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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to Spetember 2021
In-life phase: March 17, 2021, to April 22, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

401-540-3

EC Name:

-

Cas Number:

84632-65-5

Molecular formula:

C18H10Cl2N2O2

IUPAC Name:

3,6-bis(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Cinilex® DPP Red SR2P, Lot D23508919P1
- Purity, including information on contaminants, isomers, etc.: 99.6% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25ºC) protected from humidity
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S & K-LAP Kft., Császár út 135, 2173 Kartal, HUNGARY
- Age at study initiation: young adults
- Weight at study initiation: 3427-4008 g (at insemination)
- Fasting period before study: none
- Housing: individually in metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 or 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.4-21.0 °C
- Humidity (%): 30 - 64 %
- Air changes (per hr): 12 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 March 2021 To: 22 April 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water therefore 0.5% aqueous methylcellulose was used for preparing formulations. 0.5% aqueous methylcellulose has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
The test item is suitable for oral administration when suspended in methylcellulose.

- Amount of vehicle (if gavage): treatment volume: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance content was determined in 0.5 % Aqueous Methylcellulose.
Five samples were analysed from each test concentration and five samples were analysed from the control two times.
Formulation samples were diluted to fit the calibration curve and analysed by a UV photometric method.
Test substance concentrations in the samples varied in the range from 95 % to 106 % in comparison to the nominal values. Test substance was not detected in the control samples.

Details on mating procedure:

- Impregnation procedure: artificial insemination

Day of insemination was regarded as day 0 of gestation.
Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed at the test facility by the breeder. Each female was inseminated with diluted sperm and receiveds 0.2 mL Receptal hormone preparation was given with intramuscular injection into the femur region to provoke ovulation. The sperm originated from New Zealand White male rabbits from the same source as the females. The origin and quality of the sperm were certified by the breeder.

Duration of treatment / exposure:

22 days (from gestation day (GD) 6 to GD 27)

Frequency of treatment:

daily, in the morning hours, at approximately the same time each day

Duration of test:

28 days (from insemination to sacrifice)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

also refered to as low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

also refered to as mid dose

Doses / concentrations 4

Dose / conc.:

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

Remarks:

also refered to as high dose

No. of animals per sex per dose:

24 females per dose and per control

Control animals:

yes

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor with the highest dose level at 1000 mg/kg b.w./day (limit dose according to OECD TG 414) based on the results of the Tolerability Study of the test susbatnce in rabbits by oral administration (Study number 678-410-5845; Toxi-Coop Zrt.).
- Rationale for animal assignment: Females were randomly assigned to dose groups on the basis of their body weight on the day of insemination in such a way that the group averages of the body weight were as similar as possible on the first day (day 0) of gestation.
- Fasting period before blood sampling for (rat) dam thyroid hormones: none

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g).


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: neck, thorax and abdomen of the does were examined macroscopically, ovaries and uterus

OTHER: Blood was sampled for possible measurement one and three hours after the last treatment (GD 27), but not examined.

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

Blood sampling:

Blood was sampled for possible measurement one and three hours after the last treatment (GD 27), but not examined.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: not examined

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 detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was 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 will be 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 were performed using the Mann-Whitney U-test.
Chi2 test was performed if feasible.

Indices:

Pre-implantation loss: (no. of corpora lutea - no. of implantations)/no. of implantations*100

Post-implantation loss: (no. of implantations - no. of live fetuses)/no. of live fetuses*100

Historical control data:

Historical control data on eight New Zealand white rabbit studies conducted between 2016 and 2020 were available. Four of those studies used water as a solvent and for sunflower oil. Five studies included visceral and skeletal examinations, comprising three studies using water as a solvent.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of systemic toxicity.
Red coloured faeces were observed in all test item treated animals from GD 7 during the whole in-life phase. This was not considered as adverse and attributed to the red color of the test item. Before the treatment period, one female had reddish discoloration on the underlay on GD 1 in the 300 mg/kg bw/day group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

