Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November - 01 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

red powder

Specific details on test material used for the study:

Appearance, physical form: red powder
Composition: 100% pigment
Purity: > 99%, typically 99.4%
Solubility: in DMSO only (< 0.4 g/l)
Storage: Room temperature
Production Lot: A31506
Manufacturing Date: 02.03.2008
Testing Date: 03.03.2008
Expiry date: 03.03.2018
Preparation of formulations: Preparation of the test item formulation was made in the vehicle from daily up to every three days using magnetic stirrer in the formulation laboratory of the test facility.
Stability and homogeneity of formulations: A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd. Brl. Han: WIST Rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
- Age of females at arrival: 7.5-9 weeks
- Body weight of females at arrival: 130-170 g
- Age of males at arrival: 15 males arrived September 29, 2011: 7-8 weeks 20 males arrived August 04, 2011: 8.5-9 weeks, 45 males October 06, 2011: 8.5-9 weeks
- Body weight of males at arrival: ca. 230-290 g
- Age at start of mating: females 8.5-10 weeks, males at least 12 weeks
- Acclimatisation time: 8 days for females, at least 27 days for males
- Fasting period before study: no
- Hygienic level: SPF at arrival and kept in good conventional environment during the study
- Animal health: Only healthy animals were used for the study. Health status was certified by the breeder.
- Housing: pre-mating period: 2-3 females /cage, 2-3 males/cage, during mating hours: 1 male with 1- 3 females, during pregnancy: 2-3 sperm positive females /cage
- Cage type: Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 36 - 44%
- Air changes (per hr): 8-12 air exchanges/hour by central air-conditioning system.
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: From: November 02, 2011 To: December 01, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Aqueous methylcellulose

Details on exposure:

VEHICLE
- 0.5% Methylcellulosum (Batch number: N83746634) in distilled water
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulation for its test item content was performed twice during the study, on the first and last week of treatment. The deviation from the nominal concentration was within the range of -9% and + 6%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male with 1-3 females
- Length of cohabitation: 2-4 hours/d until mating
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day 5 to 19
(including the starting and last days)

Frequency of treatment:

daily

Duration of test:

20 days (after prood of pregancy)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20-22 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected with agreement of the Sponsor based on literature data presented by the Sponsor (MSDS).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20
- Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 of gestation
- by re-weighing the non-consumed diet (accuracy: 1 g)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, cervix, ovaries

OTHER:
- Examination for sign of implantation on gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses in each uterine horn: Yes

Fetal examinations:

- Caesarean section: on gestation day 20
- Number of live and dead fetuses in each uterine horn: Yes
- Bodyweight of fetuses: Yes, all per litter
- Sex of fetuses: Yes, all per litter
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No data

Statistics:

Statistical analysis was performed with SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.

Indices:

Pre-implantation loss
Post-implantation loss
Sex distribution
External abnormalities/litter
Visceral abnormalities/litter
Skeletal abnormalities/litter

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discoloration of the feces by the test item during the treatment period was recorded for all of the does in the test item treated groups.

Mortality:

no mortality observed

Description (incidence):

There was no treatment related mortality or morbidity recorded for the dams in the experimental groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significantly lower body weight gain (p<0.05) was indicated in the 500 mg/kg bw/day dose group between gestational days 17 and 20 without a biological relevance. There were no dose related differences in body weight, body weight gain, corrected body weight and corrected body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically but not biologically significantly reduced food consumption indicated in the 250 (-4%) and 500 (-5%) mg/kg bw/day groups (p<0.01 in both) between
gestational days 11 to 17 as well as (p<0.05 in both), (– 4%, -7%) in the 250 and 500 mg/kg bw/day groups respectively between gestation days 17 to 20. This 1.0 to 1.7 g difference was not attributed to the administration of the test item to the dams. There was no statistically significant difference indicated in the food consumption of the 1000 mg/kg bw/day animals.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Placental and relative placental weight was similar in the experimental groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was no necropsy findings recorded for the dams in the experimental groups.
Pinkish discoloration of the stomach and intestines was observed in the test item treated groups.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the intrauterine mortality of the conceptuses.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the preimplantation loss.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the intrauterine mortality of the conceptuses.

