Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.03.-05.06.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.03.-05.06.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test material: Reactive Red 45:1
- Appearance: red powder
- Expiry date 14 April 2021
- Storage: at a dry and dark place between 5 - 30 °C

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: approximately 7 to 8 weeks old.
- Weight at study initiation: 223 - 233 g for males and 175 - 190 g for females.
- Housing: The animals were housed in a limited access rodent facility. Animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
- Diet: laboratory rodent diet 4 RF 21, manufactured by Mucedola S.r.l, was provided ad libitum throughout the study, except as noted in section.
- Water: Water was provided ad libitum.
- Acclimation period: 26 days.

DETAILS OF FOOD AND WATER QUALITY
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Food and water was checked during the study

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

IN-LIFE DATES: FROM 26 Mar 2019 To 05 Jun 2019

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of the test item was dissolved in the vehicle. The preparations were made weekly at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item corrected for purity. Preparations of the test item were prepared as solutions in water. Concentration was assessed for all levels by taking two analytical aliquots (approximately 1 mL). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the two determinations.

VEHICLE
- Concentration in vehicle: 10, 30 100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1. Exceptions can arise in the case of occasional deaths of males.
- Length of cohabitation: until mating occurred or until 14 days had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations.
The analytical method was validated in the range from 5 to 100 mg/mL. Linearity, accuracy and precision were within the for solutions (r > 0.98; accuracy 90-110%; precision CV <5%). In the same study, a 28 hour stability at room temperature and an 8 day stability at 2 - 8 °C were verified in the range from 5 to 100 mg/mL.

Duration of treatment / exposure:

Females for at least 51 days and males for 35 - 36 days.

Frequency of treatment:

Once a day, seven days a week.

Details on study schedule:

Females dosed for two consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days).

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: Selection of dose levels Dose levels have been selected in consultation with the Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.: E0374). 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 dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Fasting period before blood sampling for clinical biochemistry: no.
- Culling and pups selection for blood collection (serum hormone) at necropsy Culling - Day 4 post partum: On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. Two pups (males or females, selected for culling) were sacrificed for blood collection.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATION
- Time schedule: All the animals were observed early in each working day and again in the afternoon for clinical signs of toxicity and twice daily for morbidity. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment and at least once daily during the study, each animal was observed for any clinical signs 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
Observations of the cage tray during the pre-mating period, after mating for males and post partum periods were performed for all groups and were recorded three times weekly. During pairing and gestation periods (females) observations of the cage tray were performed and recorded daily for all groups. These data are not tabulated in this report but will be archived together with all other raw data.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
Once before commencement of treatment and 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.
Data are reported until Week 5 for male animals and Week 7 for females. All the data not tabulated has been be archived together with all other raw data. The following parameters were measured or analysed in 5 out of 10 animals of each group randomly selected: Grip strength and sensory, reactivity to stimuli, Motor activity (MA) assessment and Clinical pathology investigations.

GRIP STRENGTH AND SENSORY REACTIVITY 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 reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males, the tests were performed on Day 29 of the study and for females on Day 12 post partum.

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 males, the test was performed on Day 29 of the study and for females on Day 12 post partum.

BODY WEIGHT
Parental animals Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20.

FOOD CONSUMPTION:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY
Blood collection was performed for hormone determination (0.8 mL) from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, by random selection from 5 males and 5 females of each group, under condition of food deprivation.
Males: Blood samples for haematological investigations, biochemical tests and hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females: As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups. The blood samples were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone assay
The following parameters were assessed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus
These parameters were analysed by Siemens Advia 1200.
Per group, 5 males were randomly selected and groups serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) were determined by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K).

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, These parameters were analysed by Siemens Advia 120.
Coagulation tests: the Prothrombin time was analysed by Instrumentation Laboratory ACL Elite PRO.

Oestrous cyclicity (parental animals):

VAGINAL SMEARS AND OESTROUS CYCLE
Stock females: Oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle. These data are not tabulated in this report, but will be archived with all raw data. No replacement occurred after allocation of the animals.
Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including not less than 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before despatch to necropsy.

Sperm parameters (parental animals):

SPERM PARAMETER (PARENTAL ANIMALS)
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 performed. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages.

Litter observations:

PUPS IDENTIFICATION, WEIGHT AND OBSERVATIONS
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters. After culling, all pups were sacrificed with the dams on Day 14 post partum.
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. Two pups (males or females, selected for culling) were sacrificed for blood collection.

NIPPLE COUNT
The presence of nipples/areolae in male pups was checked on Day 13 post partum, on the day before despatch to necropsy.

NIPPLE RETENTION AT DAY 14 POST PARTUM
The ventral region of male pups was checked for presence of nipples/areolae. No nipples were found in pups to be retained on Day 14 post partum. Data were not tabulated, but will be archived with all raw data.

ANOGENITAL DISTANCE (AGD)
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalised to the cube root of the pup’s body weight measured on Day 1 post partum.

Postmortem examinations (parental animals):

- Euthanasia: animals were killed by exsanguination under isoflurane anaesthesia. Males were killed after the mating of all females on Days 36 and 37 of the study. The females with live pups were killed on Day 14 post partum.
- Necropsy: 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.
- Tissues fixed and preserved: Samples of all the tissues (parental animals) were fixed and preserved in 10% neutral buffered formalin.
- Organ weights: from all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
- Histopathological examination: The tissues required for histopathological examination are: Adrenal glands, Bone marrow (from sternum), Brain (cerebrum, cerebellum,,medulla/pons), Caecum, Clitoral gland, Colon, Duodenum, Epididymides, Eyes, Femur with knee joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes mesenteric, Mammary gland Females, Mammary gland Males, Nasal cavity1, Oesophagus, Ovaries with Oviducts, Parathyroid glands2, Pituitary gland, Penis, Prostate gland (dorsolateral and ventral), Rectum, Sciatic nerve, Seminal vesicles with coagulating glands, Spinal column1, Spinal cord (cervical, thoracic, lumbar), Skeletal muscle, Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus cervix, Vagina.
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. The examination, in the first instance, was restricted as detailed below:
i) Tissues specified from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
ii) All abnormalities in all groups. Since differences were observed between control and high dose animals, the examination was extended to:
 Kidneys in all males and females of Groups 2 and 3 and in the remaining animals of control and high dose groups. 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 performed. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages.

