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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2018 to 08 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
From study plan regarding Terminal observation and food consumption
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP

Test material

Constituent 1
Reference substance name:
Carbonic acid, 2-[(1-amino-9,10-dihydro-4-hydroxy -9,10-dioxo-2- anthracenyl)oxy] ethyl- phenyl- ester
Cas Number:
28173-59-3
IUPAC Name:
Carbonic acid, 2-[(1-amino-9,10-dihydro-4-hydroxy -9,10-dioxo-2- anthracenyl)oxy] ethyl- phenyl- ester
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: 20140120-2
- Expiration date of the lot/batch: 25 February, 2019
- Purity: 88.5 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity in darkness; used/formulated in light

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: the males were approximately 11 weeks old, and the females were approximately 12 weeks old.
- Weight at study initiation: the males weighed 274 to 358 g, the females weighed 191 to 240 g
- Housing: solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.), ad libitum
- Water: ad libitum
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours darkness

IN-LIFE DATES: From: 12 March 2018 To: 13 May 2018

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Arachis oil BP. The stability and homogeneity of the test item formulations were determined. Results showed the formulations to be stable for at least twenty-one days when stored refrigerated. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on two occasions and analyzed for concentration of FAT93504/B. The results indicate that the prepared formulations were within 95 to 111 % of the nominal concentration.
Duration of treatment / exposure:
6 weeks for males, and 8 weeks for females including a 2-week preparing phase, pairing, gestation and early lactation.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
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:
Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work, including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase, females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group.
Adrenals, prostate, brain, seminal vesicles (with coagulating gland), epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroid (weighed after partial fixation with parathyroid), ovaries, pituitary (weighed after partial, fixation), uterus (weighed with Cervix).

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated.
Adrenals, muscle (skeletal), aorta (thoracic), ovaries, bone & bone marrow (femur including stifle joint), pancreas, bone & bone marrow (sternum), pituitary, brain (including cerebrum, cerebellum and pons) prostate, cecum, rectum, colon, salivary glands (submaxillary), cowpers glands, sciatic nerve, duodenum, seminal vesicles (with coagulating epididymides gland), esophagus, skin, eyes, spinal cord (cervical, mid-thoracic and glans penis lumbar), gross lesions, spleen, heart, stomach, Ileum (including Peyer’s patches), testes.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip strength, motor activity, body weight, body weight change, food consumption during gestation and lactation, pre-coital interval, gestation length, litter size, litter weight, sex ratio, implantation sites, post-implantation losses, viability indices, offspring body weight, offspring body weight change, offspring developmental parameters, hematology, blood chemistry, absolute organ weights, body weight-relative organ weights and thyroid hormone (Thyroxine).

