Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD guideline no. 474 adopted on September 2014
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:
A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined (218.3-232.2 g for males and 183.1-207.3 g for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 12 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C 2°C and 55% 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
In life phase: from 13 January (allocation) to 14 March (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v.
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same
dose volume.
The required amount of Disperse Green GNA was suspended in the vehicle, Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v. The formulations were prepared daily (concentrations of 6.25, 25 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. For female no. 69, the mating was inadvertently not detected in the first mating session. On the basis of this apparently negative sign, the female was allowed to have the second pairing cohabitation with proven male of the same treatment group. During the second pairing combination the female gave birth.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory (RTC Study No. A0679).
In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (Weeks 1 and 6 of treatment). Chemical analysis was 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 was Skanlt® version 2.4.2.55 (Thermo Scientific).
Duration of treatment / exposure:
Main groups (Groups 1 to 4)

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 approximately 6 weeks. 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 (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment was given during the recovery period.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
62.5, 250, 1000 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
6.25, 25 and 100 mg/mL
Basis:
nominal conc.
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats groups 1 to 4).
Two groups (control and high dose levels) included 5 animals per sex to be sacrificed after 3 weeks of recovery (Group 5 and 6).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on experience with this kind of substance and the results of the acute oral toxicity study in rats.

Examinations

Parental animals: Observations and examinations:
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.

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
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, prostate weight, seminal vescicle weight, morphological evaluation of the seminiferous epithelium
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum. As soon as possible after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and only live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
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).
Postmortem examinations (offspring):
Pups
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum)
were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
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 was 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.
Reproductive indices:
Group mean values were calculated for all parameters. Data from nonpregnant females and from female with total litter loss were excluded from group mean calculations as appropriate.
The following reproductive indices were calculated:

Males
Copulation Index (%) = no: of animals mated/no: of animals paired x 100
Fertility Index (%) = no: of males which induced pregnancy/no: of animals paired x 100

Females
Copulatory Index (%) =no: of animals mated/no: of animals paired x100
Fertility Index (%) = no: of pregnant females/no: of females paired x 100

Males and females
Pre coital Interval = Mean number of days between pairing and mating
Offspring viability indices:
Pre-birth loss was calculated as a percentage from the formula:
no: of visible implantations - total litter size at birth/no: of visible implantations x 100

Pre-implantation loss was calculated as a percentage from the formula:
no: of corpora lutea - no: of visible implantations/no: of corpora lutea x 100

Pup loss at birth was calculated as a percentage from the formula:
Total litter size - live litter size/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
Total litter size at birth - live litter size at Day 4/Total litter size at birth x100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY AND FATE OF FEMALES
No mortality occurred throughout the study. One control female (no. 13) and one low dose female (no. 35) were found not pregnant at necropsy. Unilateral implantation was observed in one control female (no. 15) and one high dose female had total litter loss on Day 0 post partum (no. 69). The number of females with live pups on Day 4 post partum was 9 in the control and low and high dose groups and 10 in the mid dose group.

CLINICAL SIGNS AND OBSERVATION OF CAGE TRAY
Staining of different regions of the body surface was the major clinical sign recorded during treatment and recovery period in all treated groups. At observation of the cage tray, green/orange staining were 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 weight gain were recorded in treated groups when compared 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 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.


OESTRUS CYCLE, REPRODUCTIVE PARAMETERS, PAIRING COMBINATION AND MATING PERFORMANCE
All males and all females mated. A total of 2 males (animal nos. 14 and 36) did not induce pregnancy. Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) did not show differences related to treatment.

IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES
Gestation periods were similar between treated animals and controls. All dams gave birth between Days 21 and 22 post coitum. Statistically significant decreases were detected in number of corpora lutea (-13%) and implantation sites (-11%) in the high dose females when compared to the controls. Consequently a significantly reduction in total litter size at birth (-12%) was also detected in the same group. No differences were observed in pre-implantation and pre-birth loss between control and treated females.

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 and the increase of liver weights in some high dose animals. In addition, a decrease in thymus weights was also 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 of the tail, the dorsum of the skin or 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 of 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 weigh/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 septaeand 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 (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no toxicological relevant adverse effects were observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES
Statistically significant decreases were detected in number of corpora lutea (-13%) and implantation sites (-11%) in the high dose females when compared to the controls. Consequently a significantly reduction in total litter size at birth (-12%) was also detected in the same group. No differences were observed in pre-implantation and pre-birth loss between control and treated females.