Postmortem examinations (offspring):

- All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed on Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection. Pups with abnormalities were retained in 10 % neutral buffered formalin.
- Pups at day 14 post partum: Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.
- Blood collection and thyroid hormone determination (T3, T4 and TSH): blood collection from pups on Days 4 and 14 post partum On Day 4 post partum as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 pups (1 male and 1 female, if possible). On Day 14 post partum as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female). Blood sample was withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots and stored at -80 °C pending.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, 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 nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric 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.

Reproductive indices:

Presentation of data / Off spring viability indices
Group mean values were calculated for all parameters. The following reproductive indices were calculated:
- Males:
Copulation Index(%) = (no. of animals with confirmed mating)/( no. of males cohabitated) x100
Fertility Index (%) = (no. of males which induced pregnancy)/ (no. of males cohabitated) x100
- Females:
Copulatory Index(%) = (no. of females with confirmed mating) / (no. of females cohabitated) x 100
Fertility Index (%) = (no. of pregnant females) /(no. of females cohabitated) x100
- Males and females
Pre coital Interval = The number of nights paired prior to the detection not mating
Pre-implantation lossa was calculated as a percentage from the formula:
(no. of corpora lutea−no. of implantations)/ (no. of corpora lutea) ×100

Pre-natal lossb was calculated as a percentage from the formula:
(no. of visible implantations−live litter size at birth)/(no. of visible implantations) x 100

Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented
as the percentage of males per litter.

Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented as the percentage of males per litter.

Offspring viability indices:

Pup loss at Day 0 post partum was calculated as a percentage from the formula: (Total litter size−live litter size) / (Total litter size) ×100

Post natal lossb at Day 4 post partum (before culling) was calculated as a percentage from the formula: (Live litter size at birth−live litter size at Day 4(before culling))/(Live litter size at birth) ×100

Post natal lossb at Day 13 post partum (after culling) was calculated as a percentage from
the formula: (Live litter size on Day 4(after culling)−live litter size on Day 13)/ (Live litter size on Day 4(after culling)) ×100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all males receiving 1000 mg/kg bw/day (Group 4), red staining in different regions of the body surface (i.e. tail, dorsum and sacral region) was noted starting from the second day of dosing and during the entire treatment period. In addition, salivation was observed in 9 out of 10 animals of the group generally starting after the first two weeks of treatment and, in the majority of animals, up to the end of the treatment period even if not in a continuous manner. Damaged tail recorded in one male only from Day 6 of the mating phase up to the end of treatment was not considered related to the treatment. In all males receiving 300 mg/kg bw/day (Group 3), red staining on the tail was observed starting from Day 9 of the mating phase up to the end of the study. No clinical signs were observed in males receiving 100 mg/kg bw/day (Group 2) and in control animals.
In all females receiving 1000mg/kg bw/day (Group 4), red staining on the tail was noted starting from the second day of dosing and during the entire treatment period (i.e. premating period, mating phase, gestation and post partum periods). In addition, salivation was generally observed in 7 out of 10 females before and during mating as well as in all females during gestation and post partum periods but not continuously. In six females receiving 300 mg/kg bw/day (Group 3) and in two females receiving 100 mg/kg bw/day (Group 2), red staining on the tail was seen during the gestation and/or post partum periods. Hair loss in different regions of the body surface noted in three females receiving 1000 mg/kg bw/day and in one control female and scab on the head observed in one female receiving 300 mg/kg bw/day were not considered treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study. Statistically significant differences in body weight gain recorded in males receiving 300 mg/kg/day in the last week of pre-mating period and first week of pairing period were considered incidental because not dose-related and not consistent in direction.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both sexes during the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes were recorded at the haematological investigation performed at the end of the study. Coagulation: No changes were recorded in prothrombin time measured at the end of the study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant findings were observed at clinical chemistry investigation performed at the end of the study. The statistically significant decreases of chloride recorded in males dosed at 1000 mg/kg bw/day (3% below controls) and creatinine recorded in females of the same group (8%) were of minimal severity, therefore they were considered to be not adverse.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone determination: for males: No changes were recorded in thyroid hormones determination performed at the end of the study

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals of both sexes, the examination was extended to the kidneys of animals of both sexes of Groups 2 and 3 and remaining animals of control and high dose groups.
Treatment-related lesions were only noted in kidneys of high dose animals of both sexes. At microscopic evaluation, the kidneys presented tubular cell vacuolation involving cortex and medulla, characterised by the presence of amorphous material in the vacuoles, with a mild to marked degree in high dose animals of both sexes. This renal finding was not observed in the mid- and low dose animals of both sexes when compared to the controls. In addition, the increased severity of hyaline droplets accumulation in kidneys from mild to marked degree was only observed in high dose males, when compared with controls. The hyaline droplets represented by eosinophilic homogeneous cytoplasmic droplets and the increased accumulation in the high dose males, evaluated respect to controls as reported in literature (Toxicologic Pathology, 36: 1014-1017, 2008 - GORDON C. HARD X1100), could represent low molecular weight protein accumulation within lysosomes, due to the disturbance of the normal balance of tubular reabsorption and hydrolysis, as a result of either increased filtered protein loads or decreased catabolism. The remaining sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

For males, no changes were recorded in thyroid hormones determination performed at the end of the study. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups in both sexes.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of oestrous cycles observed in all females before pairing (number of non sequential days in which the females were in oestrous) were similar between control and treated groups and was of 3 times (mean value). Vaginal smears examined on the day of necropsy to determine the stage of the oestrous cycle showed the phase of diestrous for the not pregnant control female and oestrous for the low dose non pregnant female and that of diestrous for the majority of females sacrificed on Day 14 post partum. The number of copulatory plugs (mean value) found in the cage were 2/3 for all groups. The majority of females mated in 1-5 days of cohabitation. Two females only, one of the low dose group and one of the high dose group mated after 14 and 6 days, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males.