None of the pregnant females died before scheduled termination due to toxicity or was considered as moribund.
Mis-gavage caused the death of 2 does in the low dose group, 2 does in the mid dose group and 1 doe in the high dose group.
One doe of the control died due to an intercurrent disease.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in the mean body weight, body weight gain, corrected body weight of the dams in the experimental groups in the different time periods, except on G.D. 28 in the 1000 mg/kg bw/day dose group (p<0.05). Between G.D. 27 and 28 the mean body weight gain was lower in the 1000 mg/kg bw/day group without attaining statistical significance. The corrected body weight gain was negative in the test item treated groups and positive in the control, however the difference was not statistically significant and the values (together with the body weight on G.D. 28) were within the historical control range. Hence, the slightly lower values in the 1000 mg/kg bw/day group were neither confirmed to have a relationship with the treatment, nor could a relation be ruled out. However, these slight reductions were considered as not adverse.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no significant differences indicated the mean food consumption of the does.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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

Gross pathological findings:

no effects observed

Description (incidence and severity):

The findings revealed at gross pathology such as point-like haemorrhages in the lungs were not attributed to the treatment of the test item considering the similar distribution in the groups including control. One dam had double gall bladder in the 100 mg/kg bw/day and one clotted blood in the uterine horn in the 1000 mg/kg bw/day group. Considering that these findings occurred in single cases, it was not attributed to the test item.
Brick-reddish or brownish discolouration of the stomach or/and intestines’ content in all mid and high dose animals and 20 of 21 low dose animal (but none in the controls) was attributed to the red colour of the test item and considered to be without any toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female each aborted in the control group (GD 27) and the 100 mg/kg bw/day dose group (GD 25) due to an intercurrent disease.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no adverse effects indicated in the percentage of pre- and post- implantation loss when comparing dose group mean values to the control group mean values.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no adverse effects indicated in the number of dead fetuses, percentage of total intrauterine mortality and number of viable fetuses when comparing dose group mean values to the control group mean values.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were three non pregnant females (no implantation and no corpora lutea) in the control group, a one each in the mid and high dose group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no effects observed

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weight and crown-rump length was slightly lower in the 1000 mg/kg bw/day group, however without a statistical significance and the values were within the historical control range. The lower values were neither confirmed to have a relationship with the treatment or the slightly lower body weight values of the does on G.D. 27 and 28, nor could a relation be ruled out.. However, these slight reductions were judged as not adverse.

Relative placental weight was statistically significantly increased (p<0.01) in male fetuses of the 300 mg/kg bw/day dose group without showing any dose response and, therefore, this alteration was considered as incidental and unrelated to treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.
Statistical significance (p<0.01) was revealed in the higher relative placental weight only belonging to male fetuses in the 300 mg/kg bw/day dose group, but there was no dose response indicated.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no significant increases in the incidence of external malformations.

Two fetuses were found with malformation at external examination, one fetus with acrania in the control group and one with umbilical hernia in the 1000 mg/kg bw/day group.
Considering that umbilical hernia was found in one single fetus, the occurrence of this malformation was considered incidental.
The neck edema which was recorded for one fetus in the 300 mg/kg bw/day group was classified as a variation. Considering that this was a single case and not in the high dose group, this was judged as incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant increases in the incidence of all over skeletal variations and malformations.
Moreover, the incidence of malformations was statistically significantly lower in the 300 mg/kg bw/day group by U test (p<0.05) and by the Chi square test (p<0.01) as well as the incidence of malformed fetuses was statistically significantly lower in the 1000 mg/kg bw/day group by the Chi square test (p<0.05).

There was no statistical significance indicated in the different type of malformations which occurred with a sporadic distribution and without a dose response.

Malformation of the skull (see below) was found in one fetus in each group except the 100 mg/kg bw/day dose group where no skull malformation was observed.

A hole was observed between parietal and frontal bones in the control fetus which was observed with acrania at external examination.
The anterior fontanelle was larger and irregular ossification was recorded for one fetus in the 300 mg/kg bw/day dose group. The anterior fontanelle was markedly larger and misshapen, and the posterior fontanelle was larger in one fetus in the 1000 mg/kg bw/day group.