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the number of viable fetuses.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the number of implantations.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed up to the limit dose.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal weight was similar in the experimental groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect related to the administration of the test substance indicated in the sex distribution.

External malformations:

no effects observed

Description (incidence and severity):

External variations i.e. growth retarded fetuses was similar in the experimental groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no dose related significant differences indicated at the skeletal evaluation of the fetuses.

Malformations such as misshapen sternebra in one fetus in the 250 mg/kg bw/day group and a hemicentric thoracic vertebral centrum in the 1000 mg/kg bw/day dose groups were recorded at the skeletal examinations. Concidering the low incidence these abnormalities were not attributed to the administration of the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no dose related significant differences indicated at the visceral evaluation of the fetuses.
Visceral malformations such as dilated lateral brain ventricles were observed in one fetus in the 500 mg/kg bw/day groups. An enlarged left ventricle of heart was recorded for one fetus in the 250 mg/kg bw/day groups. Considering the low incidence and lack of dose response, these alterations were not attributed to an effect of the test item.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral Variations:
The incidence of visceral variations (8, 5, 5 and 6 in the 0, 250, 500 and 1000 mg/kg groups respectively) was similar to the vehicle control level. Visceral variations such as an enlarged perimeningeal space, slightly dilated lateral or IIIrd brain ventricle, short brachiocephalic trunk, hydroureter with dilated renal pelvis, malpositioned kidneys or testes were recorded for the fetuses in experimental groups with similar incidences and percentages.

Skeletal Variations:
Incomplete ossification of the skull, bipartite supraoccipital bone, incompletely ossified, bipartite or misaligned sternebrae, or an extra ossification centrum between two sternebra, wavy ribs, dumb-bell shaped or bipartite vertebral centra, incomplete ossification of sacral arches and asymmetric or incomplete ossification of metacarpal, incomplete ossification of os pubis were evaluated as variations during the skeletal examination. There was no increase in the overall incidence of skeletal variations in the test item treated groups.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the limit dose.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: PREGNANCY DATA OF FEMALES, MORTALITY

 

Dose groups	Control		250 mg/kg		500 mg/kg		1000 mg/kg
No. of sperm positive females	24		24		24		24
Died	0		0		0		0
No. of pregnant females	22	92%	22	92%	21	88%	22	92%
No. of non pregnant females 0 impl	2		2		2		2
No. of females with no implantation but corpora lutea (100% preimplantation loss)	0		0		1		0
No. of dams with total intrauterine death	0		0		0		0
No. of pregnant females with viable fetuses but with 3 or less than 3 implantations	0		1		1		0
No. of evaluated dams and litters	22		21		20		22
No. of evaluated dams with malformed fetuses	0	0%	2	9%	1	5%	1	4%

 

 

Table 2: SUMMARY OF CLINICAL SIGNS AND NECROPSY FINDINGS OF DAMS

 

DESCRIPTION	DOSES:	control	250 mg/kg	500 mg/kg	1000 mg/kg
No. of animals		22	21	20	22
- none	N	22	0	0	0
	%	100	0	0	0
-reddish-brownish discolouration of faeces	N	0	21	20	22
	%	0	100	100	100
- alopecia, slight	N	0	1	0	0
	%	0	5	0	0
- alopecia and wound	N	0	1	0	0
	%	0	5	0	0
NECROPSY FINDINGS
- no macroscopic alterations	N	22	13	10	9
	%	100	62	50	41
- pinkish content of stomach, and intestines	N	0	1	1	1
	%	0	5	5	5
- pinkish content of stomach, and intestines,	N	0	3	0	3
slight	%	0	14	0	14
- pinkish content of stomach, slight	N	0	0	1	0
	%	0	0	5	0
- pinkish content of intestines	N	0	1	1	3
	%	0	5	5	14
- pinkish content of intestines, slight	N	0	3	6	6
	%	0	14	30	27
- empty stomach	N	0	0	1	0
	%	0	0	5	0
- stomach few weak content	N	0	0	0	1
	%	0	0	0	5