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulatory index was 100% for both males and females of all treated groups. Fertility index both for males and females was 100% each for mid- and high dose groups and 90% for control and low dose groups. One control male and one low dose male failed to induce pregnancy. These cases were considered incidental.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant findings were observed at clinical chemistry investigation performed at the end of the study.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No differences in the anogenital distance (normalised value) to the cube root of the body weight, performed on Day 1 post partum, were seen between control and treated groups for female pups. The mean values of anogenital distance of male pups in order of ascending dose levels were: 1.95 (control), 1.89, 1.93 and 1.72 mm/ 3√g . A slight decrease, significant at statistical analysis, in the mean values was noted in high dose male pups when compared to the control value. However this change was dose-unrelated and therefore cannot be conclusively attributed to treatment.

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

No nipples were found in male pups on Day 13 post partum.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences were noted in thyroid weight between control and treated pups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. Autolysed abdominal organs were observed in three early decedent pups belonging to control and high dose groups which died at birth or during the lactation period, while no abnormalities were observed in the other early decedent pups.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1 Macroscopic observations - Group data

	Males				Females
Group:	1	2	3	4	1	2	3	4
Number in group:	10	10	10	10	10	10	10	10
Adrenals -Abnormal colour -Abnormal size	0 0	0 0	0 0	0 0	1 0	1 0	3 2	1 0
Cervicalnodes -Abnormal colour	1	0	3	1	0	0	0	0
Epididymides -Abnormal size	2	2	0	1
Ileum Abnormal colour	0	0	0	0	0	0	0	2
Jejunum -Abnormal colour	0	0	0	0	0	0	0	1
Kidneys -Abnormal colour	0	0	0	8	0	0	0	8
Liver -Abnormal area(s)	1	0	0	0	0	0	0	0
Mesenteric nodes -Abnormal colour	0	0	0	0	1	2	0	4
Prostate -Abnormal size	1	2	4	4
Seminal vesicles -Abnormal size	2	1	3	2
Stomach -Abnormal area(s) -Abnormal colour - Abnormal size	0 0 0	0 0 0	0 0 0	0 6 0	3 0 0	0 0 0	3 0 1	1 3 2
Testes -Abnormal size	1	0	0	1
Thymus
-Abnormal area(s)	0	0	0	1	0	0	0	0
-Abnormal colour	0	1	0	0	0	0	0	0
-Abnormal size	0	0	0	0	1	0	0	0
Uterus -Not pregnant.					1	1	0	0
Forelimbs -Hairloss	0	0	0	0	1	0	0	3
Hindlimbs -Hairloss.	0	0	0	0	0	0	0	1
Skin -Hairloss -Staining	0 0	0 0	0 0	0 6	1 0	0 0	0 0	1 0
Tail -Abnormal shape. -Staining	0 0	0 0	0 10	1 10	0 0	0 2	0 5	0 10
Whole animal -No abnormalities detected	5	6	0	0	5	5	3	0

TABLE 2: Absolute organ weights (g): kidneys - group mean data - Males

Group	Control	2	3	4
Number/group	10	10	10	10
Mean	2.440	2.508	2.323	2.571
Standard deviation	0.176	0.330	0.198	0.213
Group diff. at p < 0.05		0.260	0.260	0.260
Group diff. at p < 0.01		0.329	0.329	0.329

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 2.00 Df = 3/ 36 F probability = 0.130

Note: a * indicates group mean is significantly different from control at level of significance shown.

TABLE 3: Absolute organ weights (g): kidneys - group mean data - Females

Group	Control	2	3	4
Number/group	10	10	10	10
Mean	1.806	1.799	1.874	2.011
Standard deviation	0.147	0.191	0.173	0.186
Group diff. at p < 0.05		0.192	0.192	0.192*
Group diff. at p < 0.01		0.243	0.243	0.243

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.47 Df = 3/ 36 F probability = 0.237

Note: a * indicates group mean is significantly different from control at level of significance shown.

Conclusions:

Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day both for males and females.

Executive summary:

The toxic effects on Wistar Hannover rats after repeated dosing with Reactive Red 45:1, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated in a GLP compliant OECD TG 422 study. All doses were administered orally, by gavage. The control group received purified water. The dose volume was 10mL/kg body weight. Treatments were 0, 100, 300 and 1000 mg/kg bw /day (in terms of test item corrected for purity) corresponding to Group 2, 3 and 4 respectively. Each group was composed of Males were treated daily for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 35/36 days. Females were treated for 2 weeks prior to pairing, during pairing up to Day 13 post partum. Two non pregnant females were dosed up to the day before necropsy.

The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight, food consumption, oestrous cycle, mating performance and clinical pathology investigations (haematology and clinical chemistry). Blood collection for hormone assay, anogenital distance, litter data, sex ratios, macroscopic observations and organ weights were performed. Vaginal smears in all adult female groups were investigated at termination. Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals of both sexes, the examination was extended to the kidneys of animals of both sexes of Groups 2 and 3 and remaining animals of control and high dose groups. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups. Thyroid hormone determination was performed in all adult males. As a consequence of the colour of the test item, red staining and/or red faeces were observed inside the cage of animals receiving the dose levels ≥ 300 mg/kg bw /day during the treatment period. Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw /day for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg bw /day both for males and females. No mortality occurred throughout the study. The main clinical sign noted in treated animals of both sexes receiving 1000 mg/kg bw/day was salivation. This sign appeared in the majority of the animals from the second day of dosing and it was observed for the entire treatment period even if not in a continuous manner. The red staining in different locations of the body surface observed in animals receiving the dose levels ≥ 300 mg/kg bw/day was related to the colour of the test item. The other clinical signs noted were considered incidental and not treatment-related. As a consequence of the colour of the test item, red staining and/or red faeces were observed inside the cage of animals receiving the dose levels ≥300 mg/kg bw /day during the treatment period. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study. No effects on food consumption were observed in either males or females. No relevant differences were noted in all parameters investigated between control and treated groups. No changes were recorded at haematological investigation performed at the end of the study. No changes were recorded in prothrombin time measured at the end of the study. The changes observed at clinical chemistry investigation performed at the end of the study were of minimal severity, therefore they were considered to be not adverse. No changes in thyroid hormones were recorded in parental male animals of treated groups. No changes were observed in terminal bodyweight of study animals of both sexes in control and treated animals. A statistically significant increase was observed in kidneys of high dose females when compared to controls. The most relevant change observed at necropsy was red colour of kidneys in high dose rats of both sexes when compared to controls, whereas the same finding was also noted sporadically in cervical and mesenteric lymph nodes and gastro-intestinal tract of treated animals in both sexes. Treatment-related changes, associated with the oral administration of Reactive Red 45:1, were present in the kidneys and consisting of tubular cell vacuolation involving cortex and medulla characterised by the presence of amorphous material in the vacuoles of high dose animals of both sexes and hyaline droplets accumulation only in high dose males. Regular layering in the germinal epithelium was noted. Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day both for males and female.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.03.-05.06.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test material: Reactive Red 45:1
- Appearance: red powder
- Expiry date 14 April 2021
- Storage: at a dry and dark place between 5 - 30 °C

