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

Endpoint summary

Description of key information

- Oral: NOAEL = 300 mg/kg bw/day for both sexes, based on data from a GLP compliant OECD 408 study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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 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.

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

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.

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.

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

Key result

Critical 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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 422 study.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral sub-acute repeated dose toxicity study in rats

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.

Justification for classification or non-classification

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.