 

 

Table 3: SUMMARY OF BODY WEIGHT (g) OF DAMS

 

Gestational days		Control	250 mg/kg	500 mg/kg	1000 mg/kg
0	MEAN	189.9	188.9	191.4	189.6
	SD	16.63	14.94	13.12	10.43
	n	22	21	20	22
5	MEAN	211.9	208.2	212.1	210.7
	SD	16.57	15.94	14.42	11.87
	n	22	21	20	22
11	MEAN	237.0	232.6	235.7	235.4
	SD	16.03	16.10	18.82	14.91
	n	22	21	20	22
17	MEAN	274.1	270.8	271.5	272.7
	SD	18.82	17.24	22.54	17.29
	n	22	21	20	22
20	MEAN	310.7	304.5	303.0	307.2
	SD	20.39	20.50	27.19	20.29
	n	22	21	20	22

Remarks:

Body weight (g)

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 4: SUMMARY OF BODY WEIGHT GAIN (g) OF DAMS

 

Gestational day		Control	250 mg/kg	500 mg/kg	1000 mg/kg
0-5	MEAN	22.0	19.3	20.7	21.1
	SD	4.88	5.04	3.80	4.81
	n	22	21	20	22
5-11	MEAN	25.0	24.4	23.6	24.6
	SD	5.97	5.79	8.14	6.32
	n	22	21	20	22
11-17	MEAN	37.2	38.1	35.8	37.4
	SD	5.97	6.55	6.97	5.59
	n	22	21	20	22
17-20	MEAN	36.5	33.7	31.6	34.5
	SD	6.23	5.59	6.70	5.60
	n	22	21	20 *	22	DN
0-20	MEAN	120.8	115.6	111.6	117.6
	SD	16.08	14.85	20.24	16.80
	n	22	21	20	22

Remarks:

Body weight gain (g)

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 5: SUMMARY OF GRAVID UTERINE WEIGHT, CORRECTED BODY WEIGHT AND BODY WEIGHT GAIN OF DAMS

 

		Control	250	500	1000
			mg/kg	mg/kg	mg/kg
Gravid uterine weight	MEAN	59.0	57.5	54.5	58.6
(g)	SD	9.45	10.64	12.08	11.09
	n	22	21	20	22
Corrected body weight	MEAN	251.7	247.0	248.5	248.6
(g)	SD	15.41	15.33	19.69	15.60
	n	22	21	20	22
Corrected	MEAN	61.8	58.1	57.1	59.0
body weight gain	SD	9.96	10.43	14.96	12.13
(g)	n	22	21	20	22

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 6: SUMMARY OF FOOD CONSUMPTION DATA OF DAMS

 

Gestational days		Control	250 mg/kg		500 mg/kg		1000 mg/kg
0-5	MEAN	18.3	17.7		18.1		18.4
	SD	1.22	0.89		1.02		0.77
	n	22	21		20		22
5-11	MEAN	20.8	20.2		20.2		20.8
	SD	1.35	0.94		1.62		1.02
	n	22	21		20		22
11-17	MEAN	23.0	22.0		21.8		22.4
	SD	1.88	1.06		2.21		1.34
	n	22	21	**	20	**	22	U
17-20	MEAN	23.6	22.6		21.9		22.5
	SD	1.93	1.10		2.57		1.69
	n	22	21	*	20	*	22	U

