Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jan 2011 - 25 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40849/C TE
- Physical state: blue solid powder
- Analytical purity: 65.1% all colored organic constituents, 43.6% main constituent
- Purity test date: 20.08.2010
- Lot/batch No.: BOP 02-10
- Expiration date of the lot/batch: 25.08.2015
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at receipt: Females: 184.4-210.6 g, (mean: 196.14 g, ± 20% = ± 39.23 g); Males: 276.6-318.8 g, (mean: 295.95 g, ± 20% = ± 59.19 g)
- Housing: The animals were housed individually in IVC cages (except during mating period when 2 females were paired with one male), type III H, polysulphone cages on Altromin saw fiber bedding (Lot No.: 150910 and 241110)
- Diet (ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.: 1307)
- Water (ad libitum): Free access to tap water, acidified using sulfuric acid to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sterile water (Manufacturer: Berlin Chemie)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in sterile water. The vehicle was chosen based on the test item’s solubility. The test item formulation was prepared freshly on each administration day before the administration procedure.
The test substance as well as the vehicle was administered at a dose volume of 10 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed on samples collected at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study.
All formulation samples were stored at -20 °C and analyzed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 103762.
Details on mating procedure:
After acclimatisation, females were paired with males as per the ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularize the number of animals for terminal sacrifice on particular day. The subsequent morning and the next morning onwards, the vaginal smear of female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as Gestation Day 0. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other.
Duration of treatment / exposure:
Gestation Day 5-19
Frequency of treatment:
Daily
Duration of test:
Animals were sacrificed on Gestation Day 20.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Control group: 25
Low dose group: 35
Mid dose group: 24
High dose group: 25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of the dose range finding study (BSL Study No. 103756).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily during the entire gestation period except during weekends and holidays where clinical observation was made only once. Mortality, morbidity, pertinent behavioral changes and all signs of overt toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the day of receipt to ensure that the body weights were within the ± 20% variation.
The sperm positive females were weighed during GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except on the day of receipt.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption of pregnant females was measured on GD 5, 8, 11, 14, 17 and 20. The food consumption was presented for period 0-5, 5-8, 8-11, 11-14, 14-17, and 17-20. The food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 20
- Examinations: At the time of termination, the presumed pregnant females were examined macroscopically for any structural abnormalities or pathological changes which might have influenced the pregnancy.
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
Fetal examinations:
- External examinations: Yes: All fetuses from particular dams were identified by different colored strings, weighed and sexed, based on the anogenital distance. Each fetus was examined for external anomalies.
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one interval to the next.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Chi-square test.The statistical analysis was performed with GraphPad Prism (Version V) software (p<0.05 was considered as statistical significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical Observation:
No test item related clinical signs were observed in any of the dose group females during the entire period of the treatment when compared with controls.
Few predominant clinical signs observed in control group were slight nasal discharge(1/25), Alopecia (2/25); in LD nasal discharge (1/35), alopecia (2/35); in MD slight nasal discharge 2/ 24, nasal discharge (2/24), slight piloerection (1/24); in HD group slight nasal discharge (5/25), nasal discharge (1/25), slight/moderate piloerection (5/25), slight/moderate salivation (3/25), slight vocalisation (2/25). These various clinical signs were not considered to be test item related due to lack dose dependency and the fact that they were noted immediately after the dose application.
All animals survived throughout the study period except for one animal from LD group which died during the treatment period (Gestation Day 8) due to a gavaging error.

Body Weight and Body Weight Change:
Statistical analysis of body weight and body weight change data revealed no significant difference in body weight and body weight change in treated groups when compared with controls.

Food Consumption:
No treatment related effect on food consumption was observed during treatment period in any of the treatment group when compared with controls.

Prenatal Data:
Statistical analysis of prenatal data revealed no significant effect on parameters in the treated groups when compared with controls. However, statistically significant increase in group mean number of live fetuses and female fetuses in MD group was observed when compared with controls. Due to lack of dose dependency, this significant difference in parameters was considered to be incidental and not related to the test item.

Litter Data:
Statistically significant increase in group mean total number of fetuses and total number of females was observed in MD when compared with controls. Due to lack of dose dependency, this significant difference in parameters was considered to be incidental and not related to the test item. All other parameters remained unaffected in treated groups when compared with controls.
Decrease in pregnancy rate was observed in LD group (65. 71%), HD (80.00%), and MD (87.50) as compared to control (88.00%). As this effect on pregnancy rate is marginal in MD and HD when compared with control, it was considered to be a biological varion and of no toxicological relevance.

Gross Pathology:
The terminally sacrificed animals belonging to the control and treatment groups revealed predominant lesions like red/dark spots on lungs, blue colored kidneys and digestive tract. There was increased number of incidences of blue/green discoloration of various visceral organs observed in treated groups and which could be attributed to the blue color of the test item and as such of no toxicological significance.
The pattern of gross pathological lesions at necropsy from different treatment groups suggested that the lesions were of spontaneous/ incidental in nature.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Few external abnormalities were seen among the control and treatment groups. Predominant external finding noted was haematoma on various body locations in control and treated groups.

