[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

- Oral: study available; NOAEL 750 mg/kg in OECD 421
- Dermal: no study available
- Inhalation: no study available

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 02 September 2010; Experiment start date - 24 March 2011; Experiment completion date - 14 May 2011; Study completion date - 09 August 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: (P) 10-11 weeks
- Weight at study initiation: (P) Males: 257-304 g; Females: 175-213 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 081110)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot. No. 1307 and 1324), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
- Acclimation period: at least 5 days

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The amount of test substance for each dose concentration was suspended separately in aqua ad injectionem (sterile water) on each administration day, immediately before the administration. Homogeneity was ensured by using a vortex machine.

VEHICLE
- Justification for use and choice of vehicle: Selection was based on the solubility of the test item.
- Concentration in vehicle: 5, 20, 75 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 0195A191

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Females with unsuccessful mating were allowed to mate with other male of the same group.
- Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each concentration was analysed for nominal concentration. Homogeneity in the vehicle was analysed for the low and high dose concentrations. Samples for nominal concentration verification were taken in Week 1 (first week of pre-mating period), Week 3 (first week of mating), Week 5 (gestation) and Week 7 (gestation/lactation). Samples for homogeneity were taken from the top, middle and bottom of the low and high dose preparation in Weeks 1 and 5. All concentration samples were stored frozen (approx. -20 °C) until the analysis was performed.

Duration of treatment / exposure:

Males: 14 days before mating, 14 days during mating
Females: 14 days before mating, 14 days during mating, 20 days during gestation, 3 days during lactation

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks

Dose / conc.:

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

Remarks:

Group 1 - Control group

Dose / conc.:

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

Remarks:

Group 2 - Low dose group

Dose / conc.:

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

Remarks:

Group 3 - Mid dose group

Dose / conc.:

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

Remarks:

Group 4 - High dose group

No. of animals per sex per dose:

10

Control animals:

other: yes, concurrent vehicle; the control group was shared with BSL Study no. 111000, another OECD 421 study, which was performed in parallel.

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. A descending sequence of dose levels was selected in order to demonstrate any dose-related response and a NOAEL.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily during weekend/holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before assignment to the experimental groups and on the first day of administration. Males were weighed weekly during the entire study period. Females were weighed weekly during the pre-mating period, on Gestation Day 0, 7, 14, 20 and on Post-natal Day 0 (within 24 hours of parturition) and Post-natal Day 4 along with pups.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration and was not measured during the mating period.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the completion of mating period (Day 28)
- Maternal animals: All surviving animals were sacrificed on respective PND 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: Ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), macroscopic lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age (PND 4). The animals were subjected to postmortem examinations for gross external abnormalities.

Statistics:

One-way analysis of variance (ANOVA) followed by DUNETT’s multiple comparison test (p <0.05 was considered as statistical significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated /No. of pairs) x 100
Fertility Index (%) = (No. of females pregnant/No. of females copulated) x 100
Delivery Index (%) = (No. of dams with live newborns/ No. of pregnant dams) x 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring at Day 4/ No. of live offspring at birth) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were observed. There were very few clinical signs recorded in control and treated animals during the study period but these findings were observed transiently post dose administration and had no relevance to treatment.

Mortality:

no mortality observed

Description (incidence):

None of the animals died during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effect on body weight was observed in both sexes throughout the complete study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females, statistical analysis of food consumption data revealed no effect on food consumption throughout the study period in treated groups when compared with controls.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was golden-brown pigment observed in interstitial macrophages of the testes in all males of HD group, and was also observed in the epididymis in 9/10 males of this group. In the uterus, minimal amounts of golden-brown pigment in the endometrium were seen in 6/10 females of HD group. Uterus was not evaluated in the intermediate dose groups in agreement with the sponsor. There were no test item-related histological findings noted in the other male and female reproductive organs. Kidney was evaluated histologically in those animals showing macroscopic renal discoloration. There was golden-brown pigment in the tubular epithelium of the cortex seen in all females of HD group and in a single female of MD group. All histological findings are due to test item deposition and there were no accompanying findings that would indicate organ damage.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In males, there was statistically significant increase in mean values of absolute epididymides weight, which was further supported by increased relative epididymides weight to terminal body weight. There was also statistically significant increase in relative weight of left testes when compared with the controls, but the relative weight of total testes was statistically insignificant. Histologically, there were no accompanying findings that would indicate organ damage except the test item deposition. Hence, the above epididymal finding might be to lesser extent attributable to test item and does not justify a classification on reproductive toxicity.