LITTER DATA AT BIRTH, ON DAY 1 AND ON DAY 4 POST PARTUM OF FEMALES AND SEX RATIO OF PUPS
Increase in pup loss at birth and on Day 4 post partum was recorded in the high dose group when compared to control. Consequently, statistically significant decrease of live litter size at birth (-13%) and on Day 4 post partum (-16%) was also detected in the same group. In addition, litter weight was lower than controls on Days 1 (-16%) and 4 post partum (-16%). Sex ratios at birth and on Day 4 post partum did not show differences between treated and control groups.

CLINICAL SIGNS OF PUPS
An increase in pups found dead was recorded in the high dose group: 1 pup in control group, 2 pups in the low and mid-dose groups and 6 pups in the high dose group. Other pre-weaning clinical signs were comparable between treated and control groups: cold to touch, small appearance and bruise on dorsum were the principal clinical signs noted. In addition, abnormal green colour was detected in few pups of the high dose group. Some pups were missing during the observation period in control and treated groups

NECROPSY FINDINGS IN DECEDENT PUPS AND IN PUPS SACRIFICED
ON DAY 4 POST PARTUM
All organs autolysed in abdominal and/or thoracic cavities were noted at necropsy in the decedent pups. Findings detected in pups sacrificed on Day 4 post partum were considered incidental.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

OESTRUS CYCLE – BEFORE PAIRING - GROUP SUMMARY DATA

 

 STUDY NO.: X0110

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           Group 1              Group 2             Group 3             Group 4

 Animal          Oestrus    Animal    Oestrus    Animal    Oestrus    Animal    Oestrus

 Number          Cycles   Number    Cycles     Number    Cycles     Number    Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

X0110001       1       X0110021       3       X0110041       3       X0110061       3

X0110003       3       X0110023       4       X0110043       4       X0110063       3

X0110005       4       X0110025       4       X0110045       3       X0110065       3

X0110007       4       X0110027       4       X0110047       3       X0110067        4

X0110009       1       X0110029       2       X0110049       3       X0110069       4

X0110011       3       X0110031       4       X0110051       1       X0110071       3

X0110013       1       X0110033       3       X0110053       3       X0110073       3

X0110015       3       X0110035       4       X0110055       3       X0110075       2

X0110017       1       X0110037       3       X0110057       3       X0110077       1

X0110019       3       X0110039       2       X0110059       3       X0110079       2

Means             2.4                            3.3                            2.9                            2.8

 -----------------------------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

REPRODUCTIVE PARAMETERS OF ANIMALS - SUMMARY

 

 STUDY NO.: X0110

 

 MALES

 -----------------------------------------------------------------------------------------------------------------------------------

                        Group        1          2          3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                     100.0     100.0      100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                       90.0     90.0     100.0       100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 FEMALES

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group        1          2         3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0      100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                      90.0      90.0      100.0       100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, as well as for reproductive and developmental toxicity, was considered to be 250 mg/kg body weight/day for female rats and 1000 mg/kg body weight/day for male rats.
No malformations were observed in the offspring. A slightly higher mortality rate between birth and Day 4 pp was observed in the high dose group resulting from the unspecific toxicity in high dose females and were therefore not taken into account for NOAEL determination in F1 generation.
Executive summary:

The toxic effects on Sprague Dawley rats of both sexes after repeated dosing with the test item 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 3 post 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. This 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.

All males and all females mated. Two males did not induce pregnancy. Two females (one control and one low dose) were found not pregnant at necropsy. One high dose female had total litter loss on Day 0 post-partum. Unilateral implantation was observed in one control female. The number of females with live pups on Day 4 post-partum was: 9 in each of the control and low and high dose groups and 10 in the mid dose group. Oestrus cycle and reproductive parameters did not show differences related to treatment.

Gestation periods were similar between treated animals and controls. A statistically significant decrease in number of corpora lutea and implantation sites and, consequently in total litter size at birth, was detected in the high dose group when compared to the controls. No differences were observed in pre-implantation and pre-birth loss between control and treated females

Increase in pup loss at birth and on Day 4 post-partum was recorded in the high dose group when compared to controls. Consequently, a statistically significant decrease in litter weight and live litter size at birth and on Day4 post-partum was also detected in the same group. Sex ratios at birth and on Day 4 post-partum did not show differences between treated and control groups.

An increase of pups found dead was recorded in the high dose group. Other pre-weaning clinical signs were comparable between treated and control groups.

All organs autolysed in abdominal and/or thoracic cavities were noted at necropsy in the decedent pups. Findings detected in pups sacrificed on Day4 post-partum were considered incidental.

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, as well as for reproductive and developmental toxicity, was considered to be 250 mg/kg body weight/day for female rats and 1000 mg/kg body weight/day for male rats. No malformations were observed in the offspring. A slightly higher mortality rate between birth and Day 4 pp was observed in the high dose group resulting from the unspecific toxicity in high dose females and were therefore not taken into account for NOAEL determination in F1 generation.