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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experiment start date - 12 September 2012; Experiment completion date - 05 November 2012; Study completion date - 13 May 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
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
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40827/B TE
- Substance type: Black powder
- Physical state: Powder
- Purity: 55.3 % (main constituent coloured)
- Lot/batch No.: BOP 01-12 BS-1118392
- Expiration date of the lot/batch: 17 April 2017
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required: No
- Volatile No
- pH: 7.29
- Solubility in water: >306 g/L
Specific details on test material used for the study:
Identification: FAT 40827/B TE
Description: Black powder
Batch: BOP 01-12 BS-1118392
Purity: 55.3 % (main constituent coloured)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 17 April 2017.

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Details on test animals or test system 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 328 g (males) or 208 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. 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: 14 September - 05 November 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.
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 22 days.
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 animals in the study: Approximately 13 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 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).
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 41-52 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 and two females of Group 3 were not dosed during littering. Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 28 days
Females: 41-52 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1: Control group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2: Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3: Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4: High dose group
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 Wistar rats (RCC Project A65081) in which 50, 200 and 1000 mg/kg were tested. Test item-related findings were generally restricted to dark faeces in animals treated at 1000 mg/kg. At 200 and 1000 mg/kg, the urine colour was affected by treatment, but was reversible after the recovery period. In animals treated with 1000 mg/kg, the urine pH was increased after the treatment period, but was reversible after the recovery period. The NOEL was determined to be 50 mg/kg and the NOAEL was established at 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.

Examinations

Maternal 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 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.

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.

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: Epididymites and testes

HISTOPATHOLOGY:
- According to test guidelines
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 abnomlies, 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.
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).
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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity related to test substance treatment were noted during the observation period. One female (no. 52) treated at 100 mg/kg showed piloerection and pale appearance on Day 27 postcoitum. At necropsy, several findings were noted. This was all considered to be related to pregnancy/delivery difficulties as two fetuses were still observed in utero on Day 28 post-coitum. For treated animals, purple staining of the tail or general purple staining 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). Incidental findings that were noted included alopecia and scabs. 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
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 1000 mg/kg, body weights and body weight gain of female animals were decreased during postcoitum; this was mainly considered a cause of their pregnancy status (i.e. implantation sites only or not pregnant) and therefore considered a secondary effect of treatment with FAT 40827/B TE. No data on body weight (gain) during lactation was obtained at 1000 mg/kg as no litters were born in this group. Body weights and body weight gain of treated males up to 1000 mg/kg and females up to 300 mg/kg remained in the same range as controls over the treatment period.
The statistically significantly decreased body weight gain noted on Day 1 of mating for high dose males was not considered toxicologically significant as it was a very slight decrease and occurred only on one occasion.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The lower food consumption levels at 1000 mg/kg for females during post-coitum were considered a cause of their pregnancy status (i.e. implantation sites only or not pregnant) and therefore considered a secondary effect of treatment. No food consumption values during lactation were obtained at 1000 mg/kg as no litters were born in this group. Food consumption before or after allowance for body weight was similar between treated males up to 1000 mg/kg and females up to 300 mg/kg when compared to control animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted for testes and epididymides weights and terminal body weights of treated males up to 1000 mg/kg. The statistically significant decrease in absolute epididymides weight noted for high dose males was considered not to be a sign of toxicity as the change was very minor, all values remained within normal limits and no microscopic correlate was observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. One female (no. 52) at 100 mg/kg showed several reddish foci at the glandular mucosa of the stomach, stomach with purple contents, dark red discolouration of the right lateral liver lobe, black discolouration of both kidneys, and an enlarged spleen. This was probably all related to pregnancy/delivery difficulties as two fetuses were observed in utero still on Day 28 post-coitum. One male at 100 mg/kg, all males and eight females at 1000 mg/kg showed purple 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 pelvic dilation of the kidneys, flaccid testes, nodule at the epididymites, diaphragmatic hernia of the liver, scab formation, alopecia, focus on the clitoral glands, and uterus containing fluid.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings. The microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain. There were no treatment-related microscopic findings in the reproductive organs. Spermatogenic staging profiles were normal for all males examined. The females at 1000 mg/kg that were not pregnant (females 72 – 80) all had a histologically normal cycling female reproductive tract.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, developmental toxicity could not be assessed as out of ten mated females only one showed implantations; no pups were available for examinations. At 300 mg/kg, the mean number of living pups at first litter check was decreased (not statistically significantly).
Up to 300 mg/kg, 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.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Four pups of the control group, two pups at 100 mg/kg and one pup at 300 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
One pups showed a blue spot on the back at first litter check. Macroscopic findings for pups that were found dead included absence of milk in the stomach, beginning autolysis and one pup at 100 mg/kg showed a small lower jaw. 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 (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Systemic toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
pre and post implantation loss

