	399	400	387	391
2300	269	289	300	323	343	370	416
2301	270	300	323	334	341	376	420
2302	279	305	318	344	350	382	442
2303	282	304	316	328	346	370	423
2304	287	313	331	355	372	414	462
2305	288	305	320	345	376	408	470
2306	298	318	333	355	370	400	452
2307	300	334	358	378	406	442	500
2308	349	374	389	413	436	468	526
2309	260	280	300	325	330	360	409
2310	283	300	316	320	335	370	397
2311	285	321	341	356	375	407	452
2312	297	312	330	343	358	399	440
2313	298	313	323	336	353	384	432
2314	258	289	300	313	313	312	307
2315	281	310	312	337	350	372	428
2316	257	278	294	314	330	350	406
2317	262	290	303	311	320	350	380
2318	284	310	330	351	366	399	462
2319	285	307	315	322	310	308	315
2320	291	313	320	346	350	392	344
2321	294	300	320	352	367	396	450
2322	304	316	321	342	355	387	441
2323	304	334	338	360	364	386	416
2324	330	355	378	398	406	446	493
2325	330	363	375	397	417	452	508
2326	290	326	332	357	372	401	461
2327	291	319	324	358	371	406	363
2328	345	380	392	409	424	450	486

Table 2-2. Individual body weight gains (g)

