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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 13, 2015 to December 09, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
- 8 to 9 weeks old, weighing 225 to 235 g for males and 207 to 223 g for females, from Charles River Italia S.p.A.
- acclimatisation period of 33 d (main groups), 41 d (recovery groups), and ca. 70 d (positive control group).
- temperature and relative humidity at 22°C ± 2°C and 55% ± 15%, respectively.
- approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 h each day.
- Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study, except before blood samples withdrawal.
Route of administration:
oral: gavage
Details on route of administration:
All doses were administered at a constant volume of 5 mL/kg bw.
Vehicle:
water
Remarks:
softened
Details on oral exposure:
- The test substance was administered orally, by gavage to animals at 5 mL/kg bw suspended in softened water. The oral route was selected as it is a possible route of exposure of the test substance in man. Dose levels of 62.5, 250 and 500/1000 mg/kg bw/day were selected by the Sponsor based on information from previous studies. Control animals received the vehicle alone at the same dose volume. The positive control group (Group 7) received Mitomycin-C at a concentration of 2 mg/kg bw by intraperitoneal injection at the dose volume of 10 mL/kg body weight.
(The doses were administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.)

- Test substance
1. Main groups
The required amount was suspended in the vehicle (softened water, by reverse osmosis). The formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL starting from Day 1 to Day 8 of the study. Starting from Day 9 of the study, the formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL for males and 12.5, 50 and 100 mg/mL for females. Concentrations were calculated and expressed in terms of test substance as supplied.
2. Recovery groups
The required amount of was suspended in the vehicle (softenedwater, by reverse osmosis). Starting from Day 1 of the study, the formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL for males and 12.5, 50 and 100 mg/mL for females. Concentrations were calculated and expressed in terms of test substance as supplied.
- Positive control substance
The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. Determination of the stability and concentration of solutions of the positive control substance were not undertaken.

- Each main group comprised 10 male and 10 female rats (Groups 1 to 4: 0, 62.5, 250, and 1000 mg/kg bw/day).
- Two groups (control and high dose levels) included 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6).
- For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male and 5 female rats (Group 7).
NB. In agreement with the Sponsor, the high dose level (1000 mg/kg bw/day) was reduced to 500 mg/kg bw/day in females of the main group, due to the toxicity observed during the first week of treatment (in terms of clinical signs, reduced body weight and food consumption). The dose level of 1000 mg/kg bw/day was administered to the animals (males and females) of the main groups starting from Day 1 to Day 8 of the study. Starting from Day 9 of the study for the main groups and from Day 1 for the recovery groups, the dose levels used were: 0, 62.5, 250 and 1000 mg/kg bw/day for males and 0, 62.5, 250 and 500 mg/kg bw/day for females.
Analytical verification of doses or concentrations:
yes
Remarks:
spectrophotometric analysis
Details on analytical verification of doses or concentrations:
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory (RTC Study No. A0773). The proposed formulation procedure for the test substance was checked in the range from 10 to 200 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period, to confirm that the method was suitable. Stability after 28 h (10 mg/mL) and 26 h (200 mg/mL) at room temperature and at +5°C for 8 d (range from 10 to 200 mg/mL) was also verified. In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (two occasions during the study). Results of all analyses were within the acceptability limits for the first occasion. The results of the analysis in the second occasion were within range for Groups 1, 2 and 3 and below the acceptance range for Group 4. The analysis of a new preparation for this group gave acceptable results. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0773) using spectrophotometric analysis. The software used for this activity was SkanIt® version 2.4.2.55 (ThermoScientific).
Duration of treatment / exposure:
- Main groups (Groups 1 to 4):
Males animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 d. Females animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). (During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post-coitum and on Day 1 post-partum. Thereafter individual dose volumes remained constant.)
- Recovery groups (Groups 5 and 6) animals were dosed once a day, 7 d/week, for 4 consecutive weeks (no treatment was given during the recovery period.)
- Positive control group (Group 7) animals received a single dose, approximately 24 h before sacrifice.