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities comparable with controls. Statistically significant decrease in incidence of enlarged anterior and posterior fontanelle, incomplete ossification of Frontal (Bilateral), Parietal (B), Interparietal, Supraoccipital, Temporal (B), Zygomatic (B), 4th Sternebra and Xiphoid, Absent 4th Metacarpal (B), 14th rudimentary rib (B) and increase in incomplete ossification of 4th Sternebra was onserved in HD when compared with controls. There were also statistically significant decrease in incomplete ossification of Parietal (B), Interparietal, Supraoccipital and absent 4th metacarpal (B) in LD and 14th rudimentary rib (B) was observed in MD when compared with controls. Extra ossification of 4th sacral vertebral arch (B) was significantly increased in LD and MD when compared to controls.
Due to lack of dose dependency and consistency in these findings indicate no relevance with treatment. There was no indication of a compound related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including controls. There was a statistically significant decrease in hemorrhagic adrenal glands in MD and significant increase in convoluted ureter (B) in HD group was observed when compared with controls. Since these visceral abnormalities observed in treated groups were in frequencies comparable, slightly higher or even less in numbers to controls, therefore no serious toxicological significance can be attributed to these findings and considered to be spontaneous in nature.

Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls. Statistical analysis of the data revealed no significant difference in number of incidences in treated group as compared to controls. Therefore, these findings are not to be considered as treatment related and solely spontaneous in nature.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of FAT 40489/C TE to pregnant female Wistar rats at dosages of 0, 100, 300 and 1000 mg/kg bw/day from Gestation Day 5 to 19 revealed no major toxicological findings in females and fetuses.
Based on the data generated from this study, the NOAEL for both maternal toxicity and fetal toxicity of FAT 40849/C TE in Wistar rats was 1000 mg/kg bw/day. FAT 40489/C TE had no effect on intrauterine development.
Executive summary:

This prenatal developmental toxicity study of FAT 40849/C TE was conducted in pregnant female Wistar rats to detect possible adverse effects on pregnant females and embryofetal development when administered by oral gavage from Gestation Day 5 to 19. Nulliparous and non pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smear (GD 0). Three groups of presumed pregnant females were dosed daily by oral gavage with 100, 300 and 1000 mg/kg bw/day of FAT 40849/C TE at dose volume of 10 mL/kg body weight. The control group was handled identically as treated groups and received aqua ad injectionem (sterile water) as a vehicle in similar volume as treated groups.

The test item formulation was prepared freshly and dose volumes were adjusted based on the most recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured on various gestation days.

The treated and control females were sacrificed on respective Gestation Day 20. Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of corpora lutea, implantations, resorptions (early and late), live and dead fetuses. Fetuses were identified by color strings, sexed and weighed. All fetuses were observed for external abnormalities. Half of the foetuses were observed for the visceral and craniofacial abnormalities and remaing half of the litter for skeletal abnormalities.

Uteri of the non pregnant females were processed with 10% ammonium sulphide solution and checked for the early embryonic deaths if any.

No test item related clinical observations were observed in any of the dose group females during the entire period of the treatment when compared with controls. One animal (34) from LD group was died during the treatment period (Gestation Day 8) due to gavaging error.

Body weight, body weight change and food consumption remained unaffected throughout the treatment period.

Statistical analysis of prenatal and litter data revealed no significant effect when compared with controls. However, statistically significant increase in group mean number of live fetuses and female fetuses in MD group was observed when compared with controls. Due to lack of dose dependency, this significant difference in parameters was considered to be incidental and not related to the test item. Moreover, statistically significant increase in group mean total number of fetuses and total number of females was observed in MD when compared with controls.

Decrease in pregnancy rate was observed in LD group (62.86. %), HD (80.00%), and MD (87.50) as compared to controls (88.00%).

Few gross external abnormalities like hematoma on various body locations were seen among the control and treatment groups.

Skeletal examination of fetuses revealed a range of abnormalities, which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant decrease in incidence of enlarged anterior and posterior fontanelle, incomplete ossification of Frontal (Bilateral), Parietal (B), Interparietal, Supraoccipital, Temporal (B), Zygomatic (B), 4th Sternebra and Xiphoid, Absent 4th Metacarpal (B), 14th rudimentary rib (B) and increase in incomplete ossification of 4th Sternebra was onserved in HD when compared with controls. There were also statistically significant decrease in incomplete ossification of Parietal (B), Interparietal, Supraoccipital and absent 4th metacarpal (B) in LD and 14th rudimentary rib (B) was observed in MD when compared with controls. Extra ossification of 4th sacral vertebral arch (B) was significantly increased in LD and MD when compared to controls.

Internal observation of the fetal viscera revealed a range of visceral abnormalities in all groups including control. There was statistically significant decrease in hemorrhagice adrenal glands in MD and significant increase in convoluted ureter (B) in HD group was observed when compared with controls.

Craniofacial examination revealed a range of visceral abnormalities in all groups including controls. Statistical analysis of the data revealed no significant difference in number of incidences in treated group as compared to controls.

The terminally sacrificed animals belonging to the control and treatment groups revealed predominant lesions like red/dark spots on lungs, blue colored kidneys and digestive tract. There was increased number of incidences of blue/green discoloration of various visceral organs observed in treated groups and which could be attributed to the blue color of the test item and as such of no toxicological significance.

In conclusion, the repeated dose administration of FAT 40489/C TE to pregnant female Wistar rats at dosages of 100, 300 and 1000 mg/kg bw/day from Gestation Day 5 to 19 revealed no major toxicological findings in females and fetuses.

Based on the data generated from this study, the NOAEL for both maternal toxicity and fetal toxicity of FAT 40489/C TE in Wistar rats was 1000 mg/kg body weight.