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effect on precoital interval and duration of gestation was observed.
The copulation index, fertility index and delivery index remained unaffected. No treatment-related effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, % pre- and post-implantation loss were observed.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4. Viability index was unaffected.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The statistical analysis of litter weight data of treated and control groups measured on PND 0 and PND 4 revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight.

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No substance-related gross external findings were observed in any of the treated groups.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Critical effects observed:

no

Reproductive effects observed:

not specified

Table 1: Clinical Observations (P)

Clinical Findings	Control	50 mg/kg	200 mg/kg	750 mg/kg
Males
Nasal discharge	1/10	0/10	1/10	4/10
Piloerection	0/10	0/10	0/10	1/10
Alopecia	1/10	0/10	0/10	0/10
Females
Aggressive behavior	1/10	0/10	0/10	0/10
Nasal discharge	0/10	0/10	0/10	1/10
Alopecia	0/10	1/10	0/10	0/10
Injury (right leg)	0/10	1/10	0/10	0/10
Respiratory sound	0/10	0/10	0/10	1/10

 

Table 2: Reproductive function - Organ weights (g)

Findings			Control	50 mg/kg	200 mg/kg	750 mg/kg
Testes	Left	Mean	1.732	1.809	1.736	1.879
		SD	0.106	0.189	0.122	0.123
	Right	Mean	1.729	1.798	1.723	1.811
		SD	0.120	0.232	0.1556	0.130
	Total	Mean	3.461	3.607	3.459	3.690
		SD	0.214	0.417	0.275	0.238
Epididymides	Left	Mean	0.609	0.645	0.682	0.697
		SD	0.083	0.072	0.115	0.072
	Right	Mean	0.612	0.648	0.690*	0.668
		SD	0.069	0.066	0.055	0.047
	Total	Mean	1.221	1.293	1.373*	1.365*
		SD	0.138	0.130	0.140	0.094

 

Table 3: Macroscopic Findings (P)

Findings	Control	50 mg/kg	200 mg/kg	750 mg/kg
Males
Epididymides: yellow spots	1/10	0/10	4/10	2/10
Testes: discoloration (yellowish)	0/10	0/10	0/10	6/10
Females
Kidney: discoloration (yellowish)	0/10	3/10	4/10	8/10
Kidney: discoloration (dark)	0/10	1/10	0/10	2/10
Adrenal Glands: enlarged	0/10	1/10	0/10	0/10
Uterus with oviduct and cervix: discoloration (yellowish)	0/10	0/10	0/10	5/10
Abdominal aorta: hard	0/10	0/10	0/10	1/10

 

Table 4: Reproductive Indices (%)

	Control	50 mg/kg	200 mg/kg	750 mg/kg
Copulation Index	100	100	100	100
Fertility Index	60	90	90	90
Delivery Index	100	100	100	100
Viability Index	100.00	99.21	87.65	100.00

Conclusions:

In conclusion, the repeated dose administration of FAT 40851/B in sterile water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 50, 200 and 750 mg/kg body weight revealed no major toxicological findings. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40851/B, the no observed adverse effect level (NOAEL) is believed to be 750 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

The aim of this study was to assess the possible effect of FAT 40851/B on male, female fertility and embryofetal development in Wistar rats. This study was conducted according to OECD test guideline 421 in a GLP certified laboratory. In this study, four groups comprised of 10 adult male and 10 non pregnant nulliparous female rats [WISTAR rat Crl:WI(Han)] were dosed daily by oral gavage with 50, 200 and 750 mg/kg body weight per day of FAT 40851/B at dose volume of 10mL/kg body weight. The test item was formulated in sterile water with an administration volume of 10 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups. The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both males and in females, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted weekly based on the most recent body weight measurement. Animals were examined daily for clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective day 26 after the evidence of mating or completion of the mating period. 