Frequency of treatment:
Once a day, 7 d/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
1000 to 500 mg/kg bw/day for females of the main groups; due to high toxicity
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
Yes: mitomycin-C (i.p. injection)
Observations and examinations performed and frequency:
- Mortality: throughout the study, all animals were checked early in each working day in the morning and in the afternoon.
- Clinical signs: once before commencement of treatment and at least once daily during the study. Observations were performed at the same time interval each day (the interval was selected taking into consideration the presence of post-dose reactions (15-30 min after treatment).
- Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week from the start of treatment until termination. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
- Grip strength and sensory reactivity to stimuli: once during the study, towards the end of treatment.
- Motor activity assessment: once during the study, towards the end of treatment.
- Body weight:
Main groups. Males: weekly from allocation to termination and females: weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups. On the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive control group. On the day of allocation, on the day of dosing and just prior to necropsy.
- Food consumption: weekly.
- Clinical pathology investigations: samples of blood (for haematology and coagulation assessments, and clinical chemistry) and urine samples.
Sacrifice and pathology:
- The males were killed after the mating and all females, after a total of 37 or 38 d of dosing. Animals were killed after 2 weeks of recovery.The clinical history of adult animals was studied and a detailed post mortem examination was conducted (external and internal abnormalities, examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
- The ratios of organ weight to body weight were calculated for each animal and histopathological examinations were realised for the following organs::
Adrenal glands
Bone marrow (from femur)
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Clitoral gland
Caecum
Colon
Duodenum
Epididymides
Eye (+optin nerve)
Harderian glands
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
Oesophagus
Ovaries with oviduct
Parathyroid grlands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal column
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
Other examinations:
Mammalian erythrocyte micronucleus test (main groups and positive control group):
Extraction of bone marrow and preparation of the smears for analysis (scoring) of immature polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs). This test gives information on cell division. Finally, the incidence of micronucleated PCEs provides an index of induced genetic damage.
Statistics:
Group mean values were calculated for all parameters. Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified ttest, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing phase:
– Treated males receiving 1000 mg/kg bw/day showed staining (brown) of the body surface (e.g. ventral region, tail and perigenital region) and piloerection. A few males receiving 250 mg/kg bw/day also showed piloerection.
– Before the mating period, during mating and thereafter during post coitum and post partum periods, staining (brown) of the body surface (e.g. ear, tail,perigenital region) and piloerection were recorded in females receiving the dose levels ≥ 250 mg/kg bw/day. In addition, hunched posture and emaciated aspect were recorded in a few females, before pairing,during the first week of treatment at the high dose level of 1000 mg/kg bw/day. Following these findings, the high dose level of 1000 mg/kg bw/day was reduced to 500 mg/kg bw/day and a gradual recovery from these clinical signs was noted. These signs disappeared after Day 13 of the study.
– During the gestation period, two females receiving 500 mg/kg bw/day showed hunched posture in one occasion (Day 0 post coitum).
– During the treatment period, staining (brown) of the tail and piloerection were also noted in animals of the recovery group receiving 500 (females) or 1000 (males) mg/kg bw/day.
(All occurrences of discolouration in test substance-treated animals (main and recovery groups) were related to the colour of the test substance and its properties as textile dye).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Main groups:
Slightly lower mean body weights, compared to control animals, were noted in males receiving the dose levels ≥250 mg/kg bw/day starting from Days 8 of study until termination (-5% to -10%). These changes were statistically significant in the high dose group, while they were limited to Days 15 and 22 (pairing phase) for the dose of 250 mg/kg bw/day. On Day 8 of the study, before pairing, body weight gain was significantly decreased (at statistical analysis) in all treated males (-76% to -271%). During the mating phase on Day15,a statistically significant decrease was also noted in males receiving 250 mg/kg bw/day (-41%). Statistically significant lower body weights were noted in high dose females before pairing, on Day 8 of the study (-15%) and during the gestation and post partum periods from Day 7 onwards (-10% to -20%),when compared to the control group. In addition, females receiving 250 mg/kg bw/day showed a lower body weight on Days 14 and 20 post coitum (-8% and -9%). Body weight gain of treated males and females was lower than controls on Day 8, before pairing. During theg estation and post partum periods, a decrease in body weight gain of females receiving 500 mg/kg bw/day was also noted, reaching a statistical significance on Days 7 and 20 post coitum.