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: approximately 7 to 8 weeks old.
- Weight at study initiation: 223 - 233 g for males and 175 - 190 g for females.
- Housing: The animals were housed in a limited access rodent facility. Animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
- Diet: laboratory rodent diet 4 RF 21, manufactured by Mucedola S.r.l, was provided ad libitum throughout the study, except as noted in section.
- Water: Water was provided ad libitum.
- Acclimation period: 26 days.

DETAILS OF FOOD AND WATER QUALITY
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Food and water was checked during the study

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

IN-LIFE DATES: FROM 26 Mar 2019 To 05 Jun 2019

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved in the vehicle. The preparations were made weekly at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item corrected for purity. Preparations of the test item were prepared as solutions in water. Concentration was assessed for all levels by taking two analytical aliquots (approximately 1 mL). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the two determinations.

VEHICLE
- Concentration in vehicle: 10, 30 100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations.
The analytical method was validated in the range from 5 to 100 mg/mL. Linearity, accuracy and precision were within the for solutions (r > 0.98; accuracy 90-110%; precision CV <5%). In the same study, a 28 hour stability at room temperature and an 8 day stability at 2 - 8 °C were verified in the range from 5 to 100 mg/mL.

Duration of treatment / exposure:

Females dosed for two consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days).

Frequency of treatment:

Once a day, seven days a week.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: Selection of dose levels Dose levels have been selected in consultation with the Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.: E0374). 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 dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Fasting period before blood sampling for clinical biochemistry: no.
- Culling and pups selection for blood collection (serum hormone) at necropsy Culling - Day 4 post partum: On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. Two pups (males or females, selected for culling) were sacrificed for blood collection.

Maternal examinations:

CAGE SIDE OBSERVATION
- Time schedule: All the animals were observed early in each working day and again in the afternoon for clinical signs of toxicity and twice daily for morbidity. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment and at least once daily during the study, each animal was observed for any clinical signs 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
Observations of the cage tray during the pre-mating period, after mating for males and post partum periods were performed for all groups and were recorded three times weekly. During pairing and gestation periods (females) observations of the cage tray were performed and recorded daily for all groups. These data are not tabulated in this report but will be archived together with all other raw data.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
Once before commencement of treatment and 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.
Data are reported until Week 7 for females. All the data not tabulated has been be archived together with all other raw data. The following parameters were measured or analysed in 5 out of 10 animals of each group randomly selected: Grip strength and sensory, reactivity to stimuli, Motor activity (MA) assessment and Clinical pathology investigations.

GRIP STRENGTH AND SENSORY REACTIVITY TO STIMULI
Once during the study, towards the end of treatment, 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males, the tests were performed on Day 29 of the study and for females on Day 12 post partum.

MOTOR ACTIVITY ASSESSMENT (MA)
Once during the study, towards the end of treatment, 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 males, the test was performed on Day 29 of the study and for females on Day 12 post partum.

BODY WEIGHT
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION:
The weight of food consumed by each cage of females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

NECROPSY
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.
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed on Day 4 post partum.

EUTHANASIA
Parental animals were killed by exsanguination under isoflurane anaesthesia.
Prenatal females: The females with live pups were killed on Day 14 post partum. Females were dosed for a minimum of 51 consecutive days.

Ovaries and uterine content:

OVARIES AND UTERINE CONTENT
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Clinical pathology: Blood collection was performed for hormone determination (0.8 mL) from all parental animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, by random selection from 5 females (females with viable litters) of each group, under condition of food deprivation.
Females: As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups. The blood samples were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone assay
The following parameters were assessed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorusThese parameters were analysed by Siemens Advia 1200.
The pups groups serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) were determined by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K.

- 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. These parameters were analysed by Siemens Advia 120.
- Coagulation tests:
Prothrombin time: This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

- Anogenital distance (AGD): each pup was measured on Day 1 post partum. The measure of AGD was normalised to the cube root of the pup’s body weight measured on Day 1 post partum.
- Pups at day 14 post partum : Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.
- Pups identification, weight and observations: As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters. After culling, all pups were sacrificed with the dams on Day 14 post partum.
- Nipple count: The presence of nipples/areolae in male pups was checked on Day 13 post partum, on the day before despatch to necropsy.
- Nipple retention at day 14 post partum: The ventral region of male pups was checked for presence of nipples/areolae. No nipples were found in pups to be retained on Day 14 post partum. Data were not tabulated, but will be archived with all raw data.
- Blood collection and thyroid hormone determination (T3, T4 and TSH): Blood collection from pups on Days 4 and 14 post partum On Day 4 post partum as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 pups (1 male and 1 female, if possible). On Day 14 post partum as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female). Blood sample was withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots and stored at -80 °C pending.
- Pups were subjected to an external examination and internal abnormalities.
- Sex was determined by internal gonads inspection. All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection. Pups with abnormalities were retained in 10% neutral buffered formalin.
- Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, 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 nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric 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.

