Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July - 24 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40826/B TE
- Substance type: Orange powder
- Physical state: Powder
- Purity: 53.4% Main constituent
74.4% all coloured constituents
- Lot/batch No.: TZ 5604-BS-BOP 01-12
- Expiration date of the lot/batch: 28 January 2017
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required: No
- Volatile No
- pH 6 to 8 (% w/w : 2%)
- Stability in water: Stable at pH 7.00 and HLT at 25°C >1 year
- Solubility in water: >740 g/L at 20°C

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 317 gr (males) or 212 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL 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.

IN-LIFE DATES
From: 31 July - 24 September 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
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 19 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 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Two females of Group 1, one of Group 3 and two females of Group 4 were not dosed during littering.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Details on study schedule:
- Age at mating of the animals in the study: Approximately 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 28-day toxicity study in which FAT 40826/A was dosed to Wistar rats at doses of 50, 200 and 1000 mg/kg. Test item-related findings were generally restricted to minimal to slight and reversible focal spongiosis of the stratified squamous epithelium in the forestomach of all animals treated with 1000 mg/kg. Based on the results of this study, 200 mg/kg of FAT 40826/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg of FAT 40826/A as the noobserved-adverse-effect-level (NOAEL).
- 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, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from start of treatment onwards, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: 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 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No.

HAEMATOLOGY
No.

CLINICAL CHEMISTRY
No.

URINALYSIS
No.

NEUROBEHAVIOURAL EXAMINATION
No.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis for all males of the control and high dose group and animals suspected to be infertile.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: 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 in 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 (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
- All animals were deeply anaesthetised and subsequently exsanguinated. The animals were not deprived of food overnight.
- According to test guidelines

ORGAN WEIGHTS
- All males: Epididymides and testes

HISTOPATHOLOGY:
- According to test guidelines
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

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:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. At 1000 mg/kg, orange discolouration of the faeces (all animals) and tail (two males) was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Salivation seen after dosing among all animals of the 1000 mg/kg dose group was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included alopecia and rales. 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. As one female at 1000 mg/kg was pregnant with only one pup, a lower body weight gain was noted during post-coitum. At this single occurrence it was not considered toxicologically significant. The statistically significantly increased body weight gain noted on Day 4 post-coitum for high dose females was not considered toxicologically significant as it was a slight increase and occurred only on one occasion.

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. Three males at 1000 mg/kg showed orange discolouration of the tail, which was considered due to the staining properties of the test substance and not regarded toxicologically relevant. 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 nodule at the epididymides or uterine adipose tissue, pelvic dilation of the kidney, reddish discolouration of the thymus, foci on the thymus or clitoral glands, and enlarged spleen.

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 in the reproductive organs. Orange brown pigment was present in the stratum corneum of the skin for three males treated at 1000 mg/kg. This finding was considered to be due to the deposition of the (orange) test item onto the skin excreted in the feces. There were two Group 1 and two Group 3 animals of which the females were not pregnant and were therefore selected for histopathological examination of the reproductive organs. Their infertility could not be attributed to treatment. Spermatogenic staging profiles were normal for all males examined. All microscopic findings recorded in animals surviving to the end of the assigned study period were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The slightly lower mean number of corpora lutea and implantation sites (not statistically significant) at 1000 mg/kg were due to one female which had only two corpora lutea and implantation sites. At this single occurrence it was not considered toxicologically significant.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

DEVELOPMENTAL DATA
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. At 1000 mg/kg, one female had only one pup. At this single occurrence it was not considered toxicologically significant.

MORTALITY PUPS
One pup at 100 mg/kg was found dead at first litter check and one pup at 100 mg/kg and one pup at 1000 mg/kg were missing on Day 2 of lactation. The pups that were missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

OBSERVATIONS
One pup at the control group showed a lean appearance on Day 4 of lactation (which concurred with a lower body weight) and the pup at 100 mg/kg that was found dead at first litter check showed absence of milk in the stomach. The nature and incidence of these signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with FAT 40826/B TE by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental, reproduction and developmental toxicity for treatment up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.