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Description of key information

Based on the results of the 28-day oral toxicity study, it can be concluded that the histopathological alterations observed in kidneys and liver of male animals treated at the high dose level (40 mg/kg body weight/day) were adaptive changes and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day was considered to be the No Observed Adverse Effect Level (NOAEL) in this study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase (allocation of the animals): 30 May 2017 End of experimental phase (last day of necropsy) :17 July 2017 Study completion: Study Director's signature
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many
regulatory authorities and there are ample experience and background data on this species
and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy; supplied by Envigo Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: approximately 190 g for males and 160 g for females (at the start of dosing)
- Fasting period before study: No
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): 4 RF 21, Mucedola S.r.l., ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 6 days prior to the start of treatment

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2° C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): the rooms were lit by artificial light for 12 hours each day.

IN-LIFE DATES: From: 30 May 2017 To: 17 July 2017
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared inWeeks 1 and 4 of the current study were analysed to
check the concentration. Results of the analyses were within the acceptability limits stated in
RTC SOPs for concentration of solutions (90-110%)
Duration of treatment / exposure:
minimum of 4 consecutive weeks
Frequency of treatment:
All animals were dosed once a day, 7 days a week.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group comprised 5 male and 5 female rats, plus 5 male and 5 female recovery rats in the control and high-dose group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.

Clinical signs and neurotoxicity assessment
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during the study, each animal was observed
and 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.Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a
detailed clinical examination. Each animal was observed in an open arena. The test 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, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
Once during Week 4 of treatment and once during Week 2 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual
and proprioceptive stimuli) and an assessment of grip strength were also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once duringWeek 4 of treatment and once during Week 2 of recovery by an automated activity recording.
Measurements were performed using a computer generated random order.
Body weight
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

Food consumption
The weight of food consumed by each cage of rats was recorded at weekly intervals during the treatment and recovery periods. The group mean daily intake
per rat was calculated.

Clinical pathology investigations
Prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava, under conditions of food deprivation.
Since possible treatment-related changes were observed, further blood samples were taken from all surviving animals, under identical conditions, at the end of the recovery period for
clinical chemistry tests.
Blood samples were collected and analysed in the same order. The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
Sacrifice and pathology:
Euthanasia
Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals were subjected to necropsy,
supervised by a pathologist.