Indices:

Presentation of data /Indices
Group mean values were calculated for all parameters. The following reproductive indices were calculated:

Females:
Copulatory Index(%) = (no. of females with confirmed mating) / (no. of females cohabitated) x 100
Fertility Index (%) = (no. of pregnant females) /(no. of females cohabitated) x100
Pre coital Interval = The number of nights paired prior to the detection not mating
Pre-implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea−no. of implantations)/ (no. of corpora lutea) ×100

Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations−live litter size at birth)/(no. of visible implantations) x 100

Pup loss at Day 0 post partum was calculated as a percentage from the formula:
(Total litter size−live litter size)/(Total litter size) ×100

Post natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
(Live litter size at birth−live litter size at Day 4(before culling))/(Live litter size at birth) ×100

Post natal loss at Day 13 post partum (after culling) was calculated as a percentage from
the formula:
(Live litter size on Day 4(after culling)−live litter size on Day 13)/ (Live litter size on Day 4(after culling)) ×100

Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented
as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all females receiving 1000 mg/kg bw/day (Group 4), red staining on the tail was noted starting from the second day of dosing and during the entire treatment period (i.e. premating period, mating phase, gestation and post partum periods). In addition, salivation was generally observed in 7 out of 10 females before and during mating as well as in all females during gestation and post partum periods but not continuously. In six females receiving 300 mg/kg bw/day (Group 3) and in two females receiving 100 mg/kg bw/day (Group 2), red staining on the tail was seen during the gestation and/or post partum periods. Hair loss in different regions of the body surface noted in three females receiving 1000 mg/kg bw/day and in one control female and scab on the head observed in one female receiving 300 mg/kg bw/day were not considered treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated females throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment during the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes were recorded at the haematological investigation performed at the end of the study. Coagulation: No changes were recorded in prothrombin time measured at the end of the study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant findings were observed at clinical chemistry investigation performed at the end of the study.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No changes were observed in terminal body weight of study animals, when compared to the control group. A statistically significant increase was observed in kidneys of females treated at 1000 (high dose) mg/kg bw/day (11% of absolute and relative weights), when compared to controls. Such organ weight variation was considered treatment-related also on the basis of histopathological results. No other organ weight variations were observed in treated groups, when compared to the controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The most relevant change observed at necropsy was red colour of kidneys in high dose rats when compared to controls. The same finding was also observed sporadically in low and/or mid- and/or high dose female rats in cervical and mesenteric lymph nodes and gastro-intestinal tract (stomach, jejunum and ileum). In addition, red staining on the external surface of the body [superior part of the skin (dorsum) and tail] was reported in most treated animals. This finding was considered likely due to the colour of the test item. All other observed changes in the control and treated groups are known to occur spontaneously in untreated Wistar Hannover rats of the same age, under our experimental conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals, the examination was extended to the kidneys of animals of Groups 2 and 3 and remaining animals of control and high dose groups.
Treatment-related lesions were only noted in kidneys of high dose animals. At microscopic evaluation, the kidneys presented tubular cell vacuolation involving cortex and medulla, characterised by the presence of amorphous material in the vacuoles, with a mild to marked degree in high dose animals. This renal finding was not observed in the mid- and low dose animals when compared to the controls. In addition, the increased severity of hyaline droplets accumulation in kidneys from mild to marked degree was only observed in high dose males, when compared with controls.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Gestation length was similar in treated and control groups. Pregnant dams gave birth on Day 22/23 post coitum (mean value). Corpora lutea, number of implantation sites, pre-implantation and pre-natal loss of all treated females were comparable to the control values.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratios at birth, on Days 4 and 14 post partum did not show differences between groups when calculated as the percentage of males.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Small appearance of pups were noted in a limited number of pups in low, high dose and control groups. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No differences in the anogenital distance (normalised value) to the cube root of the body weight, performed on Day 1 post partum, were seen between control and treated groups for female pups. The mean values of anogenital distance of male pups in order of ascending dose levels were: 1.95 (control), 1.89, 1.93 and 1.72 mm/ 3√g . A slight decrease, significant at statistical analysis, in the mean values was noted in high dose male pups when compared to the control value. However this change was dose-unrelated and therefore cannot be conclusively attributed to treatment.

Changes in postnatal survival:

no effects observed

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Description (incidence and severity):

Autolysed abdominal organs were observed in three early decedent pups belonging to control and high dose groups which died at birth or during the lactation period, while no abnormalities were observed in the other early decedent pups.

Other effects:

no effects observed

Description (incidence and severity):

- Clinical signs observed such as: damaged tail, hairloss, small appearance of pups were noted in a limited number of pups in low, high dose and control groups. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period.
- No nipples were found in male pups on Day 13 post partum.
- Pups sacrificed on Days 4 and 14 post partum: No findings were recorded in pups sacrificed on Day 4 post partum. No significant findings were recorded in pups sacrificed on Day 14 post partum. However, hair loss (generalised or on dorsum) was observed at sacrifice on Day 14 post partum in all pups of one control female, one low dose female and three high dose dams.
- No differences were noted in thyroid weight between control and treated pups.
- Triiodothyronine (T3) was decreased in some male pups of mid- and high dose groups. Mean group values were 43% and 57% below controls, respectively. Due to the absence of other related findings (e.g. changes of T4 and TSH), this change was not considered to be adverse.
- Left eye opacity observed in one pup only of the low dose group dam no. was considered incidental. No signs were recorded in pups of the mid-dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1 Macroscopic observations - Group data

	Males				Females
Group:	1	2	3	4	1	2	3	4
Number in group:	10	10	10	10	10	10	10	10
Adrenals -Abnormal colour -Abnormal size	0 0	0 0	0 0	0 0	1 0	1 0	3 2	1 0
Cervicalnodes -Abnormal colour	1	0	3	1	0	0	0	0
Epididymides -Abnormal size	2	2	0	1
Ileum Abnormal colour	0	0	0	0	0	0	0	2
Jejunum -Abnormal colour	0	0	0	0	0	0	0	1
Kidneys - Abnormal colour	0	0	0	8	0	0	0	8
Liver - Abnormal area(s)	1	0	0	0	0	0	0	0
Mesenteric nodes - Abnormal colour	0	0	0	0	1	2	0	4
Prostate - Abnormal size	1	2	4	4
Seminal vesicles - Abnormal size	2	1	3	2
Stomach - Abnormal area(s) - Abnormal colour - Abnormal size	0 0 0	0 0 0	0 0 0	0 6 0	3 0 0	0 0 0	3 0 1	1 3 2
Testes - Abnormal size	1	0	0	1
Thymus
- Abnormal area(s)	0	0	0	1	0	0	0	0
- Abnormal colour	0	1	0	0	0	0	0	0
- Abnormal size	0	0	0	0	1	0	0	0
Uterus - Not pregnant.					1	1	0	0
Forelimbs - Hairloss	0	0	0	0	1	0	0	3
Hindlimbs - Hairloss	0	0	0	0	0	0	0	1
Skin - Hairloss - Staining	0 0	0 0	0 0	0 6	1 0	0 0	0 0	1 0
Tail - Abnormal shape. - Staining	0 0	0 0	0 10	1 10	0 0	0 2	0 5	0 10
Whole animal - No abnormalities detected	5	6	0	0	5	5	3	0