Animal codes	GD5-8	GD8-11	GD11-14	GD14-17	GD17-20	GD5-20	Corrected body weight on GD20	Corrected body weight gain	Net body weight change
2000	20	20	13	24	47	124	317.154	61.154	34.154
2001	11	19	18	28	60	136	339.434	63.434	49.434
2002	18	13	8	30	58	127	330.362	53.362	38.362
2003	11	10	34	37	58	150	356.019	71.019	46.019
2004	5	13	21	25	50	114	325.344	39.344	20.344
2005	8	8	10	11	18	55	337.794	47.794	37.794
2006	20	12	20	30	49	131	358.555	64.555	50.555
2007	18	19	19	32	58	146	382.344	74.344	47.344
2008	11	16	8	36	48	119	385.620	62.620	36.620
2009	9	6	3	-3	0	15	-	-	-
2010	19	9	16	35	45	124	338.786	66.786	42.786
2011	6	8	-20	16	0	10	-	-	-
2012	7	7	18	29	44	105	350.332	59.332	33.332
2013	6	21	24	42	54	147	373.353	72.353	50.353
2014	2	27	20	32	56	137	381.222	75.222	30.222
2015	8	6	15	45	50	124	346.750	40.750	23.750
2016	8	-9	44	48	63	154	423.443	85.443	50.443
2017	10	24	6	43	59	142	356.779	82.779	46.779
2018	7	22	18	36	50	133	356.765	78.765	43.765
2019	7	12	17	23	38	97	330.952	43.952	29.952
2020	10	20	18	42	34	124	373.035	85.035	41.035
2021	17	23	2	30	51	123	342.738	46.738	22.738
2022	5	22	19	49	46	141	360.443	61.443	35.443
2023	6	19	13	35	60	133	366.134	61.134	31.134
2024	7	10	7	-3	1	22	-	-	-
2025	18	-4	18	36	31	99	343.857	68.857	33.857
2026	8	10	17	28	59	122	341.436	65.436	31.436
2027	14	14	14	40	48	130	351.351	58.351	37.351
2028	6	8	21	25	52	112	368.478	55.478	28.478
2100	22	4	20	28	43	117	328.945	68.945	50.945
2101	21	10	15	33	56	135	351.419	75.419	44.419
2102	25	12	12	43	49	141	329.855	52.855	38.855
2103	11	22	20	33	59	145	352.740	69.740	47.740
2104	8	31	16	31	52	138	355.453	69.453	40.453
2105	19	21	22	29	61	152	369.646	78.646	64.646
2106	11	22	18	29	57	137	371.350	76.350	51.350
2107	10	30	11	39	50	140	388.158	82.158	46.158
2108	12	16	21	27	45	121	408.609	85.609	48.609
2109	12	21	11	40	54	138	341.342	74.342	44.342
2110	12	10	22	35	51	130	346.575	72.575	38.575
2111	11	17	30	9	67	134	371.106	82.106	51.106
2112	16	8	2	4	18	48	-	-	-
2113	14	24	12	40	52	142	361.109	62.109	48.109
2114	18	-1	1	5	-16	7	-	-	-
2115	12	20	21	29	46	128	360.551	65.551	42.551
2116	18	17	19	39	58	151	445.890	72.890	42.890
2117	5	15	10	30	46	106	321.761	51.761	21.761
2118	9	17	11	27	35	99	359.109	76.109	46.109
2119	5	22	16	24	58	125	354.072	68.072	39.072
2120	0	21	18	25	57	121	347.447	58.447	22.447
2121	4	14	16	26	48	108	341.210	45.210	25.210
2122	11	20	10	40	56	137	347.571	46.571	18.571
2123	4	22	14	30	36	106	365.931	60.931	29.931
2124	0	24	7	31	44	106	370.260	59.260	30.260
2125	0	22	10	42	54	128	399.240	60.240	21.240
2126	12	29	5	39	43	128	362.240	82.240	52.240
2127	13	17	17	29	60	136	364.113	71.113	44.113
2128	19	17	14	50	64	164	405.830	90.830	55.830
2200	9	17	11	39	45	121	312.228	48.228	23.228
2201	23	16	22	27	50	138	350.727	76.727	53.727
2202	25	17	16	36	41	135	350.205	72.205	54.205
2203	20	21	14	35	56	146	356.365	73.362	51.362
2204	10	24	10	38	46	128	331.831	45.831	31.831
2205	15	30	17	27	42	131	369.951	79.951	54.951
2206	22	18	-12	27	-2	53	-	-	-
2207	20	35	14	37	52	158	380.645	78.645	60.645
2208	22	21	17	30	54	144	398.114	70.114	42.114
2209	9	15	22	28	48	122	324.661	61.661	28.661
2210	5	12	28	40	54	139	346.883	69.883	41.883
2211	9	2	5	19	12	47	354.496	67.496	39.496
2212	16	16	14	27	30	103	372.138	76.134	62.138
2213	22	20	15	39	46	142	375.962	77.962	55.962
2214	18	-1	29	39	48	133	404.845	72.845	34.845
2215	7	19	17	26	57	126	366.638	81.638	43.638
2216	18	11	27	34	51	141	384.618	176.618	57.618
2217	16	5	17	24	48	110	331.257	61.257	26.257
2218	10	23	14	29	53	129	382.504	98.504	45.504
2219	9	10	10	24	36	89	321.312	36.312	18.312
2220	9	18	15	39	55	136	357.101	66.101	337.101
2221	6	20	11	27	43	107	352.493	58.493	38.493
2222	14	22	18	46	46	146	399.534	98.534	53.534
2223	10	23	14	32	36	115	372.852	67.852	35.852
2224	6	30	16	51	55	158	406.180	86.180	46.180
2225	5	22	13	36	44	120	405.001	73.001	39.001
2226	12	7	15	40	49	123	339.320	49.320	23.320
2227	13	21	8	42	59	143	370.038	78.038	52.038
2228	7	29	1	-13	4	28	-	-	-
2300	11	23	20	27	46	127	335.075	66.075	46.075
2301	23	11	7	35	44	120	343.315	73.315	43.315
2302	13	26	6	32	60	137	345.224	66.224	40.224
2303	12	12	18	24	53	119	334.687	52.687	30.687
2304	18	24	17	42	48	149	364.354	77.354	51.354
2305	15	25	31	32	62	165	361.275	73.275	56.275
2306	15	22	15	30	52	134	379.124	81.124	61.124
2307	24	20	28	36	58	166	399.166	99.166	65.166
2308	15	24	23	32	58	152	413.790	64.790	39.790
2309	20	25	5	30	49	129	338.976	78.976	58.976
2310	16	4	15	35	27	97	327.142	44.142	27.142
2311	20	15	19	32	45	131	359.511	74.511	38.511
2312	18	13	15	41	41	128	346.103	49.103	34.103
2313	10	13	17	31	48	119	343.598	45.598	30.598
2314	11	13	0	-1	-5	18	-	-	-
2315	2	25	13	22	56	118	353.069	72.069	43.069
2316	16	20	16	20	56	128	328.755	71.755	50.755
2317	13	8	9	30	30	90	327.070	65.070	37.070
2318	20	21	15	33	63	152	365.190	81.190	55.190
2319	8	7	-12	-2	7	8	262.907	-22.093	-44.093
2320	7	26	4	42	-48	31	252.550	-38.450	-60.450
2321	20	32	15	29	54	150	368.430	74.430	68.430
2322	5	21	13	32	54	125	344.425	40.425	28.425
2323	4	22	4	22	30	82	-	-	-
2324	23	20	8	40	47	138	381.017	51.017	26.017
2325	12	22	20	35	56	145	413.683	83.683	50.683
2326	6	25	15	29	60	135	363.854	73.854	37.854
2327	5	34	13	35	-43	44	271.334	-19.666	-47.666
2328	12	17	15	26	36	106	447.454	102.454	67.454

Conclusions:

The exposure of gestating rats to different doses of CJ303 by gavage during GD5~GD19 did not result in any death or significant specific toxicity at the dose of 1000 mg/kg.d. At the same time, no treatment related effect on the body weight gain and food consumption of pregnant rats was observed. Based on these results, it is concluded that the test item had no maternal toxicity at this dose level.
In all dose groups, no adverse effect was observed in all prenatal reproductive parameters. The fetal examination showed that no adverse effect in the survival, body weight and sex distribution of live fetuses was observed in all dosed groups, additionally, no adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations. Based on these results, it is concluded that the test item had no developmental toxicity and teratogenicity at the dose of 10000 mg/kg.d.
In conclusion, under the conditions of this investigation, CJ303 administered to pregnant SD rats by gavage in water, did not produce any dose-related maternal toxicity, teratogenic and embryo-/fetotoxicity at the dose of 1000 mg/kg.d.
Based on these findings, the following effect levels, No Observed Adverse Effect Level (NOAEL) were derived:
NOAEL(maternal toxicity): 1000 mg/kg bw.d
NOAEL(embryo-/fetotoxicity): 1000 mg/kg bw.d
NOAEL(teratogenicity): 1000 mg/kg bw.d

Executive summary:

Introduction

This study is conducted to detect maternal and developmental toxicity during the “critical” phase of organogenesis, in particular focused on structural abnormalities in and altered growth of the fetus caused by CJ303 after prenatal exposure. The method of this test was designed to be compatible with OECD414.

Method

This study was conducted in SD rats and all animals were SPF grade. Based on the results of a preliminary range finding test, the animals were treated in the main study with test item at doses of 100, 300 and 1000mg/kg.d. A concurrent vehicle control group was included in the study. There were 29 mated female rats in each group, which resulted in 26, 27, 27 and 27 pregnant rats in the vehicle control group and the low-, mid- and high-, respectively. In the test, all animals were administered the test item by gavage daily during the day 5~19 of pregnancy (GD5~GD19), and were euthanized with CO2 on GD20. After that, removed the uterus of each animal immediately ascertained the pregnancy status, weighed gravid uterine and total placenta, counted the number of corpora lutea, adsorptive fetuses, dead fetuses and viable fetuses for each pregnant animal. Each fetus was examined for external alterations, and the sex and body weight of each fetus were determined at the same time. The fetus was examined for soft tissue or skeleton after being prepared.

Results

No deaths or treatment-related clinical toxicity were observed during the course of this study. All of rats in dose groups had blue faeces after being administrated, but which was considered to be caused with the color of the test item and without adverse effect.

During the administration period, the mean body weight and body weight change of pregnant rats in all dose groups had no significant difference compared with the control group. At the same time, no significant effect in food consumption of the pregnant rat during the treated period was observed in all dose groups.

In all dose groups, no adverse effect was observed in all prenatal reproductive parameters.

The fetal examination showed that no adverse effect in body weight and sex distribution of live fetuses was observed in all dosed groups. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations except that some examined fetuses had less than six sternal ossification points, but there was no difference in the men number of sternal ossification points, but there was no difference in the mean number of sternal ossification points in all dosed groups as compared with the control group.

Conclusion

In conclusion, under the conditions of this study, CJ303 administered to pregnant SD rats by gavage in water, had not produced any maternal toxicity, or teratogenic and embryo-/fetotoxicity at the dose of 1000 mg/kg.d.

Based on these findings, the following effect levels, No Observed Adverse Effect Level (NOAEL) were derived:

NOAEL(maternal toxicity): 1000 mg/kg bw.d

NOAEL(embryo-/fetotoxicity): 1000 mg/kg bw.d

NOAEL(teratogenicity): 1000 mg/kg bw.d

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Effect on developmental toxicity: via oral route

The exposure of gestating rats to different doses of CJ303 by gavage during GD5~GD19 did not result in any death or significant specific toxicity at the dose of 1000 mg/kg.d. At the same time, no treatment related effect on the body weight gain and food consumption of pregnant rats was observed. Based on these results, it is concluded that the test item had no maternal toxicity at this dose level.

In all dose groups, no adverse effect was observed in all prenatal reproductive parameters. The fetal examination showed that no adverse effect in the survival, body weight and sex distribution of live fetuses was observed in all dosed groups, additionally, no adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations. Based on these results, it is concluded that the test item had no developmental toxicity and teratogenicity at the dose of 10000 mg/kg.d.

In conclusion, under the conditions of this investigation, CJ303 administered to pregnant SD rats by gavage in water, did not produce any dose-related maternal toxicity, teratogenic and embryo-/fetotoxicity at the dose of 1000 mg/kg.d.