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: decreased fertility and conception indices, and decreased numbers of corpora lutea and implantation sites; out of ten mated females only one showed implantations

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
At 1000 mg/kg, developmental toxicity could not be assessed as out of ten mated females only one showed implantations; consequently no pups were available for examinations. At 300 mg/kg, the mean number of living pups at first litter check was decreased. This was mainly due to two out of the nine litters, which represented 22 % of the litters. Even though this decrease was not statistically significant, this finding could not be ignored as a clear effect was noted at 1000 mg/kg (no pups were born) and the mean number of living pups at first litter check (i.e. 9.8) was lower than generally noted for Wistar Han rats (historical control mean is 11.8 living pups).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with FAT 40827/B by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity up to 1000 mg/kg. Reproduction toxicity was observed at 1000 mg/kg and developmental toxicity was noted at 300 mg/kg. Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: 300 mg/kg
Developmental NOAEL: 100 mg/kg
Executive summary:

In a GLP-compliant study conducted according to OECD 421 and OPPTS 870.3550, the developmental toxicity potential of FAT 40827/B was evaluated. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 46-51 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.


Parental results:


No direct parental toxicity was observed up to the highest dose level tested (1000 mg/kg). For females at 1000 mg/kg, decreased body weight (gain) and food consumption during post-coitum were noted when compared to the concurrent control group. However, these parameters were influenced by the fact that no females delivered live pups (nine were not pregnant and one showed implantation sites only) and could therefore not be evaluated correctly for parental toxicity. In addition, no data on body weight (gain) and food consumption during lactation was obtained at 1000 mg/kg as no litters were born in this group. No direct treatment-related toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination).


Reproductive results:


Reproduction toxicity was observed at 1000 mg/kg. This consisted of decreased fertility and conception indices, and decreased numbers of corpora lutea and implantation sites; out of ten mated females only one showed implantations. The decreased number of copora lutea would indicate disruption of the reproduction process at fertilization or ovulation. Mating index and precoital time were unaffected by treatment up to 1000 mg/kg.


Developmental results:


At 1000 mg/kg, developmental toxicity could not be assessed as out of ten mated females only one showed implantations; consequently no pups were available for examinations. At 300 mg/kg, the mean number of living pups at first litter check was decreased. This was mainly due to two out of the nine litters, which represented 22 % of the litters. Even though this decrease was not statistically significant, this finding could not be ignored as a clear effect was noted at 1000 mg/kg (no pups were born) and the mean number of living pups at first litter check (i.e. 9.8) was lower than generally noted for Wistar Han rats (historical control mean is 11.8 living pups). Up to 300 mg/kg, no treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). In conclusion, treatment with FAT 40827/B by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity up to 1000 mg/kg. Reproduction toxicity was observed at 1000 mg/kg and developmental toxicity was noted at 300 mg/kg. Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:


Parental NOAEL: at least 1000 mg/kg


Reproduction NOAEL: 300 mg/kg


Developmental NOAEL: 100 mg/kg