 

Conclusions:

Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for females. The NOAEL for reproductive was considered to be 1000 mg/kg bw/day for maternal females and developmental toxicity 1000 mg/kg bw/day pups.

Executive summary:

The toxic effects on Wistar Hannover rats after repeated dosing with Reactive Red 45:1, as well as any effects of the test item development of conceptuses, parturition and early lactation of the offspring were investigated in a GLP compliant OECD TG 422 study. All doses were administered orally, by gavage. The control group received purified water. The dose volume was 10mL/kg body weight. Treatments were 0, 100, 300 and 1000 mg/kg bw/day ( in terms of test item corrected for purity) corresponding to Group 2, 3 and 4, respectively. Each group was composed females until the day before necropsy, for a total of 35/36 days. Maternal animals were treated for 2 weeks prior to pairing, during pairing up to Day 13 post partum. Two non pregnant maternal females were dosed up to the day before necropsy. The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight, food consumption and clinical pathology investigations (haematology and clinical chemistry). Blood collection for hormone assay (adult animals and pups), anogenital distance, litter data, sex ratios, macroscopic observations and organ weights were performed. Vaginal smears in all adult female groups were investigated at termination. Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals, the examination was extended to the kidneys of animals of both sexes of Groups 2 and 3 and remaining animals of control and high dose groups. Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14 post partum were determined. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum and those sacrificed on Day 14 post partum were subjected to an external examination and the sex was determined by internal gonads inspection. As a consequence of the colour of the test item, red staining and/or red faeces were observed inside the cage of animals receiving the dose levels ≥300 mg/kg bw /day during the treatment period. Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for maternal females. The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg bw /day both for maternal females. No mortality occurred throughout the study. The main clinical sign noted in treated animals of both sexes receiving 1000 mg/kg bw/day was salivation. This sign appeared in the majority of the animals from the second day of dosing and it was observed for the entire treatment period even if not in a continuous manner. The red staining in different locations of the body surface observed in animals receiving the dose levels ≥300 mg/kg bw /day was related to the colour of the test item. The other clinical signs noted were considered incidental and not treatment-related.

As a consequence of the colour of the test item, red staining and/or red faeces were observed inside the cage of animals receiving the dose levels ≥300 mg/kg bw /day during the treatment period. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No relevant differences in body weight and body weight gain were noted between control and treated maternal females throughout the study. No effects on food consumption were observed in either maternal females. No relevant differences were noted in all parameters investigated between control and treated groups.

No changes were recorded at haematological investigation performed at the end of the study. No changes were recorded in prothrombin time measured at the end of the study. The changes observed at clinical chemistry investigation performed at the end of the study were of minimal severity, therefore they were considered to be not adverse. No changes were observed in terminal bodyweight of study animals of both sexes in control and treated animals. A statistically significant increase was observed in kidneys of high dose maternal females when compared to controls. The most relevant change observed at necropsy was red colour of kidneys in high dose rats of both sexes when compared to controls, whereas the same finding was also noted sporadically in cervical and mesenteric lymph nodes and gastro-intestinal tract of treated animals in both sexes. Treatment-related changes, associated with the oral administration of Reactive Red 45:1, were present in the kidneys and consisting of tubular cell vacuolation involving cortex and medulla characterised by the presence of amorphous material in the vacuoles of high dose animals in maternal females. Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for females. The NOAEL for reproductive was considered to be 1000 mg/kg bw/day for maternal females and developmental toxicity 1000 mg/kg bw/day pups.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

EC Number:

277-616-3

EC Name:

Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Cas Number:

73816-74-7

Molecular formula:

C28H24ClN7O10S3.3Na

IUPAC Name:

trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test material: Reactive Red 45:1
- Appearance: red powder
- Expiry date 14 April 2021
- Storage: at a dry and dark place between 5 - 30 °C

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: approximately 7 to 8 weeks old.
- Weight at study initiation: 223 - 233 g for males and 175 - 190 g for females.
- Housing: The animals were housed in a limited access rodent facility. Animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
- Diet: laboratory rodent diet 4 RF 21, manufactured by Mucedola S.r.l, was provided ad libitum throughout the study, except as noted in section.
- Water: Water was provided ad libitum.
- Acclimation period: 26 days

DETAILS OF FOOD AND WATER QUALITY
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Food and water was checked during the study

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

IN-LIFE DATES: FROM 26 Mar 2019 To 05 Jun 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved in the vehicle. The preparations were made weekly at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item corrected for purity. Preparations of the test item were prepared as solutions in water. Concentration was assessed for all levels by taking two analytical aliquots (approximately 1 mL). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the two determinations.

VEHICLE
- Concentration in vehicle: 10, 30 100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations.
The analytical method was validated in the range from 5 to 100 mg/mL. Linearity, accuracy and precision were within the for solutions (r > 0.98; accuracy 90 -110%; precision CV <5%). In the same study, a 28 hour stability at room temperature and an 8 day stability at 2 - 8 °C were verified in the range from 5 to 100 mg/mL.

Duration of treatment / exposure:

Females at least for 51 days and males for 35 - 36 days.