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
From all animals completing the scheduled test period, the organs indicated in the protocol 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 listed in Annex of the protocol were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides,
which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in Annex 1. After dehydration and embedding in paraffin wax, sections of the tissues were cut
at 5 micrometre thickness and stained with haematoxylin and eosin.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal of the control group was found dead on Day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Very slight reductions in body weight were noted in animals of the treated groups when compared to controls. These decrements were statistically significant on Day 22 for males of Group 4, on Days 22 and 29 for females of Groups 2 and 3 and on Day 8 up to the end of the treatment and recovery periods for females of Group 4.
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in
relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls.
No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition,
multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.
The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery
period.
Details on results:
- Mortality
One male animal of the control group (no. A2787010) was found dead on Day 23 of the study. No clinical signs were observed before death. The macroscopic findings observed at post mortem examination (red fluid content of thoracic cavity, incomplete collapse and dark colour of lungs and red staining of muzzle) and the main changes noted at the histopathological evaluation (moderate alveolar haemorrhage, minimal oedema of lungs and minimal myocardial chronic inflammation) suggested that the cause of death was likely due to a mis-dosing.
- Clinical signs
No clinical signs were noted in animals of the control and treated groups, except for two animals of the high dose group (Group 4) that showed rales on Days 19-20 (animal no. 52) and from Day 21 to 29 (animal no. 58). One animal of the same group (no. A2787056) showed ataxia and dyspnoea between Days 12 and 17 of the dosing phase.
Rales continued to be noted in one animal (no. 58) during the first three days of the recovery period. This sign was no longer apparent fromDay 4 up to the end of the observation period. No clinical signs were recorded in control animals and in the other animals of Group 4.
- Weekly detailed clinical signs (removal from cage and open field measurements)
No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity.
- Neurotoxicity assessment (Functional Tests and Motor activity)
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
- Body weight
From Day 8 to Day 29 of the dosing period, a very slight reduction in body weight (ranging from -8% to -4%), statistically significant only on Day 22, was recorded in males of the high
dose group. No remarkable differences in body weights were noted during the recovery period, when compared to controls. A very slight, but statistically significant, decrease in body weight (included in a range of -12% to -7%) was noted in female animals of the low and mid-dose groups, on Days 22 and 29 of the treatment period, when compared to females of the control group. A very slight and statistically significant, decrease in body weight (ranging from -8% to -6%) was also recorded in females of the high dose group from Day 8 up to the end of the treatment period.
Body weights remained lower than controls also during the recovery period, with a slight, but statistically significant, reduction in body weight of -11% and -10% on Days 8 and 15, respectively.
- Food consumption
No differences in food consumption were recorded between treated and control animals during the dosing and recovery periods.
- Haematology
The statistically significant difference of leucocytes recorded between controls and males dosed at 10 mg/kg/day (37% below mean control data) was not dose-related, therefore it was considered to be incidental.
- Coagulation
No changes were recorded.
- Clinical chemistry
Dosing phase:Compared to animals of the control groups, statistically significant fluctuations of some biochemical parameters were recorded, as follows: decrease of alkaline phosphatase in males dosed at 20mg/kg/day (-21%), decrease of aspartate aminotransferase in males receiving 20 and 40 mg/kg/day (-23% and -20%, respectively), decrease of triglycerides in males dosed at 40 mg/kg/day (-45%) and increase of calcium in those receiving 20 mg/kg/day (+12%), decrease of aspartate aminotransferase in females dosed at 20 mg/kg/day (-14%), increase of glucose in those receiving 10 mg/kg/day (+19%) and 40 mg/kg/day (+26%) and decrease of chloride in females treated at 40 mg/kg/day (-3%). Some of these changes (alkaline phosphatase and calcium in males, aspartate aminotransferase in females) were not dose-related, therefore considered unrelated to treatment. The other findings were not considered to be suggestive of tissue/organ injury, due to their slight severity.
Recovery phase: Cholesterol and chloride showed a complete reversibility in females. In male animals triglycrides were lower than controls (-33%), although this change was not considered to be adverse.The other statistically significant changes recorded (sodium in males, urea, triglycerides and potassium in females) were considered incidental, since not observed during the dosing phase.
- Terminal body weight and organ weights
Final sacrifice: No differences of toxicological relevance were observed on terminal body weight of treated animals, when compared to controls. Male animals of mid- (Group 3, 20 mg/kg/day body weight) and high (Group 4, 40 mg/kg/day body weight) dose groups showed a statistically significant increase in absolute and relative mean liver weights (approximately 14% and 22%, respectively), when compared to controls. In addition, a statistically significant increase in relative liver weight was recorded in females of the high dose group (approximately 15%) and in kidneys relative weight (approximately 13%) of the same group, when compared to animals of the control group.
No other remarkable changes in absolute and relative organ weights were noted in the animals of the other treated groups, when compared to the control data.
Recovery sacrifice: No changes of toxicological relevance were observed on terminal body weight, absolute and relative organ weights in the animals of the recovery phase.
-Macroscopic observations
Final and recovery sacrifice: No relevant macroscopic alterations that could be considered treatment-related were noted in animals sacrificed at the end of the treatment and recovery period. The lesions observed (multiple dark areas of the stomach, enlarged ovaries or distended uterus) were considered spontaneous and/or incidental, commonly observed in animals of this species and age under our experimental conditions.
-Microscopic observations
Final sacrifice: Treatment-related changes were noted in male animals of the high dose group (Group 4) receiving 40 mg/kg/day body weight of Basic Blue 159. These alterations consisted in a moderate to marked increase in hyaline droplets accumulation in kidneys (consisting in eosinophilic homogeneous cytoplasmatic droplets, that could represent low molecular weight protein accumulation within lysosomes, as a result of either increased filtered protein
loads or decreased catabolism), observed in males of Group 4, when compared to males treated at 20 mg/kg/day body weight or with the vehicle. All male animals of the high dose group showed a minimal to mild multifocal centrilobular hepatocellular hypertrophy, that could be considered as adaptative changes associated to a microsomal enzyme induction, deriving from the exposure to the test item.
The remaining lesions observed in control and treated animals were considered spontaneous and/or incidental, commonly seen in animals of this species and age under our experimental conditions.
Recovery sacrifice:The pathological changes involving the liver showed a complete reversibility. The hyaline droplets observed in the kidneys of the high dose group males were comparable to those seen in the kidneys of the control animals.
Key result
Dose descriptor:
NOAEL
Effect level:
40 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
Conclusions:
Based on these results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 40 mg/kg/day in male/female rats (oral) for Basic Blue 159 trichlorozincate.
Executive summary:

The toxicity of the test item Basic Blue 159 in rats, following daily oral administration for 4 consecutive weeks and recovery from any treatment-related effects during a period of 2 weeks, were investigated in this study.