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p <0.01 **
p <0.05 *
p >0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day. Red fur staining was observed for all animals receiving 1000 mg/kg bw/day, at a lower incidence in animals receiving 300 mg/kg bw/day and in one male at 100 mg/kg bw/day. This finding was considered to be consistent with the colored nature of the Test Item and was considered to be of no toxicological significance. One male (number 29) receiving 100 mg/kg bw/day showed noisy, labored and decreased respiration on Day 25, with noisy respiration persisting to the following day. Subsequently, this animal showed pilo-erection, hunched posture and noisy respiration on Days 29 and 30. However, on Day 29, it was noted that the animal had broken front teeth and this was considered to be the underlying cause for these later adverse clinical signs. The animal was isolated and given moistened diet to assist feeding and further clinical signs were restricted to noisy respiration during Days 32, 33, 35 and, during behavioral assessments, Day 40. In the absence of any similar episodes of these adverse clinical signs at higher dosages, these findings for this isolated animal were considered incidental and unrelated to treatment. One female (number 39) receiving 100 mg/kg bw/day showed hunched posture on Day 52 (the day of termination) but, this female had escaped overnight, depriving itself of access to water, and this was considered to be the underlying cause for this observation.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on body weight and body weight gain of males throughout the study at 100, 300 or 1000 mg/kg bw/day. At 1000 mg/kg bw/day, mean body weight gain of males during Week 5 (Days 29-36) was statistically significantly higher than control, but this difference was considered to reflect normal biological variation. There was no effect of treatment on body weight and body weight gain for females during the pre-pairing treatment phase or during gestation and lactation at 100, 300 or 1000 mg/kg bw/day. At 100 and 1000 mg/kg bw/day, mean cumulative body weight gain was statistically significantly lower than control by Day 20 of gestation, but there was no dosage relationship. These differences in mean body weight at the end of gestation are considered to be due to a lower contribution from the gravid uterus, as litter weights at these dosages were also lower than control. Supporting this, mean body weights for females on Day 1 of lactation were similar to control at all dosages, indicating that there was no underlying effect on maternal body weight.
Food efficiency:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At all dosages, mean reticulocyte counts for both sexes were statistically significant higher than control. For treated males mean values showed a dosage relationship and 1/5 individual values at 100 mg/kg bw/day and all individual values at 300 and 1000 mg/kg bw/day exceeded the historical control range compared to 1/5 individual values for the control group. For treated females, although the highest mean value occurred at 1000 mg/kg bw/day, therewas no dosage relationship at lower dosages and only 1/5 individual values at both 300 and 1000 mg/kg bw/day exceeded the historical control range. These apparent increases in reticulocyte counts were not associated with any statistically significant differences from control for other erythrocyte parameters, with the possible exception of higher mean corpuscular hemoglobin concentration for females at 300 mg/kg bw/day, nor were there any supporting histopathological changes apparent in the tissues of treated animals. In view of the lack of supporting observations, the higher number of reticulocytes was considered to be of no toxicological significance and insufficient to be regarded as representing an adverse effect. For females receiving 300 and 1000 mg/kg bw/day, mean platelet counts were statistically significantly lower than control but only one individual value at each dosage was below the historical control range. This finding, in the absence of any supporting histopathological change, was considered to be incidental and unrelated to treatment. As previously discussed, for females at 300 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly higher than control, with 2/5 values exceeding the historical control range. There was no similar increase apparent for females at 1000 mg/kg bw/day and, in the absence of any supporting histopathological change, this finding was considered incidental and unrelated to treatment. For females receiving 300 mg/kg bw/day, mean activated partial thromboplastin time was statistically significantly longer than control, with 4/5 individual values exceeding the historical control range compared to only one individual control value. There was no similar increase in mean activated partial thromboplastin time apparent for females at 1000 mg/kg bw/day and, in the absence of any supporting histopathological change, this finding was considered incidental and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For males receiving 300 or 1000 mg/kg bw/day, cholesterol levels were statistically significantly higher than control but, all individual values for these treated animals were within the historical control range. Additionally, at 300 mg/kg bw/day, sodium levels were statistically significantly higher than control and again, all individual values for these treated animals were within the historical control range. In isolation, and in the absence of any supporting histopathological change, these findings were considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Three females receiving 300 mg/kg bw/day and three females receiving 1000 mg/kg bw/day showed red staining of the urine during behavioral assessments, however, this finding was considered to be consistent with the colored nature of the Test Item and to be of no toxicological significance. As previously discussed one male receiving 100 mg/kg bw/day showed noisy respiration during behavioral assessments on Day 40. This finding was consistent with similar observations for this animal and in the absence of any occurrence at higher dosages was considered to be unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed statistically significantly increased thyroid/parathyroid weights compared to control. A number of individual values for these treated animals exceeded the historical control normal range (10/12 and 8/12 for absolute and relative weights respectively). However, a number of individual values for control animals also exceeded this range (4/12 and 4/12 for absolute and relative weights respectively). Although hypertrophy of the follicular epithelium at a minimal level was noted in the thyroid of 2/12 males treated with 1000 mg/kg bw/day, this was not considered to be related to test item administration. In the absence of any true histopathological correlates, the increase in thyroid weights for these males was considered to be incidental and unrelated to treatment. All treated females had statistically significantly higher adrenal weights, both absolute and relative to terminal body weight. However, all individual values were within the historical control normal range, and this finding, in the absence of any supporting histopathological change, is therefore considered incidental and to be due to normal variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, two males at terminal necropsy showed red colored contents in the jejunum, duodenum, ileum, cecum, colon and urinary bladder, with one of these males also showing red colored content in the stomach and rectum. A further male at this dosage showed red colored contents in the urinary bladder and a female showed red discoloration of the mammary gland. At 300 mg/kg bw/day, one female showed red colored contents in the stomach and red staining of the non-glandular of the stomach and, at 100 mg/kg bw/day, two males had red colored contents in the urinary bladder. These findings were considered to be consistent with the colored nature of the test item and, in the absence of any histopathological change, these findings were considered to be of no toxicological significance. One male at 100 mg/kg bw/day had an enlarged liver with pale patches, and a female at 300 mg/kg bw/day also had an enlarged liver. No similar liver enlargement was apparent at 1000 mg/kg bw/day. In the absence of any statistically significant increase in mean liver weights or supporting histopathological change, these findings were considered incidental and unrelated to treatment. One female treated at 300 mg/kg bw/day had a mass in the adipose tissue near to the right ovary and a further female at 1000 mg/kg bw/day had a mass on the right ovary. Both females achieved pregnancy and successfully reared a litter to Day 13 termination and, in isolation, these findings were considered incidental and unrelated to treatment. Neither the type, incidence nor distribution of other macroscopic findings for adult animals indicated any effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day. Grip strength and motor activity assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day. For males at 1000 mg/kg bw/day, mean fore limb grip strength was statistically significantly lower than control during trial three and for males at 100 mg/kg bw/day mean hind limb grip strength was statistically significantly higher than control during trial one. No other statistically significant differences from control were apparent and, in isolation, these findings were considered to be incidental and unrelated to treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Hypertrophy of the follicular epithelium at a minimal level was noted in 2/12 males treated with 1000 mg/kg bw/day. The appearance of the thyroid glands in control and 1000 mg/kg bw/day males and females were varied, and at this low incidence and severity this finding is considered to be incidental and indicative of individual variation. Whilst the organ weight data showed a minor increase in males treated at 1000 mg/kg bw/day, numerous control animals were also above expected limits. There were three non-productive matings within the study in animals treated with 100 and 300 mg/kg bw/day. No significant changes were noted in the tissues examined to explain the lack of fertility in any animal. There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
Evaluation of Thyroxine (T4) in adult males at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 1000 mg/kg bw/day.

Effect levels

Key result
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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
neuropathology
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
No Observed Adverse Effect Level (NOAEL) for adult toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

This study was designed to investigate the systemic toxicity and potential adverse effects of FAT 93504/B on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guideline 422. The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks for males and throughout a two week pre-pairing phase, pairing, gestation and lactation to Day 14 for females, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only). Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4). Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. There were no unscheduled deaths and no clinical signs observed during the study. No adverse effects of treatment were seen on functional observations. Body weight, food consumption, food conversion efficiency and water consumption were also not affected. No adverse effects were recorded on hematological, clinical biochemistry, pathological, histopathological and organ weight examinations. Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 1000 mg/kg bw/day. Further no effects on reproductive and developmental parameters were seen. Based on the above findings, the No Observed Adverse Effect Level (NOAEL) for adult toxicity was considered to be 1000 mg/kg bw/day.