2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Aug 2021 - 22 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

EC Number:

226-866-1

EC Name:

2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Cas Number:

5521-31-3

Molecular formula:

C26H14N2O4

IUPAC Name:

2,9-dimethylisoquino[4',5',6':6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation: solid, powder
- Particle size distribution (TEM): 64.9 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: cubic, spherical, rod
- Surface area of particles: 29.5 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1600 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

Test animals

Species:

rat

Strain:

other: Wistar Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories CRL, Rhone, 327 Impasse du Domaine Les Oncines, 69210 Saint Germain Nuelles (France)
- Age at order: Males: at least 11 weeks; Females: 9 weeks
- Weight at order: Males: 325-350 g; Females: 200-225 g
- Housing: no more than 5 of one sex to a cage (before and after mating); 1 male and 1 female per cage (during mating)
- Diet: ad libitum, 4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 2 °C
- Humidity: 55 % +/- 15 %
- Air changes (per hr): approximately 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 2021 To: 22 Nov 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Suspensions of the test item, in 0.5 % CMC, were prepared using the following procedure:
1. the required amount of test item were weighed.
2. the required amount of vehicle was added.
3. the mixture was treated with a Silverson for 3 minutes.
4. the resulting suspension was left under magnetic stirring for at least 16 hour, at room temperature, prior to dosing and during dosing
The formulation was prepared daily at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw
- Concentration in vehicle: 0, 10, 40, 120 mg/ml
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4346 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%). A 28 hour stability at room temperature and a 9 day stability at 2-8°C (followed by one day at room temperature under magnetic stirring) were verified in the range from 10 to 100 mg/mL.
Samples of the preparations prepared on Week 1 and Last Week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: not specified
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

from Day 6 through Day 19 post coitum (14 Days)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: given by Sponsor
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for rat dam blood sampling: in the morning
- Other: on Day 20 post coitum

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twiche daily and once daily at weekends an public holidays
- Cage side observations: Check for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: all animals, on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION: Yes
- measured on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: thyroid and brain

THYROID HORMONE DETERMINATION (T3, T4, TSH): Yes
- Blood sampled in the morning on day 20 post coitum (day of necropsy)
- slight isoflurane anaesthesia

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of intra-uterine deaths: Early resorptions (only placental remnants visible), Late resorptions (placental and foetal remnants visible)
- Number, sex and weight of all live foetuses
- Number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing) and in each foetuses allocated to the skeletal examination
- Gross evaluation of placentae

- The uteri from females without visible implantations (one in group 1, 3, 4; five in group 2) were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation

- The uteri from dams showing unilateral implantations on the right horn (one in group 1 and 3), were also immersed in a 20% solution of ammonium sulphide, revealing the non-pregnant left horn

Blood sampling:

- Serum: Yes
- Volume: 1 mL
- in the morning of Day 20 post coitum

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: Yes

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:

> Pre impl. Loss [%] = (no. of corpora lutea − no. of implantations) x 100/ no. of corpora lutea

Post-implantation loss was calculated as a percentage from the formula:

> Post impl. Loss [%] = (no. of implantations − no. of live foetuses) x 100/ no. of implantations

Total implantation loss was calculated as a percentage from the formula:

> Total impl. Loss [%] = (no. of corpora lutea − no. of live foetuses) x 100/ no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males.