Frequency of treatment:

Once a day, seven days a week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Dose selection rationale: Selection of dose levels Dose levels have been selected in consultation with the Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.: E0374). 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 dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Fasting period before blood sampling for clinical biochemistry: no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATION
- Time schedule: All the animals were observed early in each working day and again in the afternoon for clinical signs of toxicity and twice daily for morbidity. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment and at least once daily during the study, each animal was observed for any clinical signs 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
Observations of the cage tray during the pre-mating period, after mating for males and post partum periods were performed for all groups and were recorded three times weekly. During pairing and gestation periods (females) observations of the cage tray were performed and recorded daily for all groups. These data are not tabulated in this report but will be archived together with all other raw data.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
Once before commencement of treatment and 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.
Data are reported until Week 5 for male animals and Week 7 for females. All the data not tabulated has been be archived together with all other raw data. The following parameters were measured or analysed in 5 out of 10 animals of each group randomly selected: Grip strength and sensory, reactivity to stimuli, Motor activity (MA) assessment and Clinical pathology investigations.

GRIP STRENGTH AND SENSORY REACTIVITY 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 reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males, the tests were performed on Day 29 of the study and for females on Day 12 post partum.

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 males, the test was performed on Day 29 of the study and for females on Day 12 post partum.

BODY WEIGHT
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20.

FOOD CONSUMPTION:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY
Blood collection was performed for hormone determination (0.8 mL) from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, by random selection from 5 males and 5 females of each group, under condition of food deprivation.
Males: Blood samples for haematological investigations, biochemical tests and hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females: As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups. The blood samples were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone assay
The following parameters were assessed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus.
These parameters were analysed by Siemens Advia 1200.
Per group, 5 males were randomly selected and groups serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) were determined by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K)

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, These parameters were analysed by Siemens Advia 120.
Coagulation tests:
Prothrombin time: This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.

Sacrifice and pathology:

EUTHANASIA
Animals were killed by exsanguination under isoflurane anaesthesia.
Males were killed after the mating of all females on Days 36 and 37 of the study.
The females with live pups were killed on Day 14 post partum.

NECROPSY
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.

ORGAN WEIGHTS ANIMALS
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

TISSUES FIXED AND PRESERVED
Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin.

HISTOPATHOLOGICAL EXAMINATION
The tissues required for histopathological examination are listed the 'Any other information on materials and methods incl. tables' field. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. The examination, in the first instance, was restricted as detailed below:
i) Tissues specified from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
ii) All abnormalities in all groups. Since differences were observed between control and high dose animals, the examination was extended to: Kidneys in all males and females of Groups 2 and 3 and in the remaining animals of control and high dose groups. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, 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 nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all males receiving 1000 mg/kg bw/day (Group 4), red staining in different regions of the body surface (i.e. tail, dorsum and sacral region) was noted starting from the second day of dosing and during the entire treatment period. In addition, salivation was observed in 9 out of 10 animals of the group generally starting after the first two weeks of treatment and, in the majority of animals, up to the end of the treatment period even if not in a continuous manner. Damaged tail recorded in one male only from Day 6 of the mating phase up to the end of treatment was not considered related to the treatment. In all males receiving 300 mg/kg bw/day (Group 3), red staining on the tail was observed starting from Day 9 of the mating phase up to the end of the study. No clinical signs were observed in males receiving 100 mg/kg bw/day (Group 2) and in control animals.
In all females receiving 1000 mg/kg bw/day (Group 4), red staining on the tail was noted starting from the second day of dosing and during the entire treatment period (i.e. premating period, mating phase, gestation and post partum periods). In addition, salivation was generally observed in 7 out of 10 females before and during mating as well as in all females during gestation and post partum periods but not continuously. In six females receiving 300 mg/kg bw/day (Group 3) and in two females receiving 100 mg/kg bw/day (Group 2), red staining on the tail was seen during the gestation and/or post partum periods. Hair loss in different regions of the body surface noted in three females receiving 1000 mg/kg bw/day and in one control female and scab on the head observed in one female receiving 300 mg/kg bw/day were not considered treatment-related.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study. Statistically significant differences in body weight gain recorded in males receiving 300 mg/kg/day in the last week of pre-mating period and first week of pairing period were considered incidental because not dose-related and not consistent in direction.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both sexes during the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes were recorded at the haematological investigation performed at the end of the study. Coagulation: No changes were recorded in prothrombin time measured at the end of the study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant findings were observed at clinical chemistry investigation performed at the end of the study. The statistically significant decreases of chloride recorded in males dosed at 1000 mg/kg bw/day (3% below controls) and creatinine recorded in females of the same group (8%) were of minimal severity, therefore they were considered to be not adverse.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone determination: for males: No changes were recorded in thyroid hormones determination performed at the end of the study

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No changes were observed in terminal body weight of study animals of both sexes, when compared to the control group. A statistically significant increase was observed in kidneys of females treated at 1000 (high dose) mg/kg bw/day (11% of absolute and relative weights), when compared to controls. Such organ weight variation was considered treatment-related also on the basis of histopathological results. No other organ weight variations were observed in treated groups of both sexes, when compared to the controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The most relevant change observed at necropsy was red colour of kidneys in high dose rats of both sexes, when compared to controls. The same finding was also observed sporadically in low and/or mid- and/or high dose male and/or female rats in cervical and mesenteric lymph nodes and gastro-intestinal tract (stomach, jejunum and ileum). In addition, red staining on the external surface of the body [superior part of the skin (dorsum) and tail] was reported in most treated animals of both sexes. This finding was considered likely due to the colour of the test item. All other observed changes in the control and treated groups are known to occur spontaneously in untreated Wistar Hannover rats of the same age, under our experimental conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals of both sexes, the examination was extended to the kidneys of animals of both sexes of Groups 2 and 3 and remaining animals of control and high dose groups.
Treatment-related lesions were only noted in kidneys of high dose animals of both sexes. At microscopic evaluation, the kidneys presented tubular cell vacuolation involving cortex and medulla, characterised by the presence of amorphous material in the vacuoles, with a mild to marked degree in high dose animals of both sexes. This renal finding was not observed in the mid- and low dose animals of both sexes when compared to the controls. In addition, the increased severity of hyaline droplets accumulation in kidneys from mild to marked degree was only observed in high dose males, when compared with controls. The hyaline droplets represented by eosinophilic homogeneous cytoplasmic droplets and the increased accumulation in the high dose males, evaluated respect to controls as reported in literature (Toxicologic Pathology, 36: 1014-1017, 2008 - GORDON C. HARD X1100), could represent low molecular weight protein accumulation within lysosomes, due to the disturbance of the normal balance of tubular reabsorption and hydrolysis, as a result of either increased filtered protein loads or decreased catabolism. The remaining sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