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 7: INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION (MEAN, SD)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
NUMBER OF DAMS:		22	21	20	22
Corpora Lutea	Mean:	13.4	12.8	12.6	13.3
	SD:	1.22	2.09	1.76	1.64
Preimplantation Loss %	Mean:	12.3	14.5	15.0	13.2
	SD:	11.75	10.00	12.05	18.29
Implantation	Mean:	11.7	11.0	10.7	11.3
	SD:	1.70	2.20	2.06	2.01
Early Embryonic Death %	Mean:	6.0	4.9	7.4	5.0
	SD:	7.02	7.73	9.33	7.12
Late Embryonic Death %	Mean:	0.8	0.4	1.6	1.5
	SD:	2.67	1.82	4.11	4.19
Dead Fetuses %	Mean:	0.0	0.3	0.6	0.0
	SD:	0.00	1.45	2.48	0.00
Postimplantation Loss %	Mean:	6.8	5.7	9.5	6.5
	SD:	8.34	8.03	9.37	8.87
Total Intrauterine Mortality %	Mean:	18.3	19.3	22.8	18.2
	SD:	13.03	11.36	15.27	19.65
Viable fetuses	Mean:	10.9	10.3	9.7	10.6
	SD:	1.77	1.98	2.41	2.22
Male fetuses %	Mean:	47.8	46.7	50.8	45.6
	SD:	15.94	15.76	16.13	15.75
Female fetuses %	Mean:	52.2	53.3	49.2	54.4
	SD:	15.94	15.76	16.13	15.75

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 8: INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION (SUM.,%)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
NUMBER OF DAMS:		22	21	20	22
Corpora Lutea	Sum:	295	269	251	292
Preimplantation Loss	Sum:	37	39	38	43
(Data compared to no. of corpora lutea)	%:	13	14	15	15
Implantation	Sum:	258	230	213	249
Early Embryonic Death	Sum:	16	12	15	12
(Data compared to no. of implantations)	%:	6	5	7	5
Late Embryonic Death	Sum:	2	1	3	3
(Data compared to no. of implantations)	%:	1	0	1	1
Dead Fetuses	Sum:	0	1	1	0
(Data compared to no. of implantations)	%:	0	0	0	0
Postimplantation Loss	Sum:	18	14	19	15
(Data compared to no. of implantations)	%:	7	6	9	6
Total Intrauterine Mortality	Sum:	55	53	57	58
(Data compared to no. of corpora lutea)	%:	19	20	23	20
Viable fetuses	Sum:	240	216	194	234
Male fetuses	Sum:	115	101	99	105
(Data compared to no. of viable fetuses)	%:	48	47	51	45
Female fetuses	Sum:	125	115	95	129
(Data compared to no. of viable fetuses)	%:	52	53	49	55

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

 

 

Table 9: LITTER MEANS OF FETAL AND PLACENTAL WEIGHT, (Mean, SD)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
Fetal weight	MEAN	3.3	3.4	3.4	3.3
(g)	SD	0.21	0.22	0.22	0.24
	n	22	21	20	22
Placental weight	MEAN	688.2	726.5	679.2	683.2
(mg)	SD	52.22	76.10	66.10	83.03
	n	22	21	20	22
Relative placental weight	MEAN	210.0	215.9	201.5	206.6
(mg/g)	SD	22.44	26.31	19.11	28.34
	n	22	21	20	22

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

 

 

Table 10: RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS (Percentile litter means and SD)

 