Clinical Observation and Mortality: There were no predominant clinical signs considered to be due to treatment observed in treated groups compared to the controls. There were no mortalities observed in males or females during the study period.

Body Weight Development: In males and females, statistical analysis of body weight data revealed no effect on body weight throughout the study period in treated groups when compared with controls.

Food Consumption: In males and females, statistical analysis of food consumption data revealed no effect on food consumption throughout the study period in treated groups when compared with controls.

Litter Weight: Data Statistical analysis of litter weight data of control and treated groups measured on PND 0 and PND 4 revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight.

Precoital interval and duration of gestation: There was no treatment related effect observed for duration of gestation and precoital interval in treated groups when compared with controls. All females in control and treated groups showed evidence of copulation during 14 days mating period. Successful mating of females resulted in 90 % pregnancy rate each in LD, MD and HD groups, as compared to 60% pregnancy rate in Control group.

Pre and post natal data: Group mean of corpora lutea, implantation sites, live pups born on PND 0, percent preimplantation loss and post implantation loss in treated groups remained unaffected due to treatment when compared with controls.

Litter data: There was no treatment related effect observed on total number of pups born, number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4in treated groups compared to corresponding control.

Reproductive indices: The copulation index, fertility index and viability index in treated groups remained unaffected due to treatment when compared to the control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups.

Pup survival data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control.

Pup External findings: There were no treatment related gross external findings observed in pups from the treated groups on PND 0 and 4.

Gross Pathology: At necropsy, there were few macroscopic findings recorded in males and females of both control and treated groups. In males, the findings were yellow spots on epididymides (C 1/10; MD 4/10; HD 2/10 animals) and yellowish discoloration of testes (HD 6/10 animals). In females, the findings were discoloration of kidneys(LD 4/10; MD 4/10; HD 10/10 animals), enlarged adrenal glands (LD 1/10 animals), discoloration of uterus/oviduct/cervix (HD 5/10 animals), hardening of abdominal aorta (1/10 animals).

Organ Weight: In males, there were statistically significant increase in mean values of absolute epididymides weight (Right side-MD group, Total-MD and HD groups), relative epididymides weight to terminal body weight (right side-MD group, left side-HD group, Total-MD and HD groups) and relative testes weight to terminal body weight (Left side-HD group) of the treated groups when compared with the controls. In females, there were no statistically significant differences observed in the absolute and relative organ weights of the treatment groups when compared with the controls.

Histopathology: There was golden-brown pigment observed in interstitial macrophages of the epididymis and/or testes in males, and in the endometrium of the uterus in females of HD group. These changes were considered to be related to test item deposition. Based on the extent of histopathological evaluation as defined by the study plan, there was no histomorphological indication of any functional impairment of the organs and tissues concerned.

In conclusion, the repeated dose administration of FAT 40851/B in sterile water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 50, 200 and 750 mg/kg body weight revealed no major toxicological findings. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40851/B, the no observed adverse effect level (NOAEL) is believed to be 750 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality database with Klimisch rating 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OCED 421 Oral study:

The aim of this study was to assess the possible effect of FAT 40851/B on male, female fertility and embryofetal development in Wistar rats. This study was conducted according to OECD test guideline 421 in a GLP-certified laboratory. In this study, four groups comprised of 10 adult male and 10 non pregnant nulliparous female rats [WISTAR rat Crl:WI(Han)] were dosed daily by oral gavage with 50, 200 and 750 mg/kg body weight per day of FAT 40851/B at dose volume of 10mL/kg body weight. The test item was formulated in sterile water with an administration volume of 10 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups. The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both males and in females, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted weekly based on the most recent body weight measurement. Animals were examined daily for clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective day 26 after the evidence of mating or completion of the mating period. Clinical Observation and Mortality: There were no predominant clinical signs considered to be due to treatment observed in treated groups compared to the controls. There were no mortalities observed in males or females during the study period. 