- Recovery groups:
During treatment, a lower body weight, compared to control animals, was observed in males receiving 1000 mg/kg bw/day between Days 8 and 29 of the study. These changes did not exceed -15% but were statistically significant. Body weight of treated females was comparable to the control group. During the recovery phase, a slightly lower body weight persisted in males previously receiving 1000 mg/kg bw/day on Days 36 and 43, although they did not exceed -10%. No changes were noted in the body weight of females. Body weight gain in treated males was decreased starting from Day 8 until termination, while in treated females a decrease was limited to Day 8 of the study. During the recovery period, no particular differences in body weight gain were recorded in animals of both sexes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Main groups
On Day 8, before pairing, food consumption was decreased compared to the control group, in animals of both sexes receiving the dose levels ≥ 250 mg/kg bw/day. The alteration was more evident in high dose animals (up to -34% in males and -57% in females). On Day15, food consumption of treated males receiving the dose levels ≥ 250 mg/kg bw/day was still slightly lower than the control group (-11% and -9%). Thereafter during the gestation period, a statistically significant decrease was also noted on Days 14 and 20 post coitum (-16%, -14%) and on Day 4 post partum (-34%) in females receiving 500 mg/kg bw/day.
- Recovery groups:
During the treatment period, on Day 8 of the study, a decrease in food consumption was recorded in animals of both sexes receiving 1000 or 500 mg/kg bw/day (-30% and -40%). In addition, high dose females showed a decrease on Day 15. During the recovery period, a decrease in food consumption persisted on Day 36 in the males previously treated at 1000 mg/kg bw/day, while in females no effects were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing Phase - Main groups
Slight macrocytic anaemia, associated with moderate reticulocytosis, was recorded in almost all animals dosed at 1000/500 mg/kg bw/day. When compared with controls, changes were approximately -16% for erythrocytes, -12% for haemoglobin, -8% for haematocrit, +12% for mean corpuscular volume, +9% for mean corpuscular haemoglobin (males only) and +148% for reticulocytes (210% in males, 85% in females). Reticulocytosis was also recorded in most of the males dosed at 250 mg/kg bw/day (+52%) and in one female of the same group (+101% compared with mean control data). In addition, neutrophilia and monocytosis were recorded in males receiving 250 and 1000 mg/kg bw/day, with no dose-relation (approximately +63% for neutrophils and +106% for monocytes). The statistically significant increase of platelets recorded in males dosed at 62.5 mg/kg bw/day (13%) and the decrease of mean corpuscular haemoglobin concentration observed in all treated females (3% to 6%) were of minimal severity and/or not dose-related, therefore considered irrelevant.
- Recovery groups:
The anaemia recorded during the dosing phase showed an almost complete reversibility. Males dosed at 1000 mg/kg bw/day showed minimal, although statistically significant, reduction of erythrocytes (-7%) and mean corpuscular haemoglobin concentration (-2%), and increases of mean corpuscular volume (+12%) and mean corpuscular haemoglobin (+9%). Females from the same group showed a statistically significant increase of mean corpuscular volume (+12%) and reduction of reticulocytes (-39%). Due to the limited severity, the above changes were not considered adverse.
- Coagulation: no changes were recorded.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Main groups:
Fluctuations of some biochemical parameters were recorded in treated animals, mainly those receiving 1000 mg/kg bw/day. In particular, males dosed at 1000 mg/kg bw/day showed increase of cholesterol (+83%), protein (+9%), albumin (+13%) and calcium (+5%) and decrease of glucose (-28%). Cholesterol was also increased in males treated with 250 mg/kg bw/day. Females from all treated groups showed increase of cholesterol (+30% to +40%). These changes could be related with the thyroid and liver findings recorded at histopathological examination. The alterations of bilirubin recorded in many treated animals, mainly those receiving 1000/500 mg/kg bw/day,could be due to the interference of the test substance with the analytical colorimetric method. The fluctuations of bile acids were not dose-related, therefore considered incidental. One female showed augmented alanine aminotransferase (72%) and aspartate aminotransferase (+80%). Due to the low severity, they were not considered to be suggestive of liver injury.