	Control		250	500	1000
			mg/kg	mg/kg	mg/kg
EXTERNAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	240	216	194	234
Fetuses	Mean	3.3	1.5	1.9	3.8
with abnormalities	SD	6.07	4.89	6.87	8.06
Variation	Mean	3.3	1.5	1.9	3.8
	SD	6.07	4.89	6.87	8.06
Malformation	Mean	0.0	0.0	0.0	0.0
	SD	0.00	0.00	0.00	0.00
Retarded in body weight	Mean	3.3	1.5	1.9	3.8
	SD	6.07	4.89	6.87	8.06
VISCERAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	119	105	97	116
Fetuses	Mean	7.2	6.5	5.9	4.6
with abnormalities	SD	14.54	11.02	12.50	11.02
Variation	Mean	7.2	4.9	4.6	4.6
	SD	14.54	9.21	8.45	11.02
Malformation	Mean	0.0	1.6	1.3	0.0
	SD	0.00	7.27	5.59	0.00
SKELETAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	121	111	97	118
Fetuses	Mean	12.3	5.2	9.8	11.4
with abnormalities	SD	15.90	10.60	19.69	13.35
Variation	Mean	12.3	4.5	9.8	10.6
	SD	15.90	10.44	19.69	11.38
Malformation	Mean	0.0	0.7	0.0	0.8
	SD	0.00	3.12	0.00	3.55

 

 

Table 11: RESULT OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS (Sum, %)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
EXTERNAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	240	216	194	234
Fetuses	N	8	3	4	8
with abnormalities	%	3	1	2	3
Variation	N	8	3	4	8
	%	3	1	2	3
Malformation	N	0	0	0	0
	%	0	0	0	0
Retarded in body weight	N	8	3	4	8
	%	3	1	2	3
VISCERAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	119	105	97	116
Fetuses	N	8	6	6	6
with abnormalities	%	7	6	6	5
Variation	N	8	5	5	6
	%	7	5	5	5
Malformation	N	0	1	1	0
	%	0	1	1	0
SKELETAL EXAMINATION
Litters examined	N	22	21	20	22
Fetuses examined	N	121	111	97	118
Fetuses	N	16	6 *	11	13
with abnormalities	%	13	5	11	11
Variation	N	16	5 *	11	12
	%	13	5	11	10
Malformation	N	0	1	0	1
	%	0	1	0	1

Remarks:

* = p<0.05 CH2

** = p<0.01 CH2

 

 

 

Table 12: Types of External Abnormalities, (Sum., %)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
Number of Dams	N	22	21	20	22
Number of Fetuses examined	N	240	216	194	234
Number of Fetuses	N	8	3	4	8
with abnormalities	%	3	1	2	3
Variation	N	8	3	4	8
	%	3	1	2	3
Malformation	N	0	0	0	0
	%	0	0	0	0
Fetal variations
- Retarded in body weight	N	8	3	4	8
	%	3	1	2	3
Placental alterations
- Fibrinoid degeneration	N	0	0	0	1
	%	0	0	0	0
- Edema or edema slight	N	0	1	0	0
	%	0	0	0	0

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

 

 

Table 13: TYPES OF VISCERAL ABNORMALITIES (Sum., %)

 

		Control	250 mg/kg	500 mg/kg	1000 mg/kg
Number of Dams	N	22	21	20	22
Number of Fetuses examined	N	119	105	97	116
Number of Fetuses	N	8	6	6	6
with abnormalities	%	7	6	6	5
Variation	N	8	5	5	6
	%	7	5	5	5
Malformation	N	0	1	1	0
	%	0	1	1	0
Fetal variations
Brain
- Perimeningeal space enlarged	N	1	0	0	0
	%	1	0	0	0
- Slightly dilated lateral brain ventricles	N	3	0	2	0
	%	3	0	2	0
- Slightly dilated IIIrd brain ventricles	N	1	0	0	0
	%	1	0	0	0
Great vessels
- Short brachiocephalic trunk	N	1	2	1	3
	%	1	2	1	3
Ureters, kidneys
- Hydroureter and dilated renal pelvis unilateral	N	2	2	2	3
	%	2	2	2	3
Gonads
- Malpositioned testes	N	0	1	0	0
	%	0	1	0	0
Fetal malformations
Brain
- Dilated lateral ventricles	N	0	0	1	0
	%	0	0	1	0
Heart
- Left ventricle enlarged	N	0	1	0	0
	%	0	1	0	0

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

 

 

Table 14: TYPES OF SKELETAL ABNORMALITIES (sum, %)

 