Body Weight Development: In males and females, statistical analysis of body weight data revealed no effect on body weight throughout the study period in treated groups when compared with controls.

Food Consumption: In males and females, statistical analysis of food consumption data revealed no effect on food consumption throughout the study period in treated groups when compared with controls.

Litter Weight: Data Statistical analysis of litter weight data of control and treated groups measured on PND 0 and PND 4 revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight.

Precoital interval and duration of gestation: There was no treatment related effect observed for duration of gestation and precoital interval in treated groups when compared with controls. All females in control and treated groups showed evidence of copulation during 14 days mating period. Successful mating of females resulted in 90% pregnancy rate each in LD, MD and HD groups, as compared to 60 % pregnancy rate in Control group.

Pre and post natal data: Group mean of corpora lutea, implantation sites, live pups born on PND 0, percent preimplantation loss and post implantation loss in treated groups remained unaffected due to treatment when compared with controls.

Litter data: There was no treatment related effect observed on total number of pups born, number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4in treated groups compared to corresponding control.

Reproductive indices: The copulation index, fertility index and viability index in treated groups remained unaffected due to treatment when compared to the control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups.

Pup survival data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control.

Pup External findings: There were no treatment related gross external findings observed in pups from the treated groups on PND 0 and 4.

Gross Pathology: At necropsy, there were few macroscopic findings recorded in males and females of both control and treated groups. In males, the findings were yellow spots on epididymides (C 1/10; MD 4/10; HD 2/10 animals) and yellowish discoloration of testes (HD 6/10 animals). In females, the findings were discoloration of kidneys(LD 4/10; MD 4/10; HD 10/10 animals), enlarged adrenal glands (LD 1/10 animals), discoloration of uterus/oviduct/cervix (HD 5/10 animals), hardening of abdominal aorta (1/10 animals).

Organ Weight: In males, there were statistically significant increase in mean values of absolute epididymides weight (Right side-MD group, Total-MD and HD groups), relative epididymides weight to terminal body weight (right side-MD group, left side-HD group, Total-MD and HD groups) and relative testes weight to terminal body weight (Left side-HD group) of the treated groups when compared with the controls. In females, there were no statistically significant differences observed in the absolute and relative organ weights of the treatment groups when compared with the controls.

Histopathology: There was golden-brown pigment observed in interstitial macrophages of the epididymis and/or testes in males, and in the endometrium of the uterus in females of HD group. These changes were considered to be related to test item deposition. Based on the extent of histopathological evaluation as defined by the study plan, there was no histomorphological indication of any functional impairment of the organs and tissues concerned.

In conclusion, the repeated dose administration of FAT 40851/B in sterile water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 50, 200and 750 mg/kg body weight revealed no major toxicological findings. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40851/B, the no observed adverse effect level (NOAEL) is believed to be 750 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL 1000 mg/kg for both maternal and fetal toxicity/teratogenicity (OECD TG 414); study "Rudragowda, BSL, 103761, 2011, RL1"
- Oral: NOAEL 750 mg/kg in OECD 421
- Dermal: no study available
- Inhalation: no study available

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

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

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.

Dose / conc.:

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

Remarks:

Group 1 - Control group

Dose / conc.:

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

Remarks:

Group 2 - Low dose group

Dose / conc.:

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

Remarks:

Group 3 - Mid dose group

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).

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).

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.

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 %.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

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.

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality database Klimisch rating 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal development study OECD 414

 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 injection (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 remaining 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.

Justification for classification or non-classification

- Effects on fertility:

As no data on fertility is available for FAT 40851/B a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.

 

- Effects on developmental toxicity/teratogenicity:

Based on the above stated assessment of the developmental toxicity/teratogenicity potential of FAT 40851/B (absence of substance related adverse effects at 1000 mg/kg bw) the substance is deemed not to be toxic to the developmental toxicity/teratogenic according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.