All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Historical control data:

Historical control data from 2014 to 2022 was provided. Data included: foetal external abnormalities, skeletal examination, fixed visceral examination, fate of females, macroscopic observation of females at final cesarean section, litter data and sex ratio of dams with live foetuses at necropsy. Tables were extracted from reproductive toxicology historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss was observed in one control, two mid-dose and one high dose females. Considering the low incidence of hairloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the treatment period a presence of couloured faeces (red) in all treated group. Considering that the test item is a red solid the colour indicated the presence of the substance in the faeces and not an abnormalities.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and weight gain were unaffected in females treated up to 1000 mg/kg/day over the entire administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The statistically significant increase in food consumption (29.7 g of high dose group versus 27.4 g of controls) was considered incidental and unrelated to treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were observed in the determination of T3, T4 and TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus weight, corrected maternal body weight and weight gain were not affected by treatment.
There were no treatment-related organ weight changes (brain and thyroid gland) at the end of the treatment period. Any variations were considered to be within the range of expected spontaneous changes in rats of the same age and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations in the thyroid gland at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Litter data of treated females were comparable to controls.
Presence of foetuses with a weight less that 2.7 g and classified as small were noted in control, mid and high dose groups. Considering that the incidence of the high dose group is lower than controls and in the absence of the dose relation trend the presence is consider incidental and unrelated to treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus with malformation was observed in the mid- dose group. The foetus showed unilateral fusion of thoracic arches no. 10th and 11th in combination with the fusion of the 10th and 11th ribs. Considering that no other observations were noted related to the thoracic arches or ribs and in the absence of the relationship the changes were considered spontaneous in origin.
The other changes noted were comparable between the control and the treated groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

The variations observed occurred with similar incidence across all groups including controls.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

 

 

Table 1: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE
Interval: 0 - 20 Days Group	1		2		3		4
Observation	(25)		(25)		(25)		(25)
APPEARANCE	a	b	a	b	a	b	a	b
Hairloss	1	4.0	0	0.0	2	8.0	1	4.0
() = Number of animals alive at start of interval a = Number of animals affected b = Percent of animals with observation during interval

 

 

Table 2: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA
Organ: Brain	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.853	1.848	1.860	1.851
Standard deviation	0.073	0.066	0.091	0.070
Group diff. at p < 0.05		0.051	0.051	0.051
Group diff. at p < 0.01		0.064	0.064	0.064
Analysis of variance: F ratio = 0.12; Df = 3/ 96; F probability = 0.942 Note: a * indicates group mean is significantly different from control at level of significance shown.
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	0.0218	0.0236	0.0219	0.0232
Standard deviation	0.0048	0.0037	0.0045	0.0044
Group diff. at p < 0.05		0.0030	0.0030	0.0030
Group diff. at p < 0.01		0.0037	0.0037	0.0037
Analysis of variance: F ratio = 1.09; Df = 3/ 96; F probability = 0.358 Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

Table 3: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.181	1.280	1.181	1.256
Standard deviation	0.268	0.198	0.254	0.240
Group diff. at p < 0.05		0.163	0.163	0.163
Group diff. at p < 0.01		0.204	0.204	0.204
Analysis of variance: F ratio = 1.11; Df = 3/ 96; F probability = 0.349 Note: a * indicates group mean is significantly different from control at level of significance shown. ° = expressed as % organ to brain weight ratio

 

 

Table 4: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE
Group	1	2	3	4
Number in group	25	25	25	25
Caecum
-        Abnormal contents	0	0	0	1
Forelimbs
-        Hairloss	1	0	1	1
Uterus
-        Unilateral implantation	1	0	1	0
-        Not pregnant	1	4	1	1
-        Total resorption	0	1	0	0
Whole animal
-        No abnormalities detected	22	20	22	23

 

 

Table 5: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Group	Organ	Cat	Observation(s)	No. Observed	Foetuses Affected	%	No.             Observed	Litters Affected	%
1	Whole foetus		No abnormalities detected	285	280	98.25	24	-	-
	Whole foetus	AN	Small	285	5	1.75	24	4	16.67
2	Whole foetus		No abnormalities detected	228	228	100.00	20	20	100.00
3	Whole foetus		No abnormalities detected	272	265	97.43	24	-	-
	Whole foetus	AN	Small	272	7	2.57	24	5	20.83
4	Whole foetus		No abnormalities detected	290	289	99.66	24	-	-
	Whole foetus	AN	Small	290	1	0.34	24	1	4.17