Three groups, each of 5 male and 5 female Sprague Dawley rats, received the test item by gavage at dosages of 10, 20 and 40 mg/kg body weight/day. A fourth similarly constituted group received the vehicle alone (water) and acted as a control. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli, assessment of grip strength and motor activity, body weight, food consumption,

clinical pathology investigations, terminal body weight, organ weights, macroscopic observations and histopathological examination.

One male animal of the control group was found dead on Day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.

No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period. No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods. No differences between treated animals and controls which could be considered treatment related were observed at functional tests (sensory reactivity, landing footsplay, grip strength) and motor activity measurements performed at the end of treatment and recovery periods.

Very slight reductions in body weight were noted in animals of the treated groups when compared to controls. These decrements were statistically significant on Day 22 for males of Group 4, on Days 22 and 29 for females of Groups 2 and 3 and on Day 8 up to the end of the treatment and recovery periods for females of Group 4. No changes in food consumption were noted.

No treatment-related changes were recorded in haematology parameters. Alterations of some biochemical parameters observed during the treatment or recovery phase were considered unrelated to the treatment or, due to their slight entity, they were not deemed to be suggestive of tissue/organ injury or adverse.

No alterations of toxicological relevance were observed on terminal body weights. Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls. No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase.

No treatment-related changes were noted during necropsy. Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition, multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.

The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery period.

On the basis of the above results, it can be concluded that the Basic Blue 159 histopathological alterations observed in kidneys and liver of male animals treated at the high dose level (40 mg/kg body weight/day) were adaptive changes and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day could be considered the No Observed Adverse Effect Level (NOAEL) in this study.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Start of experimental phase (allocation of the animals): 30 May 2017 End of experimental phase (last day of necropsy) :17 July 2017 Study completion: Study Director's signature
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Basic Blue 159 trichlorozincate / CAS 93783-70-1 / EC 298-265-2
Target: Basic Blue 159 methyl sulfate / CAS 83969-12-4 / EC 281-589-3