Sternebral malformations such as misshapen xiphoid cartilage, small or thin split in the cartilage (between 5th and 6th or in 6th), different degree of fusions or wider sternum was observed. 

Ribs with fusion or disconnection to sternum (if other than 7th) were found with low incidences (one in the 100 and one in the 300 mg/kg bw/day group).

Malformed vertebrae were found in single cases.

In a few cases (three fetuses in the control and one fetus in the 100 mg/kg bw/day group) more regions (sternum, ribs and vertebrae, sternum and vertebrae or ribs and vertebrae) were affected.

The incidence of the variation 'misaligned sternebra' was statistically significantly higher (p<0.01) by Duncan’s test and (p<0.05) for the fetal and litter incidence by Chi square test in the 100 mg/kg bw/day group. However, this variation was not present in the higher dose groups, hence this was not attributed to the treatment.
There was no increase seen in the incidence of other skeletal variations.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no significant increases in the incidence of visceral variations and malformations.

There was one malformed fetus (with larger peri-meningeal space and slightly pushed brain) found in the control group and none in the test item treated groups.

Fetal variations such as slightly enlarged peri-meningeal space, slightly dilated IIIrd brain ventricle, larger hole between cerebral hemisphere and thalamus, smaller lung lobes, full and distended urinary bladder, convoluted ureter/s and slightly mal-positioned kidney were found sporadically, in single cases or with a distribution similar in the experimental groups or only in the control group.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The following table summarises detailed maternal and fetal data of the control and the three test groups:

		test doses
	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
number of inseminated does	24	24	24	24
number of pregnant does	21	24	23	23
number of does with no corpora lutea	3	0	1	1
number of females with no implantation but corpora lutea (100% preimplantation loss)	0	0	0	0
number of does dead due to misgavage	0	2	2	1
Number of does dead due to an intercurrent disease	1	0	0	0
number of does with abortions	2	1	0	0
number of evaluated does and litters	18	21	21	22
number of dams with early deliveries	0	0	0	0
number of dams with early embryonic death	15	20	10	11
number of dams with late embryonic death	5	4	10	3
number of dams with dead featuses	1	0	0	1
corpora Lutea	251	281	283	290
preimplantation loss (% as compared to no. of corpora lutea)	39 (16%)	24 (9%)	24 (8%)	33 (11%)
postimplantation loss (% as compared to no. of corpora lutea)	21 (10%)	24 (9%)	19 (7%)	15 (6%)
Implantations	212	257	259	258
mean number of implantations	11.8	12.2	12.3	11.7
early embryonic death (% as compared to no. of implantations)	15 (7%)	20 (8%)	10 (4%)	11 (4%)
late embryonic death (% as compared to no. of implantations)	5 (2%)	4 (2%)	10 (4%)	3 (1%)
dead fetuses	1	0	0	1
total intrauterine mortality (% as compared to no. of corpora lutea)	60 (24%)	48 (17%)	44 (16%)	48 (17%)
clinical signs: red coloured faeces (no. of does)	0	21	21	22
clinical signs: reddish discolouration of the underlay (no. of does)	0	0	1	0
clinical signs: reduced activity	0	0	0	0
clinical signs: dyspnoea	0	0	0	0
body weight (in g): gestational day 0 (mean +/- standard deviation)	3691.9 +/- 159.45	3695.1 +/- 162.92	3690.7 +/- 155.70	3681.0 +/- 124.33
body weight (in g): gestational day 28	4734.7 +/- 193.98	4652.5 +/- 180.64	4732.8 +/- 209.09	4592.0 +/- 184.58
body weight gain (in g): from gestational day 0 to day 28	1042.8 +/- 186.60	957.4 +/- 206.23	1042.10 +/- 184.70	911.1 +/- 202.85
gravid uterine weight (in g) (mean +/- standard deviation)	632.7 +/- 165.66	657.7 +/- 93.23	672.4 +/- 188.34	616.6 +/- 150.36
corrected body weight (in g)	4102.1 +/- 256.66	3994.8 +/- 214.34	4060.4 +/- 281.25	3975.5 +/- 242.94
corrected body weight gain (in g)	22.8 +/- 222.84	-71.7 +/- 169.13	-45.0 +/- 220.20	-42.6 +/- 235.57
necropsy findings: no. of does with no macroscopic alterations (disclouration of digestive system excluded)	16	17	18	18
necropsy findings: no. of does with discoloration (brick-reddish or brownish) in the digestive system content (stomach or/and intestines)	0	20	21	22
necropsy findings: no. of does with point-like haemorrhages in the lungs	2	3	2	2
necropsy findings: no. of does with dark red liver	0	0	0	1
necropsy findings: no. of does with double gall bladder	0	1	0	0
necropsy findings: no. of does with clotted blood in the uterine horn	0	0	0	1
viable fetuses (total)	191	233	239	243
viable fetuses (% as compared to no. of corpora lutea)	76.1	82.9	84.5	83.8
sex ratio (male/female)	89/102 = 0.87	108/125 = 0.86	114/125 = 0.91	102/141 = 0,72
viable fetuses (mean)	10.6	11.1	11.4	11.0
fetal weight (in g) (mean +/- standard deviation)	38.5 +/- 7.70	38.0 +/- 7.25	37.2 +/- 7.26	35.9 +/- 7.58
fetal weight (in g): male (mean +/- standard deviation)	38.5 +/- 7.79	39.0 +/- 6.73	38.1 +/- 6.84	36.2 +/- 7.33
fetal weight (in g): female (mean +/- standard deviation)	38.6 +/- 7.66	37.1 +/- 7.59	36.3 +/- 7.55	35.7 +/- 7.78
crown-rump length (in mm) (mean +/- standard deviation)	90.7 +/- 7.40	90.6 +/- 6.64	90.2 +/- 6.46	89.0 +/- 7.11
crown-rump length (in mm): male (mean +/- standard deviation)	90.4 +/- 7.78	91.3 +/- 6.12	91.2 +/- 6.04	89.8 +/- 6.74
crown-rump length (in mm): female (mean +/- standard deviation)	90.9 +/- 7.08	90.0 +/- 7.02	89.3 +/- 6.72	88.5 +/- 7.35
fetuses with malformations (total number): external/visceral/skeletal	1/1/12	0/0/11	0/0/2	1/0/6
type of malformation: external/visceral/skeletal	arcrania/enlarged perimeningeal space and brain slightly pushed /skull, sternebra, vertebrae	-/-/sternebra, ribs, vertebrae	-/-/skull, sternebra, ribs	umbilicial hernia/-/skull, sternebra, ribs
fetuses with malformations (%): external/visceral/skeletal	1/1/6	0/0/5	0/0/1	0.5/0/2
litters with malformations (total number): external/visceral/skeletal	1/1/7	0/0/9	0/0/2	1/0/4
litters with malformations (%): external/visceral/skeletal	6/6/39	0/0/43	0/0/10	5/0/18
fetuses with variations (total number)	11/10/27	7/6/39	7/5/38	12/14/37