- Recovery groups:
Changes recorded during the dosing phase showed complete reversibility, with the exception of calcium in males,which was still slightly higher than controls (+6%). The other statistically significant differences between control and treated animals (decrease of creatinine and increase of phosphorus in males, increase of triglycerides in females) were not observed during the dosing phase, therefore they were considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal body weight and organ weights:
- Main groups:
Terminal body weight was lower, compared to controls, in males receiving the dose levels ≥250 mg/kg bw/day and in all treated females. This change ranged between -8% and -13% in males and between -9% and -17% in females, and was statistically significant in males receiving 250 and 1000 mg/kg bw/day and in females dosed at 500 mg/kg bw/day.
The major changes observed in treated males were an increase in absolute and relative spleen weights (all groups) and in relative kidneys (high dose), liver (high dose) and thyroid weights (mid- and high dose groups). In treated females an increase in absolute and relative spleen weights (high dose) and an increase in relative kidneys weights (high dose) were also recorded. Relative spleen weight was also increase in females receiving 250 mg/kg bw/day. All the above mentioned changes were significant at statistical analysis.
Some other changes were also noted in males receiving 250 or 1000 mg/kg bw/day:
– decrease in absolute adrenals (-21%), epididymides (-17%), prostate (-18%) and thymus (-26%) weights;
– increase in relative heart (+9/10%), seminal vesicles (+16%) and brain (+13%) weights.
In addition, the following modifications were observed in treated females:
– increase in relative brain (+13%, +9%, +16%) and thyroid (+22%) weights.
These alterations were not accompanied by histopathological findings and therefore were considered unrelated to treatment.
- Recovery groups:
After 2 weeks of recovery period, terminal body weight of males which received 1000 mg/kg bw/day was lower (-9%), compared to the control group. In addition, some changes compared to the control group were also noted in the absolute and relative organ weights, sometimes with statistical significance:
– lower absolute brain (-8%), epididymides (-13%), heart (-11%) and higher liver weights (+14%) in males previously dosed at 1000 mg/kg bw/day. Lower absolute adrenals (-21%) weight in females previously dosed at 500 mg/kg bw/day
– increase in relative brain, kidneys (+14%), liver (+25%), spleen (+19%) and testes (+10%) weights in males and a decrease in relative adrenals weight in females (-23%).
The modification (increase) in the spleen and liver weights noted in the high dose males was associated with microscopic observations recorded after 2 weeks of recovery period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Final sacrifice:
The most remarkable changes observed at post mortem examination in treated animals, when compared with controls, were:
– abnormal colour (dark discolouration) and abnormal shape (swollen) of the spleen in most females treated at all doses (1000/500, 250 and 62.5 mg/kg bw/day) and mainly in high dose males which correlated at histopathology with up to marked splenic congestion. In addition, enlarged spleen was noted in the majority of high dose males.
– brown/yellow/orange discolouration of the tail, of the ventral region of the skin and/or the abdominal cavity and/or mesenteric lymph nodes of several males and females receiving the test substance at ≥62.5 mg/kg bw/day. Such staining was considered related to the colour of the test substance.
- Recovery sacrifice:
After 2 weeks of recovery period, brown staining of the tail was still detected in all males (5/5) previously given 1000 mg/kg bw/day. Abnormal shape (swollen) of the spleen was recorded in one male previously given 1000 mg/kg bw/day. Ther emaining macroscopic observations were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Neurotoxicity assessment (removal of animals from the home cage and open arena) with motor activity, grip strength and sensory reaction to stimuli.
