[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 02 September 2010; Experiment start date - 17 November 2010; Experiment completion date - 13 April 2011; Study completion date - 03 May 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Name: FAT 40851/B TE
Batch no: BOP 03-10
Expiry Date: 30.06.2015
Physical state: Solid powder
Colour: Orange
Density:1.717 g/cubic cm at 20 °C
Purity: 84.3 % all coloured organic constituents; 29.3 % main constituent
Date of Certificate of Analysis: September 9th 2010
Storage Conditions: room temperature
Solubility in Water: 88.3 g/L at 20 °C
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Females: 175-225 g, (mean: 195.27 g, ± 20 % = ± 39.05 g); Males: 276-306 g, (mean: 290.54 g, ± 20% = ± 58.11 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.: 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: 28 Nov 2010 To: 05 Apr 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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. 103764.

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

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 - Control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2 - Low dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 - Mid dose group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4 - High dose group

No. of animals per sex per dose:

Control: 26
100 mg/kg bw/day: 25
300 mg/kg bw/day: 25
1000 mg/kg bw/day: 26

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. 103758).

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 dam 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

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were few clinical signs observed in females of control and treatment groups during the period of the treatment. The clinical signs observed were increased sponstaneous activity (control 1/26), piloerection (Control: 1/26; LD 1/25; MD: 3/25), aelopecia (Control: 1/26; LD: 3/ 25; HD: 2/26), moving the bedding (MD: 1/25) and respiratory sound (HD: 1/26). These various clinical signs in treatment and control group were not considered to be test item related due to lack dose dependency in severity of the signs and number of animals affected from various treatment groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistical analysis of body weight and body weight change data revealed no significant difference when compared with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

The terminally sacrificed animals belonging to the control or treatment groups revealed lesions like spleen enlarged (1/26 Control), intestine gas filled (1/25 MD group), caecum discolored (1/25 MD group), ovary with cyst (1/26 HD group). The pattern of gross pathological lesions at necropsy in very few animals from control or treatment groups suggested that the lesions were of spontaneous/ incidental in nature.

Maternal developmental toxicity

Details on maternal toxic effects:

Prenatal Data:
Statistical analysis of prenatal data revealed no significant effect on parameters when compared with controls.

Litter Data:
Statistical analysis of litter data revealed no significant effect on parameters. The pregnancy rate (No. of pregnancies achieved /No. of females mated or spear positive x 100) observed were as follows- Control- 76.92 %, LD- 92 %, MD- 88 % and HD- 80.77 %.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Few gross 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. Other isolated findings such as thin tail (one individual pup in control group) and gastroschisis (one individual pup each in LD and MD groups) were observed without reaching any statistical significance. 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 unossification of 4th sternebrum (MD and HD groups), Xiphoid (HD group), 4th metatarsal-bilateral (HD group) and presence of left rudimentary rib 14th (MD and HD groups) and significant decrease in incidences was observed for wavy ribs (LD and HD group) and incomplete ossification of supraoccipital (LD, MD and HD groups), parietal (HD group), interparietal (LD, MD and HD groups) and 4th sternebrum (MD and HD groups) compared to control. The unossified structures are the conditions, which is assumed to be transient and hence is classified variation. The incidence of 14th rudimentary rib is also classified as variation. The other 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. 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 ureter-convoluted (Bilateral) in HD group. This type of incidence (ureter-convoluted) is classified as variation. 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. Therefore, these findings are not to be considered as treatment related and solely spontaneous in nature.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No material toxicity or fetal toxicity observed in the study

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the repeated dose administration of FAT 40851/B to pregnant female Wistar rats at dosages of 100, 300 and 1000 mg/kg bw/day from GD 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 40851/B in Wistar rats was 1000 mg/kg bw/day.

Executive summary:

This prenatal developmental toxicity study of FAT 40851/B 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 (GD) 5 to 19. This study was conducted according to OECD test guideline 414 in a GLP study. 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 smears (GD 0). Four groups of presumed pregnant females were dosed daily by oral gavage with 100, 300 and 1000 mg/kg bw/day of FAT 40851/B at dose volume of 10 mL/kg bw. Control animals were handled identically as treated groups and received aqua ad injectionem (sterile water) as a vehicle in similar volume as treated groups. 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 GD 20. Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of 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 fetuses were observed for the visceral abnormalities, craniofacial examination and remainnig 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. These various clinical signs in treatment and control groups lacked dose dependency in severity of the signs and number of animals affected from various treatment groups. 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 the parameters when compared with controls. The pregnancy rate (No. of pregnancies achieved /No. of females mated or spear positive x 100) observed were as follows- Control- 76.92 %, LD- 92 %, MD- 88 % and HD- 80.77 %. The terminally sacrificed animals belonging to the control or treatment groups revealed lesions like spleen enlarged (1/26 Control), intestine gas filled (1/25 MD group), caecum discoloured (1/25 MD group), ovary with cyst (1/26 HD group). The pattern of gross pathological lesions at necropsy in very few animals from control or treatment groups suggested that the lesions were of spontaneous/ incidental in nature. Few gross external abnormalities were seen among the control and treatment groups, but without statistical significance. 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. There was statistically significant increase in skeletal variations of unossification of 4th sternebrum (MD and HD groups), Xiphoid (HD group), 4th metatarsal-bilateral (HD group) and presence of left rudimentary rib 14th (MD and HD groups) compared to control. 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 variation of ureter- convoluted (Bilateral) in HD group compared to control. Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls, but without statistical significance. In conclusion, the repeated dose administration of FAT 40851/B to pregnant female Wistar rats at dosages of 100, 300 and 1000 mg/kg bw/day from GD 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 40851/B in Wistar rats was 1000 mg/kg bw/day.