Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: 218.3-232.2 g for males, 183.1-207.3 g for females
- Fasting period before study: no
- Housing: 5 animal/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology investigation
- Water (e.g. ad libitum):ad libitum except for clinical pathology investigation
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/-2°C
- Humidity (%):55% +/- 15%
- Air changes (per hr):15 to 20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To: 13 january (allocation) to 14 March 2015 (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400 in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE: Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v.
- Justification for use and choice of vehicle (if other than water): test item is insoluble in water
- Concentration in vehicle: 12.5, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Weeks 1 and 6 of treatment
Chemical analysis will be carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0679) by a spectrophotometer analysis. The software used for this activity: Skanlt® version 2.4.2.55 (Thermo Scientific). The software used for this activity was Empower Probuild No. 2154.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter until the
day before necropsy for a total of 44-45 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post
partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last
recorded body weight.
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
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment were given during the recovery period.
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
62.5, 250, 1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
12.5, 50 and 200 mg/mL
Basis:
other: test material in vehicle
No. of animals per sex per dose:
MAIN GROUPS
4 groups of 10 males and 10 females each dosed at 0, 62.5, 250, 1000 mg/kg bw/day.
RECOVERY GROUPS
2 group of 5 amles and 5 females dosed at 0, 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
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. The test item was administered orally, by gavage. The oral route was selected as it is a
possible route of exposure of the test item in man. Dose levels of 62.5, 250 and 1000 mg/kg body weight/day were selected by the Sponsor based on
information from previous studies.

Examinations

Observations and examinations performed and frequency:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CLINICAL SIGNS AND OBSERVATION OF THE CAGE TRAY
Main and Recovery groups
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
Observations of the cage tray during the pre-mating period were performed for all groups and were recorded three times weekly. During mating period these observations were performed for all main groups and were recorded daily. After mating (only males in main groups) and during gestation (only females in main groups), the observations were performed for all groups and were recorded three times weekly.
Positive Control group
Once before commencement of treatment and at least once daily during the study each animal was observed and any clinical signs were recorded.

NEUROTOXICITY ASSESMENT (removal of animals from the home cage and open arena)
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. 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). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes.
Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 42 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups the tests were performed on Day 28 of the study (during treatment) and once during Week 3 of recovery (Day 21).
Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 42 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups the tests were performed on Day 28 of the study (during treatment) and once during Week 3 of recovery (Day 21).

BODY WEIGHT
Main groups
Males were weighed weekly from allocation to termination. Females were weighed 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
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION
Main groups
The weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
Recovery groups
The weight of food consumed by each cage of rats were recorded at weekly intervals following allocation.

CLINICAL PATHOLOGY INVESTIGATIONS
During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation. Since some changes were detected at the clinical chemistry investigations performed during treatment, blood samples were taken under identical conditions during Week 3 of the recovery period (see below).


Main Group Animal Number
Males Females
1 2, 10, 12, 18, 20 1, 3, 9, 11, 17
2 24, 28, 34, 36, 40 25, 29, 31, 33, 39
3 50, 52, 54, 58, 60 41, 47, 49, 51, 55
4 64, 66, 72, 74, 76 61, 65, 67, 71, 77

Recovery Groups Animal Number
Males Females
5 82 84 86 88 90 81 83 85 87 89
6 92 94 96 98 100 91 93 95 97 99

The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
The measurements performed on blood samples are listed below:

Haematology
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
Neutrophils
Lymphocites
Eosinophils
Basophils
Monocytes
Large unstained cells
– Platelets

Coagulation
– Prothrombin time
– Activated partial thromboplastin time
Coagulation tests in males were repeated during Week 3 of the recovery
period.

Clinical chemistry
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride
Sacrifice and pathology:
EUTHANASIA
Main and Recovery groups
Animals were killed under carbon dioxide asphyxiation. Animals that had completed the scheduled test period and selected for blood collection, were killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood collection, were killed under carbon dioxide asphyxiation.
Parental males (Main groups).
The males were killed after the mating of all females after 38 or 39 days of treatment period.
Parental females (Main groups).
The females with live pups were killed on Day 4 post partum. The female with total litter loss were killed on the Day 2 post partum. The females which do not give birth 25 days after positive identification of mating were killed shortly after (Days 26 and 27 post coitum).
Males and females (Recovery groups)
Animals were killed after 3 weeks of recovery.

NECROPSY- Main and Recovery groups
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including 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.

Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals)
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS - Main and Recovery groups
From all animals completing the scheduled test period, the organs indicated in Annex 1 of Study Protocol (section 4.5.6). were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

TISSUES FIXED AND PRESERVED - Main and Recovery groups
Samples of all the tissues (all parental animals) listed in Annex 1 of Study Protocol (section 4.5.6). were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol). The tissues required for histopathological examination are listed Annex 1 of Study Protocol (section 4.5.6). After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
1. Tissues specified in Annex 1 of Study Protocol (section 4.5.6) from 5 males and 5 females randomly selected (animals evaluated for clinical pathology, see section 4.4) in the control and high dose group killed at term.
2. issues specified in Annex 1 of Study Protocol (section 4.5.6) from all animals killed or dying during the treatment period.
3. All abnormalities in all groups
On the basis of the histopathological differences observed between control and high dose groups (main groups), the examination was extended to the to the thymus (only females) and liver (both sexes) of:
- the remaining animals (not evaluated for clinical pathology) of the control
and high dose groups (main groups).
- the animals of the mid-dose group (main group) - the females of the low dose group (only thymus).
- the animals killed after 3 weeks of recovery period (recovery groups).

Statistics:
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 t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5. The nonparametric 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
MORTALITY
No mortality occurred throughout the study.

CLINICAL SIGNS AND OBSERVATION OF CAGE TRAY
Staining of different regions of the body surface was the main clinical sign recorded during treatment and recovery period in all treated groups.
At observation of the cage tray, green/orange staining was recorded in all treated groups during treatment. Moreover treated animals of the recovery
group (Group 6) showed green staining in the cage tray also during the recovery period

NEUROTOXICITY ASSESSMENT (REMOVAL OF ANIMALS FROM THE HOME CAGE AND OPEN ARENA) WITH MOTOR ACTIVITY AND SENSORY REACTIVITY TO STIMULI
Neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the test item.
No relevant differences in motor activity or sensory reactivity to stimuli were noted between control and treated groups.

BODY WEIGHT AND BODY WEIGHT GAIN
In males, no relevant differences in body weight and body weigh gain were recorded in treated groups when compare to controls.
In females, statistically significant lower body weight and body weight gain compared to control rats were detected in the high dose group starting from the second week of treatment to post partum period.
No difference in body weight gain was recorded in animals of both sexes during the recovery phase.

FOOD CONSUMPTION
Slight decrease in food consumption was recorded in high dose females starting from pairing up to post coitum period. No changes were noted in post partum period.
No changes were noted in treated males during treatment. No differences were noted during the recovery period in both sexes.

HAEMATOLOGY
In males, slight leukopenia was recorded in high dose group without reaching statistical significance. In addition, slight thrombocytosis was recorded in mid- and high dose groups, with no dose-relation.
In females, increase of reticulocytes was detected in high dose group and was not considered adverse due to the absence of changes in the other red blood cell parameters. Changes recorded at the coagulation test in mid- dose males and mid- and high dose females were considered of no toxicological relevance.
No differences that could be considered treatment related were detected in the recovery groups, confirming complete reversibility.

CLINICAL CHEMISTRY
In females, relevant changes of liver/cholestasis markers were recorded in 2 out of 5 animals of the high dose group. The above findings could reflect
liver impairment/cholestasis, even though changes were not confirmed at histopathological examination. Due to the limited incidence, it is unlikely that changes are treatment-related. In addition, a slight increase of creatinine was recorded in almost all females of the same group. In males, a decrease of liver markers and urea and an increase of total bilirubin and bile acids – which were due to an interference of the dye with the photometric measurement – were the most changes detected in the high dose group. Considering the severity and/or the direction of the findings observed in males were not considered to be suggestive of tissue/organ injury.
In the recovery groups, changes recorded during the dosing phase completely recovered.

TERMINAL BODY WEIGHT AND ORGAN WEIGHTS
No significant differences in terminal body weight were noted between controls and treated animals, both in main and recovery groups.
The most relevant change noted in the absolute and/or relative organ weight, between treated and control groups was the increase of thyroid weights in high dose females (main and recovery groups) and the increase of liver weights in some high dose animals. In addition, a decrease in thymus weights was detected in high dose females.

MACROSCOPIC OBSERVATIONS
Final sacrifice
The most remarkable changes observed at post mortem examination in treated animals, when compared with controls, were:
- reduced size of the thymus in most females dosed at 1000 mg/kg body weight/day;
- dark staining in the tail, in the dorsum of the skin or in the head. Such staining was considered related to the colour of the test item.
Recovery sacrifice
The only relevant changes noted at post mortem examination in treated animals when compared with controls were dark staining in the tail.