- Main groups:
Treated males at removal from the cage and in an open arena: no changes. Four females of Group 4: hunched posture in Week 2 of treatment. Statistically significant decrease in mean motor activity values, performed towards the end of treatment, was noted in Group 4 males (1000 mg/kg bw/day). This was a consequence of a low value noted in one male. No differences were noted in treated females compared to control group. Grip strength and sensory reaction to stimuli: no relevant differences between control and treated groups in both sexes. However, statistically significant lower body weights compared to control weights were noted in treated animals of both sexes when the grip strength test was performed. Changes were evident in males receiving ≥250 mg/kg bw/day (up to-15%) and in females receiving 62.5 and 500 mg/kg bw/day (up to-14%). Statistically significant increase in landing foot splay measurement (first trial), recorded in the individual and mean values of Group 2 (62.5 mg/kg bw/day), was considered of no toxicological significance since it was not dose-related and observed in only one sex (males).
- Recovery groups:
No alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment. A statistically significant lower (-13%) in body weights compared to control weights were noted in the high dose males. At the end of the recovery period, some variations in the sensory reaction to stimuli measurements were noted in treated animals compared to the control group, such as a lower landing foot splay measurement in males(with a statistical significance) and an increase in the same measurement in treated females. Moreover a statistically significant increase in grip strength value in females and a decrease in males were also noted. However, due to the inconsistent presence of the changes between sexes and considering that they occurred at the end of the recovery phase, these variations were considered of no toxicological relevance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At microscopic observation, treatment-related changes in the liver (hepatocytic hypertrophy, associated with increased cytoplasmic eosinophilia), spleen (diffuse congestion, extramedullary haemopoiesis and yellow-brown pigment deposition) and kidneys (yellowbrown pigment granules, multifocally, observed in the cytoplasm of cortical tubular epithelial cells) were recorded in animals of both sexes and in the thyroids of only males receiving 1000/500 mg/kg body weight/day. At 250 mg/kg body weight/day, changes were noted in liver and thyroid in males and in the spleen in animals of both sexes. The findings observed in the liver of the high dose males (1000 mg/kg body weight/day) showed increased severity levels in comparison to mid dose males (250 mg/kg body weight/day) and occasionally they also showed hepatocytic degenerative changes (fatty change, hepatocytic necrosis, inflammatory reaction), often associated with follicular hypertrophy in the thyroid.
In recovery group animals, minimal centrilobular hepatocytic hypertrophy and fatty change were still observed in the liver of males previously given 1000 mg/kg body weight/day although a lesser severity degree. Yellow/brown pigment deposition was still present in both sexes. Capsular inflammation and an increased severity degree of yellow/brown pigmentation were still noted in the spleen of the high dose animals. In the thyroids, three high dose males (3/5) displayed follicular hypertrophy.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Observations of the cage tray: red (slight to marked) or brown (marked) staining of the bedding and faeces. This was related to the red/brown colour of the test substance which was eliminated by the animals.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Under the study conditions, the oral NOAEL of the test substance for systemic toxicity in rats was established at 250 mg/kg bw/day for males and females.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance according to OECD Guideline 422, in compliance with GLP. Rats were administered the test substance orally by gavage. Main group animals received doses of 62.5, 250, 500, 1000 and 1000/500 mg/kg body weight/day. Males were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Females were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Recovery groups (0 or 1000, 1000/500 mg/kg body weight/day test substance) animals were dosed once a day, 7 d/week, for 4 consecutive weeks. No treatment was given during the recovery period. Lower body weights, body weight gains and food consumption were recorded in males and in females during the study at the dose levels ≥250 mg/kg body weight/day, however at the end of recovery period, only the highest dose group displayed lower body weights. At microscopic observation, changes were noted in the liver, spleen and kidneys in animals of both sexes and in the thyroids of males only at 1000/500 mg/kg body weight/day. At 250 mg/kg body weight/day, changes were noted in liver and thyroid in males and in the spleen in animals of both sexes. At 62.5 mg/kg body weight/day, changes were noted in the thyroid in two males and in the spleen in several females. The findings observed in the liver of the high dose males (1000 mg/kg body weight/day) showed increased severity levels in comparison to mid-dose males (250 mg/kg body weight/day) and occasionally they also showed hepatocytic degenerative changes (fatty change, hepatocytic necrosis, inflammatory reaction), often associated with follicular hypertrophy in the thyroid. Considering the pathological picture of the high dose males, the treatment-related