 

 

 

Table 6: SKELETAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
				No. Foetuses			No. Litters
Group	Organ	Cat	Observation(s)	Obs	Aff	%	Obs	Aff	%
1	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	70	46.67	24	23	95.83
	Lumbar vertebrae	VA	Centrum incomplete ossification	150	1	0.67	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification	150	1	0.67	24	1	4.17
	Ribs	AN	Wavy	150	10	6.67	24	8	33.33
	Ribs	VA	Short 14th	150	65	43.33	24	22	91.67
	Ribs	VA	Rudimentary 14th	150	11	7.33	24	9	37.50
	Ribs	VA	14 ribs	150	15	10.00	24	9	37.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	8	5.33	24	3	12.50
	Skull	AN	Hyoid no ossification	150	4	2.67	24	3	12.50
	Skull	AN	Temporal incomplete ossification	150	16	10.67	24	10	41.67
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	General incomplete ossification	150	5	3.33	24	4	16.67
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	20	83.33
	Skull	VA	Interparietal incomplete ossification	150	41	27.33	24	17	70.83
	Skull	VA	Parietal incomplete ossification	150	28	18.67	24	15	62.50
	Sternebrae	AN	Fused	150	1	0.67	24	1	4.17
	Sternebrae	AN	No ossification	150	2	1.33	24	2	8.33
	Sternebrae	VA	No ossification 5th	150	7	4.67	24	6	25.00
	Sternebrae	VA	Incomplete ossification 5th	150	22	14.67	24	11	45.83
	Sternebrae	VA	Incomplete ossification 6th	150	43	28.67	24	17	70.83
	Thoracic vertebrae	AN	Centrum bipartite and asymmetical	150	2	1.33	24	2	8.33
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	6	4.00	24	3	12.50
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	150	1	0.67	24	1	4.17
2	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	119	1	0.84	20	1	5.00
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	119	37	31.09	20	15	75.00
	Ribs	AN	Wavy	119	9	7.56	20	6	30.00
	Ribs	VA	14 ribs	119	10	8.40	20	6	30.00
	Ribs	VA	Short 14th	119	52	43.70	20	18	90.00
	Ribs	VA	Rudimentary 14th	119	12	10.08	20	11	55.00
	Sacral vertebrae	AN	Arch(es) incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Frontal incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Temporal incomplete ossification	119	6	5.04	20	4	20.00
	Skull	AN	Hyoid no ossification	119	1	0.84	20	1	5.00
	Skull	VA	Interparietal incomplete ossification	119	26	21.85	20	11	55.00
	Skull	VA	Parietal incomplete ossification	119	11	9.24	20	8	40.00
	Skull	VA	Supraoccipital incomplete ossification	119	42	35.29	20	19	95.00
	Sternebrae	VA	Incomplete ossification	119	1	0.84	20	1	5.00
	Sternebrae	VA	Incomplete ossification 6th	119	21	17.65	20	10	50.00
	Sternebrae	VA	No ossification 5th	119	3	2.52	20	3	15.00
	Sternebrae	VA	Incomplete ossification 5th	119	20	16.81	20	11	55.00
	Thoracic vertebrae	AN	Centrum bipartite	119	1	0.84	20	1	5.00
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	119	4	3.36	20	2	10.00
3	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	144	4	2.78	24	1	4.17
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	144	65	45.14	24	20	83.33
	Ribs	AN	Wavy	144	7	4.86	24	5	20.83
	Ribs	MA	Fused	144	1	0.69	24	1	4.17
	Ribs	VA	14 ribs	144	16	11.11	24	10	41.67
	Ribs	VA	Rudimentary 14th	144	11	7.64	24	9	37.50
	Ribs	VA	Short 14th	144	78	54.17	24	22	91.67
	Sacral vertebrae	AN	Arch(es) incomplete ossification	144	5	3.47	24	3	12.50
	Skull	AN	Frontal incomplete ossification	144	1	0.69	24	1	4.17
	Skull	AN	Hyoid no ossification	144	4	2.78	24	1	4.17
	Skull	AN	General incomplete ossification	144	2	1.39	24	2	8.33
	Skull	AN	Temporal incomplete ossification	144	14	9.72	24	6	25.00
	Skull	VA	Parietal incomplete ossification	144	28	19.44	24	12	50.00
