Registration Dossier

Administrative data

developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
04 October - 26 November 2012
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.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
according to guideline
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
Limit test:

Test material

Constituent 1
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40571/C TE
- Substance type: Black powder
- Physical state: Powder
- Purity: Unknown
- Lot/batch No.: DER 8107 / BOP 01-12 (Lot: MHC-0016088200)
- Expiration date of the lot/batch: 25 January 2017
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required: No
- Volatile No
- pH: 4.6 at concentration of 27.7% (%w/w)

Test animals

other: Wistar (Han)
Details on test animals or test system and environmental conditions:
- 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 325 gr (males) or 211 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental 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.

From: 04 October - 26 November 2012

Administration / exposure

Route of administration:
oral: gavage
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:
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 20 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).
- 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 and/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 animal at 100 mg/kg 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 - 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. 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 - 53 days
Doses / concentrations
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose levels were based on a 28-day toxicity study in Wistar rats (RCC Project 706397) in which 50, 200 and 1000 mg/kg were tested. At 1000 mg/kg, treatment-related findings were restricted to microscopical changes in the kidneys (hyaline droplets) for males, stomach (nonglandular stomach erosion associated with inflammation, squamous cell hyperplasia and moderate hyperkeratosis) and lung (interstitial fibrosis associated with pigment-laden alveolar macrophages) for some females. It is probable that these findings were related to the mode of application rather than a direct effect of the test article. All test article-related findings noted at the end of the treatment period were fully reversible during the 14-day recovery period.
- 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:
- Time schedule: At least twice daily.

- 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.

- 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.

- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

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

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






- 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.

- All animals were deeply anaesthetised and subsequently exsanguinated. The animals were not deprived of food overnight.
- According to test guidelines

- All males: Epididymides and testes

- 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:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Yes, if possible, defects or cause of death were evaluated.
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.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring 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

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortality occurred during the study period. During the anaesthesia procedure (just before necropsy) one female receiving 100 mg/kg died by accident. Deaths due to anaesthesia are considered to be of incidental nature.

No clinical signs of toxicity related to test substance treatment were noted during the observation period. Dark faeces noted for all animals treated at 1000 mg/kg was considered due to the staining properties of the test substance (black), and not regarded toxicologically relevant. Alopecia was noted for two females. This 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 and body weight gain of treated animals remained in the same range as controls over the treatment period.

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

All males at 300 mg/kg and all animals at 1000 mg/kg showed discolouration (reddish, dark red, purple or black) of the skin, testes, kidneys, epididymides, and/or spleen. One male treated at 100 mg/kg showed both testes and epididymides reduced in size. This was confirmed at organ weight determination and at microscopic examination. 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, nodule at the epididymides or clitoral glands, focus at the preputial glands, enlarged preputial glands or epididymides, preputial glands reduced in size, diaphragmatic hernia of the liver and alopecia.

Due to the fatty characteristics of the test substance, the discolorations in the epididymides, testes, spleen and skin(subcutis) were most likely to be present in adipose tissue. The discolourations disappeared during the histology staining process and therefore could not be microscopically correlated. Recorded microscopic findings in these organs were considered not to be treatmentrelated. Because of these reasons, the macroscopic discolouration of these organs were not considered to be toxicologically adverse.

No toxicologically relevant changes were noted for testes and epididymides weights and terminal body weights of treated males when compared to controls. One male treated at 100 mg/kg showed a reduced weight of the testes and epididymides, which was confirmed at macroscopic and microscopic examination. The statistically significant change noted for epididymides weight at 300 mg/kg was not considered toxicologically relevant as all individual values were within the normal range and no dose response relationship was noted.

Treatment-related microscopic findings were noted in the kidneys of rats treated at 1000 mg/kg and consisted of:
- the presence of hyaline droplets at minimal (8/10) to slight degree (2/10) in males. The hyaline droplets were considered to represent alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation. This protein is not present in higher mammals, including man. As the presence of hyaline droplets was not accompanied by degenerative tubular alterations this finding was considered to be a non-adverse finding.
- the presence of intracellular tubular (red)brown pigment in 10/10 males (minimal) and 7/7 females (up to moderate). This was not accompanied by other indicators of kidney pathology. The remainder of the microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

There were two couples treated at 100 mg/kg, three couples treated at 300 mg/kg and two couples treated at 1000 mg/kg that failed to sire or deliver healthy offspring and were therefore selected for histopathological examination of the reproductive organs. For male no. 20 the cause of infertility consisted of marked seminiferous atrophy with absence of elongating spermatids in the testes and massive oligospermia in the epididymides and testes. Based on the absence of these specific findings or treatment–related findings in the remaining rats, these findings were considered to be within background. Spermatogenic staging profiles were normal for all males examined, besides one male at 100 mg/kg. The females that were not pregnant all had a histologically normal cycling female reproductive tract.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Two pups of the control group, three pups at 300 mg/kg and four pups at 1000 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 incidences remained within the range considered normal for pups of this age.

At first litter check, one pup of the control group showed a pale appearance and missing tail and right hindleg. This pup was missing on Day 3 of lactation. As it concerned a control pup it was not treatment related. Incidental findings consisted of blue spot on the nose or abdomen, a wound on the back, scabbing of the back or nose. For the two pups that were found dead at first litter check absence of milk in the stomach was noted at macroscopic examination. 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.

Applicant's summary and conclusion

No developmental effects were seen in this study and thus the substance is considered non-teratogenic. The NOAEL was set at least to the highest doese tested being 1000 mg/kg bw/d in this study.
Executive summary:

In conclusion, treatment with FAT 40571/C TE by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental findings in the kidneys at 1000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg. Based on the absence of functional or morphological disturbances supporting the changes noted in the kidneys for this duration of treatment (males 28 days and females 41 - 53 days), a parental NOAEL of at least 1000 mg/kg was established. In addition, a reproduction and developmental NOAEL of at least 1000 mg/kg was derived.