changes observed in the liver could be considered adverse, while in males dosed at 250 mg/kg body weight/day, as well as in females dosed at 1000/500 mg/kg body weight/day, being not associated with any degenerative change, they were considered an adaptive change rather than an adverse effect. Follicular hypertrophy in the thyroids observed in most treated males, often associated with hepatocytic hypertrophy, was considered related to the liver drug-metabolizing enzyme induction. Females dosed at >62.5 mg/kg body weight/day and males dosed at >250 mg/kg body weight/day showed extramedullary haematopoiesis, yellow/brown pigmentation and/or congestion and/or capsular inflammation in the spleen. In the recovery animals, minimal centrilobular hepatocytic hypertrophy and fatty change were still observed in the liver of males previously given 1000 mg/kg body weight/day although a lesser severity degree. Yellow/brown pigment deposition was still present in both sexes. Capsular inflammation and an increased severity degree of yellow/brown pigmentation were still noted in the spleen of the high dose animals. In the thyroids, three high dose males (3/5) displayed follicular hypertrophy. Based on these observations and considering the effects observed at the end of recovery period, the oral NOAEL of the test substance for systemic toxicity in rats was established at 250 mg/kg body weight/day for males and females (Sisti, 2015).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: Mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate
EC Number:
226-070-4
EC Name:
2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate
Cas Number:
5261-31-4
Molecular formula:
C19H17Cl2N5O4
IUPAC Name:
2-[(2-cyanoethyl)({4-[2-(2,6-dichloro-4-nitrophenyl)diazen-1-yl]phenyl}) amino]ethyl acetate
Test material form:
solid: particulate/powder
Remarks:
brown/black powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Males - Animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 d.
Females - Animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for at least 40 d).
Duration of treatment / exposure:
Males - 37-38 d
Females - 40 d
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
reduced from high dose of 1000 mg/kg bw/day due to toxicity
No. of animals per sex per dose:
5 per sex per gourp
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C

Examinations

Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
Extraction of bone marrow
The last two treatments were performed at approximately 24 hour interval. Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from the same 5 males and 5 females of the main groups randomly selected for clinical pathology investigation. Samples of bone marrow were also collected approximately 24 hours after the single treatment from all animals of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end, and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.

Preparation of the smears
The suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally, slides were rinsed in distilled water and allowed to dry.

Scoring of the slides and data analysis
In the first instance, only slides from males were evaluated. In addition, since inter-sex differences of toxicity were observed, also slides from females were evaluated. For each animal, four slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring. The adequate quality and a sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and highpower objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCEs). Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. Four thousand polychromatic erythrocytes(PCEs) per animal were scored for the presence of micronuclei. At the same time the number of normal and normochromatic erythrocytes (NCEs) were also recorded. The proportion of immature
erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCEs provides an index of induced genetic damage.
Evaluation criteria:
The assay is considered valid if the following criteria are met:
1. The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range.
2. The positive control item results falls within the historical control range and are significantly increased, at statistical analysis, when compared with the concurrent negative control
3. 5 animals per sex and per group are available for slide analysis.

Evaluation of results
The test item is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes (at p<0.05) is observed in any treatment group and a dose-effect relationship is demonstrated.
Where statistically significant increases in the incidence of micronucleated PCEs are observed, but all results are inside the distribution of negative control values within this laboratory, then historical control data are used to demonstrate that these increases do not have any biological significance.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was considered to be non-genotoxic in the rat.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 474, in compliance with GLP. The micronucleus test was conducted on 5 male and 5 female rats in order to assess the ability of the test substance to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes. Rats were administered the test substance orally by gavage. Main group animals were exposed to concentrations of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Female animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Mitomycin C was used as the positive control substance. No relevant inhibitory effect on erythropoietic cell division was observed at any dose level. Based on these observations, the test substance was not considered to be genotoxic in rats (Sisti, 2015).