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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

The parental and reproduction No Observed Adverse Effect Level (NOAEL) of test substance was 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March 2012 to 21 May 2012
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
Qualifier:
according to guideline
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 (incl. QA statement)
Limit test:
no
Justification for study design:
The purpose of this study was to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. This study should provide part of a rational basis for toxicological risk assessment in man.
Specific details on test material used for the study:
Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at WIL Research Europe B.V.)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Solubility in Water: More than 80 g/L
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han).
Details on species / strain selection:
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 general and reproduction toxicity studies. WIL Research Europe B.V. has general and 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.
Sex:
male/female
Details on test animals or test system 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 g (males) or 183 g (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
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 post-coitum.
- 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
Based on the results of a dose range finding study. The same dose levels were tested in a 28-day toxicity study and during in life no clear treatment related toxicity was noted up to 1000 mg/kg.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Middle dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose
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
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 abnormalities, 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: Epididymites 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
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality 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
Description (incidence and severity):
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 males 15 and 29.
All microscopic findings were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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: Based on the study findings
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality / viability:
no mortality observed
Description (incidence and severity):
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.
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
Description (incidence and severity):
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
Histopathological findings:
not examined
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.
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.
Critical effects observed:
no
Reproductive effects observed:
not specified
Conclusions:
The parental and reproduction No Observed Adverse Effect Level (NOAEL) of FAT 40854/A was 1000 mg/kg/day.
Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40854/A in rats by oral gavage was carried out according to OECD guideline 421. Based on the results of a dose range finding study. The same dose levels were tested in a 28-day toxicity study and during in life no clear treatment related toxicity was noted up to 1000 mg/kg. Dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. 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. The observations and examinations were evaluated includes mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity. 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. 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 index and duration of gestation were unaffected for all groups. 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. 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. No parental and reproductive toxicity was observed up to 1000 mg/kg. Based on the results, a parental, reproduction No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.

Effect on fertility: 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:
Guideline and GLP-compliant study. Klimisch Code 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

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40854/A in rats by oral gavage was carried out according to OECD guideline 421. Based on the results of a dose range finding study. The same dose levels were tested in a 28-day toxicity study and during in life no clear treatment related toxicity was noted up to 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. 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. 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. 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. 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. 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. No parental or reproductive toxicity was observed up to 1000 mg/kg. Based on the results, a parental and reproduction No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.

Effects on developmental toxicity

Description of key information
The devlopmental No Observed Adverse Effect Level (NOAEL) of test substance was 1000 mg/kg/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March 2012 to 17 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
according to guideline
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 (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at WIL Research Europe B.V.)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Solubility in Water: More than 80 g/L
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system 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 g (males) or 183 g (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
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.
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 %).
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
- Age at mating of the mated animals in the study: Approximately 12 weeks
- 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).
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.
Duration of test:
Males: 28 days
Females: 41-55 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control (G1)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose (G2)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Middle dose (G3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose (G4)
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.
Maternal 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

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: Epididymites 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnormalities, 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. 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.
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).
Indices:
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 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
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality 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
Description (incidence and severity):
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 (no. 49) 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.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
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 males 15 and 29. All microscopic findings were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
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 epididymites 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.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The statistical significant changes noted for sex ratio were due to slightly high numbers of male pups compared to female pups in the control group, and were therefor not treatment-related.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
not specified
Skeletal malformations:
not specified
Other effects:
no effects observed
Description (incidence and severity):
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.

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.

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. The statistical significant changes noted for sex ratio were due to slightly high numbers of male pups compared to female pups in the control group and were therefor not treatment related.

Mortality:
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
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 weights
Body weights of pups were considered to have been unaffected by treatment.

Macroscopy
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.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.
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: Based on study findings
Abnormalities:
not specified
Developmental effects observed:
not specified

Reproductive 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.


 


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.


 


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. The statistical significant changes noted for sex ratio were due to slightly high numbers of male pups compared to female pups in the control group and were therefor not treatment related.


 


Mortality:


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


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 weights:


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


 


Macroscopy:


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.

Conclusions:
The developmental No Observed Adverse Effect Level (NOAEL) of FAT 40854/A was 1000 mg/kg/day.
Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40854/A in rats by oral gavage was carried out according to OECD guideline 421, in which four groups of ten male and ten female Wistar rats received the test substance at 0, 100, 300 and 1000 mg/kg via oral gavage. Apart from investigations into reproductive parameters, the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment related effects on pup development such as mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

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:
Guideline and GLP-compliant study. Klimisch Code 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

In the reproduction/developmental toxicity screening test discussed under 'Reproductive toxicity section', the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment related effects on mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Justification for classification or non-classification

Based on the findings of the absence of adverse effect on reproductive organs or tissues in the reproduction/developmental toxicity screening test, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information