MICROSCOPIC OBSERVATIONS
Treatment-related changes were noted in the thymus of most treated females dosed at >250 mg/kg body weigh/day, sacrificed at the end of treatment period. The incidence of this change was statistically significant in the high dose females with respect to controls.
No histopathological changes were noted in the high dose females sacrificed after 3 weeks of recovery period or in females dosed at 62.5 mg/kg body weight/day. The histopathological change detected in the thymus was atrophy, represented by: a minimal to moderate reduction in cortical lymphocytes, shrinkage of the thymic lobules, increased prominence of interlobular septae and an inverse cellular density ratio cortex/medulla. In addition, in some instances, an increased number of apoptotic bodies “tingible body macrophages” was also reported.
In control and high dose males, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: sign of unspecific toxicity at 1000 mg/kg bw/day in females
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse or toxicological relevant effects were noted in males

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 250 mg/kg body weight/day in female rats and 1000 mg/kg body weight/day in male rats.
Executive summary:

The toxic effects on Sprague Dawley rats of both sexes after repeated dosing with the substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring, were investigated. A 3-week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The animals received the test item, dissolved in Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v, at a constant volume of 10 mL/kg body weight as indicated in the scheme below.

 

Main groups

Group

Treatment (mg/kg body weight/day)

Number of animals

1

0

10M+10F

2

62.5

10M+10F

3

250

10M+10F

4

1000

10M+10F

 

 Recovery groups

Group

Treatment (mg/kg body weight/day)

Number of animals

5

0

5M+5F

6

1000

5M+5F

 

Main groups

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for approximately 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination.

Body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery groups

Animals of the recovery groups were treated for 4 consecutive weeks and sacrificed after 3 weeks of recovery (Groups 5, 6).

The following parameters were evaluated in these animals: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological examination.

Results

No mortality in parental animals occurred throughout the study.

Staining of different regions of the body surface was the main clinical sign recorded during treatment and recovery period in all treated groups. At observation of the cage tray, green/orange staining was recorded in all treated groups during treatment. Moreover, treated animals of the recovery group (Group 6) showed green staining in the cage tray also during the recovery period. These discolorations were due to the colour of the test item and its properties as textile dyes.

Neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity or sensory reactivity to stimuli were noted between control and treated groups.

In males, no relevant differences in body weight and body weight gain were recorded in treated groups when compared to controls. In females, statistically significant decreases in body weight and body weight gain were detected in the high dose group starting from the second week of treatment up to post partum period. No difference in body weight gain was recorded in animals of both sexes during the recovery phase.

Slight decrease in food consumption was recorded in high dose females starting from pairing up to post coitum period. No changes were noted in post partum period. No changes were noted in treated males during treatment. No differences were noted during the recovery period in both sexes.

In males, slight leucopenia was recorded in the high dose group. In addition, slight thrombocytosis was recorded in mid- and high dose groups, with no dose-relation. In females, increase of reticulocytes was detected in the high dose group. These changes were not considered adverse due to the absence of changes in the other blood cell parameters and histopathology. Changes recorded at the coagulation test in mid-dose males and mid- and high dose females were considered of no toxicological relevance. No differences that could be considered treatment-related were detected in the recovery groups, confirming complete reversibility.

In females, relevant changes of liver/cholestasis markers were recorded in 2 out of 5 animals of the high dose group. The above findings could reflect liver impairment/cholestasis, even though changes were not confirmed at histopathological examination. Due to the limited incidence, it is unlikely that changes are treatment-related. In addition, a slight increase of creatinine was recorded in almost all females of the same group. In males, a decrease of liver markers and urea and an increase of total bilirubin and bile acids – which were due to an interference of the dye with the photometric measurement – were the most changes detected in the high dose group. Considering the severity and/or the direction of the findings observed in males were not considered to be suggestive of tissue/organ injury. In the recovery groups, changes recorded during the dosing phase completely recovered.

No significant differences in terminal body weight were noted between controls and treated animals, both in main and recovery groups. The most relevant change noted in the absolute and/or relative organ weight, between treated and control groups, was the increase of thyroid weights in high dose females (main and recovery groups) and the increase of liver weights in high dose males. In addition, a decrease in thymus weights was also detected in high dose females.

The most remarkable changes observed at post mortem examination in treated main group animals, when compared with controls, were reduced size of the thymus in most females dosed at 1000 mg/kg body weight/day and dark staining on the tail, on the dorsum of the skin or on the head. Such staining was considered related to the colour of the test item. In recovery animals, the only relevant changes noted at post mortem examination in treated animals, when compared with controls, were dark staining of the tail.

Treatment-related changes were noted in the thymus of most treated females dosed at > 250 mg/kg body weight/day, sacrificed at the end of treatment period. The incidence of this change was statistically significant in the high dose females with respect to controls. No histopathological changes were noted in the high dose females sacrificed after 3 weeks of recovery period or in females dosed at 62.5 mg/kg body weight/day. The histopathological change detected in the thymus was atrophy, represented by: a minimal to moderate reduction in cortical lymphocytes, shrinkage of the thymic lobules, increased prominence of interlobular septae and an inverse cellular density ratio cortex/medulla. In addition, in some instances, an increased number of apoptotic bodies “tingible body macrophages” was also reported. In control and high dose males, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 250 mg/kg body weight/day for female rats and 1000 mg/kg body weight/day for male rats.