	Control		250 mg/kg	500 mg/kg		1000 mg/kg
Number of Dams	N	22	21	20		22
Number of Fetuses examined	N	121	111	97		118
Number of Fetuses	N	16	6 *	11		13
with abnormalities	%	13	5	11		11
Variation	N	16	5 *	11		12
	%	13	5	11		10
Malformation	N	0	1	0		1
	%	0	1	0		1
Fetal variations
Skull
- incomplete ossification marked	N	5	3	7		1
one bone or more	%	4	3	7		1
- retarded	N	0	0	0		2
	%	0	0	0		2
Sternebra
- 3 or less	N	1	0	2		3
	%	1	0	2		3
- bipartite	N	1	0	2		3
	%	1	0	2		3
- misaligned	N	0	0	3		1
	%	0	0	3		1
- extra ossification centrum	N	0	0	0		1
	%	0	0	0		1
Ribs
- wavy	N	1	1	0		1
	%	1	1	0		1
Vertebrae
Thoracic and/or lumbar
- dumb-bell shaped or dumb-bell shaped and	N	1	0	0		5
asymmetric more than 3	%	1	0	0		4
Thoracic
- bipartite or bipartite and asymmetric one or more	N	2	0	0		0
	%	2	0	0		0
- from SII arches incomplete ossification or	N	0	0	2		0
not ossified	%	0	0	2		0
Pelvic girdle
- os pubis incomplete ossification or not ossified	N	1	0	1		0
	%	1	0	1		0
Forepaw, hindpaw
- metacarpal or metatarsal asymmetric and/or	N	7	2	4		3
less than 3/3.5	%	6	2	4		3
Fetal malformations
Sternebra
- misshapen	N	0	1	0	0
	%	0	1	0	0
Vertebrae
- hemicentric	N	0	0	0	1
	%	0	0	0	1

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

Applicant's summary and conclusion

Conclusions:

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to the day before Caesarean section with the test substance at the dose levels of 250, 500 and 1000 mg/kg bw/day did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams and on the intrauterine development of the fetuses. Under the conditions applied in this study and from the observations made in the dams and their fetuses the following no-observable-adverse-effect levels were derived: NOAEL (maternal toxicity) = 1000 mg/kg bw/day, NOAEL (developmental toxicity) = 1000 mg/kg bw/day.

Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with the test substance by oral administration daily at three dose levels of 250, 500 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group was included, and the animals were given the vehicle 0.5% Methylcellulose. The treatment volume was 10 ml/kg/bw.

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. Visceral and skeletal examination of the fetuses was performed during the study. In total, there were 22, 21, 20 and 22 evaluated pregnant females at termination on gestation day 20 in the control, 250, 500 and 1000 mg/kg groups, respectively.

There were no clinical signs observed in the experimental groups. Discoloration of the feces by the test item during the treatment period was recorded for all of the does in the test item treated groups. There was no treatment related mortality, morbidity or necropsy findings recorded for the dams in the experimental groups. Pinkish discoloration of the stomach and intestines was observed in the test item treated groups. There was no indication of an effect of the test item on body weight, body weight gain and corrected body weight during the entire study. There was no dose related reduction of food consumption in the dose groups if compared to the control group. There was no effect related to the administration of the test substance indicated in the preimplantation loss and the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. Fetal, placental and relative placental weight, incidence of external variations i.e. growth retarded fetuses was similar in the experimental groups. There were no dose related significant differences indicated at the visceral and skeletal evaluation of the fetuses.

Conclusively, oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to the day before Caesarean section with the test substance at the dose levels of 250, 500 and 1000 mg/kg bw/day did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams and on the intrauterine development of the fetuses. Under the conditions applied in this study and from the observations made in the dams and their fetuses the following no-observable-adverse-effect levels were derived: NOAEL (maternal toxicity) = 1000 mg/kg bw/day, NOAEL (developmental toxicity) = 1000 mg/kg bw/day.