For males, no changes were recorded in thyroid hormones determination performed at the end of the study. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups in both sexes.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1 Macroscopic observations - Group data

	Males				Females
Group:	1	2	3	4	1	2	3	4
Number in group:	10	10	10	10	10	10	10	10
Adrenals -Abnormal colour -Abnormal size	0 0	0 0	0 0	0 0	1 0	1 0	3 2	1 0
Cervicalnodes -Abnormal colour	1	0	3	1	0	0	0	0
Epididymides -Abnormal size	2	2	0	1
Ileum Abnormal colour	0	0	0	0	0	0	0	2
Jejunum -Abnormal colour	0	0	0	0	0	0	0	1
Kidneys -Abnormal colour	0	0	0	8	0	0	0	8
Liver -Abnormal area(s)	1	0	0	0	0	0	0	0
Mesenteric nodes -Abnormal colour	0	0	0	0	1	2	0	4
Prostate -Abnormal size	1	2	4	4
Seminal vesicles -Abnormal size	2	1	3	2
Stomach -Abnormal area(s) -Abnormal colour - Abnormal size	0 0 0	0 0 0	0 0 0	0 6 0	3 0 0	0 0 0	3 0 1	1 3 2
Testes -Abnormal size	1	0	0	1
Thymus
-Abnormal area(s)	0	0	0	1	0	0	0	0
-Abnormal colour	0	1	0	0	0	0	0	0
-Abnormal size	0	0	0	0	1	0	0	0
Uterus -Not pregnant.					1	1	0	0
Forelimbs -Hairloss	0	0	0	0	1	0	0	3
Hindlimbs -Hairloss.	0	0	0	0	0	0	0	1
Skin -Hairloss -Staining	0 0	0 0	0 0	0 6	1 0	0 0	0 0	1 0
Tail -Abnormal shape. -Staining	0 0	0 0	0 10	1 10	0 0	0 2	0 5	0 10
Whole animal -No abnormalities detected	5	6	0	0	5	5	3	0

 

Table 2: Absolute organ weights (g): kidneys - group mean data - Males

Group	Control	2	3	4
Number/group	10	10	10	10
Mean	2.440	2.508	2.323	2.571
Standard deviation	0.176	0.330	0.198	0.213
Group diff. at p < 0.05		0.260	0.260	0.260
Group diff. at p < 0.01		0.329	0.329	0.329

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 2.00 Df = 3/ 36 F probability = 0.130

Note: a * indicates group mean is significantly different from control at level of significance shown.

TABLE 3: Absolute organ weights (g): kidneys - group mean data - Females

Group	Control	2	3	4
Number/group	10	10	10	10
Mean	1.806	1.799	1.874	2.011
Standard deviation	0.147	0.191	0.173	0.186
Group diff. at p < 0.05		0.192	0.192	0.192*
Group diff. at p < 0.01		0.243	0.243	0.243

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.47 Df = 3/ 36 F probability = 0.237

Note: a * indicates group mean is significantly different from control at level of significance shown.

Applicant's summary and conclusion

Conclusions:

Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw/day for males and females.

Executive summary:

The toxic effects on Wistar Hannover rats after repeated dosing with Reactive Red 45:1 were investigated in a GLP compliant OECD TG 422 study. All doses were administered orally, by gavage. The control group received purified water. The dose volume was 10 mL/kg body weight. Treatments were 0, 100, 300 and 1000 mg/kg bw /day (in terms of test item corrected for purity) corresponding to Group 2, 3 and 4 respectively. Males were treated daily for 2 weeks until the day before necropsy, for a total of 35/36 days. Females were treated for 2 weeks prior to pairing, during pairing up to Day 13 post partum. Two non pregnant females were dosed up to the day before necropsy. The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight, food consumption and clinical pathology investigations (haematology and clinical chemistry). Blood collection for hormone assay (adult animals), macroscopic observations and organ weights were performed. Routine histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the kidneys were observed in high dose animals of both sexes, the examination was extended to the kidneys of animals of both sexes of Groups 2 and 3 and remaining animals of control and high dose groups.

No mortality occurred throughout the study. The main clinical sign noted in treated animals of both sexes receiving 1000 mg/kg bw /day was salivation. This sign appeared in the majority of the animals from the second day of dosing and it was observed for the entire treatment period even if not in a continuous manner. The red staining in different locations of the body surface observed in animals receiving the dose levels ≥300 mg/kg bw /day was related to the colour of the test item. The other clinical signs noted were considered incidental and not treatment-related. As a consequence of the colour of the test item, red staining and/or red faeces were observed inside the cage of animals receiving the dose levels ≥300 mg/kg bw /day during the treatment period. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.

No effects on food consumption were observed in either males or females. No relevant differences were noted in all parameters investigated between control and treated groups. No changes were recorded at haematological investigation performed at the end of the study. No changes were recorded in prothrombin time measured at the end of the study. The changes observed at clinical chemistry investigation performed at the end of the study were of minimal severity, therefore they were considered to be not adverse.

No changes in thyroid hormones were recorded in parental male animals of treated groups. No changes were observed in terminal body weight of study animals of both sexes in control and treated animals. A statistically significant increase was observed in kidneys of high dose females when compared to controls. The most relevant change observed at necropsy was red colour of kidneys in high dose rats of both sexes when compared to controls, whereas the same finding was also noted sporadically in cervical and mesenteric lymph nodes and gastro-intestinal tract of treated animals in both sexes. Treatment-related changes, associated with the oral administration of Reactive Red 45:1, were present in the kidneys and consisting of tubular cell vacuolation involving cortex and medulla characterised by the presence of amorphous material in the vacuoles of high dose animals of both sexes and hyaline droplets accumulation only in high dose males.

Based on the occurrence of the histopathological findings observed in the kidneys of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg bw /day for males and females.