[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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

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

Administration / exposure

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

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.:

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

Remarks:

Group 2 - Low dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 - Mid dose group

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.