fetuses with variations (%)	6/5/14	3/3/17	3/2/16	5/6/15
External variation: fetuses with retarded body weight (total number/%)	7/4	5/2	6/3	8/3
External variation: litters with retarded body weight (total number/%)	3/17	4/19	3/14	5/23
External variation: fetuses retarded in crown-rump length (total number/%)	8/4	6/3	2/1	11/5
External variation: litters with retarded crown-rump length (total number/%)	4/22	5/24	2/10	6/27
External variation: fetuses with neck edema (total number/%)	0/0	0/0	1/0	0/0
Visceral variations: fetuses with slightly enlarged perimeningal space (total number/%)	1/1	0/0	0/0	2/1 (two litters)
Visceral variations: fetuses with dilated 3rd ventricle (total number/%)	1/1	0/0	0/0	0/0
Visceral variations: fetuses with slightly larger hole between cerebral hemisphere and thalamus (total number/%)	1/1	0/0	0/0	0/0
Visceral variations: fetuses with smaller lung lobes (total number/%)	0/0	0/0	1/0.5	0/0
Visceral variations: fetuses with urinary bladder distended (total number/%)	0/0	1/0.5	0/0	0/0
Visceral variations: fetuses with ureter convoluted (total number/%)	7/4 (from 5 litters)	5/2 (from 5 litters)	3/1 (from 3 litters)	12/5 (from 9 litters)
Visceral variations: fetuses with slightly malpositioned kidney (total number/%)	0/0	0/0	1/0.5	0/0
Skeletal variations: fetuses with slightly larger anterior or posterior fontanelle (total number/%/no. of litters)	2/1/2	9/4/3	5/2/5	8/3/3
Skeletal variations: fetuses with slightly larger anterior and posterior fontanelle (total number/%/no. of litters)	2/1/2	3/1/2	0/0/0	2/1/1
Skeletal variations: fetuses with less than 5 sternebrae ossified total (number/%/no. of litters)	1/1/1	0/0/0	0/0/0	1/0.5/1
Skeletal variations: fetuses with dumb-bell shaped ossified sternebrae (number/%/no. of litters)	8/4/6	7/3/5	8/3/5	8/3/5
Skeletal variations: fetuses with bipartite ossified sternebrae (number/%/no. of litters)	3/2/3	9/4/7	3/1/3	6/2/5
Skeletal variations: fetuses with hole in xiphoid cartilage (number/%/no. of litters)	6/3/3	6/3/5	7/3/5	7/3/4
Skeletal variations: fetuses misshapen ossification of sternebrae (number/%/no. of litters)	2/1/1	3/1/2	0/0/0	1/0.51
Skeletal variations: fetuses with misalinged sternebae (number/%/no. of litters)	0/0/0	5/2/5	0/0/0	0/0/0
Skeletal variations: fetuses with sternebrae with fusing tendency (number/%/no. of litters)	1/1/1	0/0/0	0/0/0	0/0/0
Skeletal variations: fetuses with slightly wider sternebrae (number/%/no. of litters)	4/2/1	0/0/0	0/0/0	2/1/2
Skeletal variations: fetuses with 7th rib not connected to sternum (number/%/no. of litters)	2/1/1	1/0.5/1	6/3/4	3/1/2
Skeletal variations: fetuses with dumb-bell shaped vertebrae (number/%/no. of litters)	4/2/3	5/2/4	4/2/3	5/2/4
Skeletal variations: fetuses with assymetric vertebrae (number/%/no. of litters)	0/0/0	0/0/0	1/0.5/1	0/0/0
Skeletal variations: fetuses with pubis not ossified (number/%/no. of litters)	0/0/0	1/0.5/1	0/0/0	3/1/3
Skeletal variations (limbs): fetuses with pollex not ossified (number/%/no. of litters)	2/1/1	1/0.5/1	0/0/0	3/1/3
Skeletal variations (limbs): fetuses with talus not ossified (number/%/no. of litters)	4/2/3	0/0/0	0/0/0	5/2/4
Skeletal variations (limbs): fetuses with small pubic (number/%/no. of litters)	3/2/2	0/0/0	0/0/0	0/0/0
Skeletal variations: fetuses with less ossified phalanges (number/%/no. of litters)	1/1/1	2/1/2	2/1/1	4/2/4
Skeletal variations: fetuses with asymmetrically ossified phalanges (number/%/no. of litters)	2/1/2	7/3/6	6/3/5	5/2/5

 

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD test guideline 414 with rabbits, the substance, orally applied, did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the does and on the intrauterine development of the fetuses. Therefore, both the NOAEL maternal toxicity and the NOAEL developmental toxicity were 1000mg/kg bw/day.

Executive summary:

The developmental toxicity of the substance was investigated in a GLP-study according to OECD test guideline 414. Oral treatment of pregnant New Zealand white rabbit does from gestation day 6 up to the day before Caesarean section on gestation day 28 with the substance at the dose levels of 100, 300 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 does and their fetuses the following no-observable-adverse-effect levels were derived:

 

NOAEL_{maternal toxicity}:1000mg/kg bw/day

NOAEL_{developmental toxicity}:1000mg/kg bw/day