	Skull	VA	Interparietal incomplete ossification	144	36	25.00	24	14	58.33
	Skull	VA	Supraoccipital incomplete ossification	144	58	40.28	24	21	87.50
	Sternebrae	AN	Bipartite 5th	144	1	0.69	24	1	4.17
	Sternebrae	AN	No ossification	144	1	0.69	24	1	4.17
	Sternebrae	VA	Incomplete ossification	144	5	3.47	24	3	12.50
	Sternebrae	VA	Incomplete ossification 6th	144	36	25.00	24	13	54.17
	Sternebrae	VA	No ossification 5th	144	10	6.94	24	5	20.83
	Sternebrae	VA	Incomplete ossification 5th	144	22	15.28	24	16	66.67
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	144	2	1.39	24	2	8.33
	Thoracic vertebrae	AN	Centrum asymmetrical ossification	144	1	0.69	24	1	4.17
	Thoracic vertebrae	AN	Centrum bipartite	144	1	0.69	24	1	4.17
	Thoracic vertebrae	MA	Arch(es) fused	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	144	1	0.69	24	1	4.17
4	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	52	34.67	24	19	79.17
	Ribs	AN	Wavy	150	12	8.00	24	6	25.00
	Ribs	VA	Rudimentary 14th	150	6	4.00	24	6	25.00
	Ribs	VA	14 ribs	150	11	7.33	24	8	33.33
	Ribs	VA	Short 14th	150	71	47.33	24	21	87.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	5	3.33	24	4	16.67
	Skull	AN	General incomplete ossification	150	2	1.33	24	2	8.33
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	Hyoid no ossification	150	4	2.67	24	4	16.67
	Skull	AN	Temporal incomplete ossification	150	17	11.33	24	9	37.50
	Skull	VA	Interparietal incomplete ossification	150	46	30.67	24	18	75.00
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	19	79.17
	Skull	VA	Parietal incomplete ossification	150	30	20.00	24	14	58.33
	Sternebrae	AN	Asymmetrical ossification 5th	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 5th	150	32	21.33	24	17	70.83
	Sternebrae	VA	No ossification 5th	150	4	2.67	24	3	12.50
	Sternebrae	VA	Incomplete ossification	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 6th	150	33	22.00	24	13	54.17
	Thoracic vertebrae	AN	Centrum bipartite	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	4	2.67	24	4	16.67

Applicant's summary and conclusion

Conclusions:

On the basis of the results, the dosage of >=1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.

Executive summary:

The effects of the test material were investigated, in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19 in a GLP compliant study according to OECD TG 414. 

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The plan for this study was to investigate doses of 100, 300 and 1000 mg/kg bw/day of the test item with a dose volume of 10 mL/kg body weight, during the gestation period from Day 6 through Day 19 post coitum. Control females received the vehicle (0.5 % carboxymethyl cellulose (CMC)) at the same dose volume during the same treatment period.
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms from the treatment with the test item were observed. No macroscopic findings were noted during necropsy of the females. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of foetuses per dam were not affected by treatment with the test item at all dose levels. No test item-related effects on foetal body weights were noted. No test item-related effects on foetal sex ratios or anogenital distance were noted in any dose group.
During the external examination of the foetuses, no test item-related abnormal findings were noted. Hormone levels of dams were comparable between groups. Females did not show any macroscopic or microscopic (thyroid) changes related to treatment. Skeletal and visceral examinations were comparable between groups.