Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
Reproduction/Developmental Toxicity Screening test (OECD 421)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from Oct. 15, 2012 to Mar.13, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification FAT 20306/B
Molecular formula C26H19N3O5S2
Molecular weight 517.59
CAS Number 119822-74-1
Description Red powder
Batch HT 2025/50
Purity/Composition Unknown
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 28 August 2015 (Retest date) (taken from label)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Rationale This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible
to the effects of reproductive toxicants.

Source F0 Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment Approximately 11 weeks.

Number of F0-animals 40 females and 40 males.

Acclimatization F0 At least 5 days prior to start of treatment.

Health inspection F0 Upon receipt of the animals.

Randomization F0 Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Identification F0 Earmark and tattoo.
Parturition The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Number of pups 445 pups.

Identification of pups On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Animal husbandry
Room number A0.13 (until Day 10 of study). A0.20 (from Day 10 of study onwards).

Conditions Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on
the outcome of the study.

Accommodation
Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were
individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm.
Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Water Free access to tap-water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 70 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70 °C for at least 9 days.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
Parental animals
Method Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Frequency Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Exposure period Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 43, 46 (Group 1), 55, 57 (Group 2), 72 and 79 (Group 4) were not dosed during littering.

Dose volume 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Pups
Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 and 400 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose levels
Dose levels were based on a 28-day toxicity study in Wistar rats (RCC Project 092441) in which 50, 200 and 1000 mg/kg were tested. Starting with day 17 of treatment one male and one female rat at 50 mg/kg showed slight sedation and ruffled fur. In addition, four males and two females at 200 mg/kg as well as nine males and ten females at 1000 mg/kg showed dyspnea, ruffled fur, alopecia, sedation, rales, hunched posture, stiff gait and loss of weight. These symptoms were classified as slight to moderate. They were different in duration and intensity among the animals. A clear dose-relation was observed. During recovery period most of the described symptoms had recovered between days 2 to 10.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
Mortality / Viability At least twice daily.

Clinical signs Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Food consumption Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Oestrous cyclicity (parental animals):
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Sperm parameters (parental animals):
no data
Litter observations:
Mortality / Viability
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs
At least once daily, detailed clinical observations were made for all animals.
Body weights
Live pups were weighed on Days 1 and 4 of lactation.
Sex
Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
Necropsy parental animals
The animals were not deprived of food overnight.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:

Condition

Day of necropsy
Females which delivered Lactation Days 5-7.
Females which failed to deliver (nos. 63, 73 and 77)
Postcoitum Days 26-27 (females with evidence of mating)
Males Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Organ weights: The following organ weights and terminal body weight were recorded from all F0-males on the scheduled day of necropsy:
Epididymides
Testes

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The
Netherlands).
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5 - 7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
Observations/measurements in the study were recorded electronically using the following programs:
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).
- TOXDATA version 8.0 (WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands): Mortality / Clinical signs / Body weights / Food consumption / Reproduction parameters / Observations pups / Organ weights.
Reproductive indices:
mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care,
Offspring viability indices:
sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
No mortality occurred during the study period.

Clinical signs
No clinical signs of toxicity related to test substance treatment were noted during the observation period.

Orange faeces noted for all animals treated at 400 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant.

Chromodacryorrhoea of the left eye was noted for one low dose male and alopecia was noted for one high dose female. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Body weights
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
The statistically significantly increased mean body weight for females at 150 mg/kg on Day 14 post-coitum and decreased mean body weight gain for males at 50 mg/kg were not regarded toxicologically relevant as no dose response relationship was noted and the changes were very slight.

Food consumption
Food consumption before or after allowance for body weight was similar between treated and control animals.

Macroscopic examination
Necropsy did not reveal any toxicologically relevant alterations.

There was one animal with nodule(s) in the epididymides in the control group, one at 150 mg/kg and one at 400 mg/kg.

The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no
toxicological relevance, and included foci on the thymus, pelvic dilation of the kidneys, alopecia, and discolouration of the clitoral glands.

Organ weights
Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.

Microscopic examination
There were no treatment related microscopic findings.

Sperm granuloma were recorded in the epididymides of two control group males, one male at 150 mg/kg and three males at 400 mg/kg, and were the histologic correlates to the nodules noted at necropsy in this organ. There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.

The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all
males evaluated.

Reproduction data
No toxicologically relevant effects on reproductive parameters were noted.

There was one female at 150 mg/kg and two females at 400 mg/kg out of the ten mated animals per group which were not pregnant. These numbers were within normal limits. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.





Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental, reproduction and developmental toxicity was observed up to 400 mg/kg.
Remarks on result:
other: Generation: F0 and P (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

The statistically significantly effect on sex ratio at 50 mg/kg was not considered toxicologically relevant as the sex ratio was considered normal and no dose response relationship was noted.

Mortality
Two pups of the control group and five pups at 400 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

Observations
Incidental clinical symptoms of pups consisted of blue snout, pale appearance, no milk in the stomach, and cold appearance. Incidental macroscopic findings of pups that were found dead included moderate autolysis, and no milk in the stomach. There was one pup (litter 79; Group 4) with missing tail on Day 4 of lactation, which was confirmed at macroscopic examination during planned necropsy on Day 5 of lactation.
The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
other: sex

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.
Executive summary:

This study was conducted to evaluate the potential toxic effects of the test substance in accordance with OECD guideline 421 under the GLP conditions.

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 150 and 400 mg/kg. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. No parental, reproduction and developmental toxicity was observed up to 400 mg/kg.

In conclusion, treatment with FAT 20306/B by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 400 mg/kg revealed no parental, reproduction and developmental toxicity for treatment up to 400 mg/kg.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.