Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2010 - 23 Dec 2010
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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40850/B TE
- Physical state: red solid powder
- Analytical purity: 70.6% all colored organic constituents, 32.4% main constituent
- Purity test date: 07.09.2010
- Lot/batch No.: BOP 01-10
- Expiration date of the lot/batch: 15.07.2015
- 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 study initiation: Females: 167-201 g, (mean: 184.78 g, ± 20% = ± 36.96 g); Males: 286-318 g, (mean: 297.98 g, ± 20% = ± 59.60 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.: 050810, 040810, 150910, 021010 and 070111)
- Diet (ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.: 1315 and 1013)
- 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 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: From: 29 Nov 2010 To: 23 Dec 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sterile water (Manufacturer: Delta Select)
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 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. 103763.
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:
25 females
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. 103757).

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 behavioural 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. The clinical signs observed in control group were wet nose and mouth (2/25), alopecia (1/25); in LD group was alopecia (1/25); in MD group were wet nose and mouth (1/25), alopecia (2/25), piloerection (2/25), nasal discharge (1/25); in HD group were alopecia (3/25), piloerection (7/25), nasal discharge (1/25), salivation (7/25), moving the bedding (10/25), respiratory sound (2/25). These symptoms were observed transiently occurring immediately after dose administration and are not assumed to be related to systemic toxicity. All animals survived throughout the study period.

Body Weight and Body Weight Change:
No test item related effect on body weight and body weight change was observed during the treatment period. However, statistical analysis of body weight and body weight change data revealed significant decrease for body weight in HD group on GD 5 and body weight change in LD group between GD 8-11. The statistically significant decrease observed in HD and LD groups were considered incidental and did not have any toxicological relevance.

Food Consumption:
Statistical analysis revealed no treatment related effect on food consumption during treatment period in any of the treatment groups when compared with corresponding controls.

Prenatal Data:
Statistical analysis of prenatal data revealed no significant effect on parameters.

Litter Data:
Statistical analysis of litter data revealed no significant effect on parameters.
The pregnancy rate observed in different groups is as follows, Control group (96%), LD (84%), MD (100%) and HD (88%).

Gross Pathology:
The terminally sacrificed animals belonging to the control and LD group revealed no findings, but there were few lesions observed in MD or HD groups like spleen enlarged (MD group: 1/25; HD group 0/25), lung with red spots ( MD group: 1/25 and HD groups: 1/25) and discolored kidney (MD group: 0/25; HD group 3/25). These finding were observed in few individual animals and were not considered treatment related.
The pattern of gross pathological lesions at necropsy in very few animals 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. These finding in treatment group were statistically insignificant compared to controls.

Skeletal examination of the Alizarin red stained 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 increase in incidence was observed for incomplete ossification of 4th metacarpal-bilateral (HD group), Xiphoid (MD group) and parietal (LD group) and significant decrease in incidences was observed for dumbbell shaped-4th sternebrum (HD group) and rudimentary 14th rib–bilateral (HD group). The incidence incomplete ossification of 4th metacarpal-bilateral was observed in one isolated animal of HD group and was considered incidental. However, the statistical significant difference observed for the findings revealed lack of dose dependency and indicated 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 control. There was statistically significant increase in heart hyperemia (without the aneurysmal changes) in MD and HD groups compared to controls. This finding is reflected as the consequence of a functional disorder and do not represent the developmental anomalies. However, due to very less severity and lack of dose dependency this incidence was classified as variation. The other visceral abnormalities observed in treated groups were in frequencies comparable or even less in numbers to controls, therefore no 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. These findings were in frequencies comparable or even less in numbers 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 40850/B 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 40850/B TE in Wistar rats was 1000 mg/kg bw/day.
Executive summary:

This prenatal developmental toxicity study of FAT 40850/B 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). Four groups of presumed pregnant females were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of FAT 40850/B TE at dose volume of 10 mL/kg body weight. Control animals were 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.

There were few clinical signs observed in females of control and treatment groups during the period of the treatment. The clinical signs recorded during the study in the treatment groups were observed transiently and were not considered to be due to systemic effect of the test item.

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

Statistical analysis of prenatal data and litter data revealed no significant effect on parameters like terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantations, live fetuses, group mean number of male and female fetuses, total fetuses, sex ratio, fetal positions, dead fetuses, group litter mean weight, male and female litter weight, resorptions, percent preimplantation loss and post implantation loss in the treated groups when compared with controls.

The pregnancy rate observed in different groups is as follows, Control group (96%), LD (84%), MD (100%) and HD (88%).

Few gross external abnormalities were seen among the control and treatment groups. Haematoma on various body locations were the predominant findings observed in both control and treatment groups.

Skeletal examination of the Alizarin red stained 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 increase in incidence was observed for incomplete ossification of 4th metacarpal-bilateral (HD group), Xiphoid (MD group) and parietal (LD group) and significant decrease in incidences was observed for dumbbell shaped-4th sternebrum (HD group) and rudimentary 14th rib –bilateral (HD group).

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant increase in heart hyperemia (without the aneurysmal changes) in MD and HD groups. This finding was observed with very less severity and showed lack of dose dependency.

Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls.

The terminally sacrificed animals belonging to the control and LD group revealed no findings, but there were lesions like spleen enlarged, lung with red spots and discolored kidney observed in MD or HD groups. These finding were observed in few individual animals and were not considered treatment related.

In conclusion, the repeated dose administration of FAT 40850/B 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 40850/B TE in Wistar rats was 1000 mg/kg bw/day.