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EC number: 201-344-6 | CAS number: 81-33-4
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:
- 2 022
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
- 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
Constituent 1
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 / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Low
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Mid
- 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)
open allclose all
- 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
- 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 |
|
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|
|
|
|
|
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 |
|
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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 |
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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.
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