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:
27 March - 21 May 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 Environmental Protection Agency (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 40854/A TE
- Substance type: Reddish-brown powder
- Physical state: Powder
- Purity: 46.2% (4 main constituents)
- Lot/batch No.: TZ 5719 / BOP 02-11
- Expiration date of the lot/batch: 01 April 2016
- Storage condition of test material: At room temperature in the dark
- Hygroscopic: Yes, store in well-sealed container
- pH: 5.4 at concentration of >80g/L
- Stability in water: Stability for at least 6 hours at room temperature is confirmed over the concentration range 20-200 mg/mL, Project 497840
- Solubility in water: More than 80 g/L

Test animals

Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: mean body weight at start of treatment was 278 gr (males) or 183 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.
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: 27 March - 21 May 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity 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. Detection of mating was not confirmed for one female of Group 3 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 postcoitum.
- 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:
Analyses were conducted on a single occasion during the treatment phase (30 March 2012), according to a validated method (Project 497818). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). 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%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
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. Female nos. 63 (Group 3) and 71 (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 mated animals in the study: Approximately 12 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:
Based on the results of a dose range finding study (Project 497845). The same dose levels were tested in a 28-day toxicity study (Project 497840), and during in life no clear treatment related toxicity was noted up to 1000 mg/kg.

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, detailed clinical observations were made in all animals, at least once daily from start of treatment onwards. 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, for males and females. Food consumption was not recorded during the mating period. 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.

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
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (all males of the control and high dose group, and all males 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. During the lactation period, no clinical observations were registered in the computer for one pup of a Control Group litter on Day 3, one pup of a litter of Group 3 on Day 4 and for one pup 13 of a litter of Group 4 on Day 3. Sufficient information available from other observations.
- 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:
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated. The animals were not deprived of food overnight.
According to test guidelines

ORGAN WEIGHTS
All F0-males on the scheduled day of necropsy: Epididymides and testes

HISTOPATHOLOGY:
According to test guidelines
From one animal of group 1 the prostate was trimmed by accident (as not needed to be examined). Slide was not examined. Block will be archived.
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. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.
At necropsy, external examinations were not recorded in the raw data for the pups of a litter the control group. Sufficient information available from other litters in this group. Moreover, it concerned a litter from the control group.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)

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 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
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 animals)

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
Gross pathological findings:
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 toxicologically relevant clinical signs were noted during the observation period. Red discolouration/staining of the faeces and/or several body parts was noted for the animals of the treated groups. This was due to the staining properties of the test substance (reddish-brown colour), and not regarded toxicologically relevant. Incidental findings that were noted included salivation, alopecia, broken tail apex, and chromodacryorrhoea. 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
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg.
The statistical significant changes noted for body weight gain of males of the treated groups was not considered toxicologically relevant as these were caused by slightly high control values.

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. Reddish discolouration of the tail was noted for almost all high dose animals. This was due to the staining properties of the test substance (reddish-brown colour), and not regarded toxicologically relevant. For one female of the control group, two fetuses with moderate autolysis were noted in utero. One fetus had a misshapen (asymmetric) mouth and the other showed proboscis with the left eye bulge absent. No clinical signs were noted for this female; slight body weight loss and reduced food consumption were noted during lactation. She delivered 5 healthy pups which survived until planned necropsy on Day 6 of lactation. As this concerned a control animal, it was not treatment related.
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 seminal vesicles reduced in size, tan discolouration of the thymus, uterus containing fluid, nodule on the genital region, and pelvic dilation of the kidneys.

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. Three males and three females failed to mate or conceive. No cause of infertility was found for these animals. Spermatogenic staging profiles were normal for all Group 1 and Group 4 males as well as for one male of Group 2 and one of Group 3. All microscopic findings 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. One female of the control group and 100 mg/kg group were not pregnant, and one female of the 300 mg/kg group did not mate.

GESTATION
Gestation index and duration of gestation were unaffected for all groups.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

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

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS
One pup of the control, low and high dose groups and three pups of the mid dose group 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 did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS
One pup at 100 mg/kg showed a black tail apex from Day 4 of lactation onwards. At this single occurrence, the nature and incidence of clinical signs remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment.

MACROSCOPY PUPS
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach and/or beginning autolysis. Incidental macroscopic findings among surviving pups included dark tail apex and blue spot on the right hindleg. 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)

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 40854/A TE by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental, reproduction and developmental toxicity.
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.
Executive summary:

Rats (5/sex/dose) were treated with 0, 100, 300 or 1000 mg/kg bw/d of FAT 40854/A TE by gavage according to OECD 421. Based on no effects observed on the parameters measured (mortality, clinical signs, body weight, food consumption, macroscopic examination, organ weights, microscopic examination, mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration of gestation) the NOAEL for parental toxicity and reproduction toxicity was established to be 1000 mg/kg bw/d.