3. ANALOGUE APPROACH JUSTIFICATION
see attached justification document in section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
minimum of 4 consecutive weeks
Frequency of treatment:
All animals were dosed once a day, 7 days a week.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group comprised 5 male and 5 female rats.
Control animals:
yes, concurrent vehicle
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal of the control group was found dead on Day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Very slight reductions in body weight were noted in animals of the treated groups when compared to controls. These decrements were statistically significant on Day 22 for males of Group 4, on Days 22 and 29 for females of Groups 2 and 3 and on Day 8 up to the end of the treatment and recovery periods for females of Group 4.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in
relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls.
No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition,
multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.
The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery
period.
Details on results:
- Mortality
One male animal of the control group (no. A2787010) was found dead on Day 23 of the study. No clinical signs were observed before death. The macroscopic findings observed at post mortem examination (red fluid content of thoracic cavity, incomplete collapse and dark colour of lungs and red staining of muzzle) and the main changes noted at the histopathological evaluation (moderate alveolar haemorrhage, minimal oedema of lungs and minimal myocardial chronic inflammation) suggested that the cause of death was likely due to a mis-dosing.
- Clinical signs
No clinical signs were noted in animals of the control and treated groups, except for two animals of the high dose group (Group 4) that showed rales on Days 19-20 (animal no. 52) and from Day 21 to 29 (animal no. 58). One animal of the same group (no. A2787056) showed ataxia and dyspnoea between Days 12 and 17 of the dosing phase.
Rales continued to be noted in one animal (no. 58) during the first three days of the recovery period. This sign was no longer apparent fromDay 4 up to the end of the observation period. No clinical signs were recorded in control animals and in the other animals of Group 4.
- Weekly detailed clinical signs (removal from cage and open field measurements)
No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity.
- Neurotoxicity assessment (Functional Tests and Motor activity)
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
- Body weight
From Day 8 to Day 29 of the dosing period, a very slight reduction in body weight (ranging from -8% to -4%), statistically significant only on Day 22, was recorded in males of the high
dose group. No remarkable differences in body weights were noted during the recovery period, when compared to controls. A very slight, but statistically significant, decrease in body weight (included in a range of -12% to -7%) was noted in female animals of the low and mid-dose groups, on Days 22 and 29 of the treatment period, when compared to females of the control group. A very slight and statistically significant, decrease in body weight (ranging from -8% to -6%) was also recorded in females of the high dose group from Day 8 up to the end of the treatment period.
Body weights remained lower than controls also during the recovery period, with a slight, but statistically significant, reduction in body weight of -11% and -10% on Days 8 and 15, respectively.
- Food consumption
No differences in food consumption were recorded between treated and control animals during the dosing and recovery periods.
- Haematology
The statistically significant difference of leucocytes recorded between controls and males dosed at 10 mg/kg/day (37% below mean control data) was not dose-related, therefore it was considered to be incidental.
- Coagulation
No changes were recorded.
- Clinical chemistry
Dosing phase:Compared to animals of the control groups, statistically significant fluctuations of some biochemical parameters were recorded, as follows: decrease of alkaline phosphatase in males dosed at 20mg/kg/day (-21%), decrease of aspartate aminotransferase in males receiving 20 and 40 mg/kg/day (-23% and -20%, respectively), decrease of triglycerides in males dosed at 40 mg/kg/day (-45%) and increase of calcium in those receiving 20 mg/kg/day (+12%), decrease of aspartate aminotransferase in females dosed at 20 mg/kg/day (-14%), increase of glucose in those receiving 10 mg/kg/day (+19%) and 40 mg/kg/day (+26%) and decrease of chloride in females treated at 40 mg/kg/day (-3%). Some of these changes (alkaline phosphatase and calcium in males, aspartate aminotransferase in females) were not dose-related, therefore considered unrelated to treatment. The other findings were not considered to be suggestive of tissue/organ injury, due to their slight severity.
Recovery phase: Cholesterol and chloride showed a complete reversibility in females. In male animals triglycrides were lower than controls (-33%), although this change was not considered to be adverse.The other statistically significant changes recorded (sodium in males, urea, triglycerides and potassium in females) were considered incidental, since not observed during the dosing phase.
- Terminal body weight and organ weights
Final sacrifice: No differences of toxicological relevance were observed on terminal body weight of treated animals, when compared to controls. Male animals of mid- (Group 3, 20 mg/kg/day body weight) and high (Group 4, 40 mg/kg/day body weight) dose groups showed a statistically significant increase in absolute and relative mean liver weights (approximately 14% and 22%, respectively), when compared to controls. In addition, a statistically significant increase in relative liver weight was recorded in females of the high dose group (approximately 15%) and in kidneys relative weight (approximately 13%) of the same group, when compared to animals of the control group.
No other remarkable changes in absolute and relative organ weights were noted in the animals of the other treated groups, when compared to the control data.
Recovery sacrifice: No changes of toxicological relevance were observed on terminal body weight, absolute and relative organ weights in the animals of the recovery phase.
-Macroscopic observations
Final and recovery sacrifice: No relevant macroscopic alterations that could be considered treatment-related were noted in animals sacrificed at the end of the treatment and recovery period. The lesions observed (multiple dark areas of the stomach, enlarged ovaries or distended uterus) were considered spontaneous and/or incidental, commonly observed in animals of this species and age under our experimental conditions.
-Microscopic observations
Final sacrifice: Treatment-related changes were noted in male animals of the high dose group (Group 4) receiving 40 mg/kg/day body weight of Basic Blue 159. These alterations consisted in a moderate to marked increase in hyaline droplets accumulation in kidneys (consisting in eosinophilic homogeneous cytoplasmatic droplets, that could represent low molecular weight protein accumulation within lysosomes, as a result of either increased filtered protein
loads or decreased catabolism), observed in males of Group 4, when compared to males treated at 20 mg/kg/day body weight or with the vehicle. All male animals of the high dose group showed a minimal to mild multifocal centrilobular hepatocellular hypertrophy, that could be considered as adaptative changes associated to a microsomal enzyme induction, deriving from the exposure to the test item.
The remaining lesions observed in control and treated animals were considered spontaneous and/or incidental, commonly seen in animals of this species and age under our experimental conditions.
Recovery sacrifice:The pathological changes involving the liver showed a complete reversibility. The hyaline droplets observed in the kidneys of the high dose group males were comparable to those seen in the kidneys of the control animals.
Key result
Dose descriptor:
NOAEL
Effect level:
40 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
Conclusions:
Based on these results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 40 mg/kg/day in male/female rats
Executive summary:

The toxicity of the test item Basic Blue 159 in rats, following daily oral administration for 4 consecutive weeks and recovery from any treatment-related effects during a period of 2 weeks, were investigated in this study.

Three groups, each of 5 male and 5 female Sprague Dawley rats, received the test item by gavage at dosages of 10, 20 and 40 mg/kg body weight/day. A fourth similarly constituted group received the vehicle alone (water) and acted as a control. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli, assessment of grip strength and motor activity, body weight, food consumption,

clinical pathology investigations, terminal body weight, organ weights, macroscopic observations and histopathological examination.

One male animal of the control group was found dead on Day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.

No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period. No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods. No differences between treated animals and controls which could be considered treatment related were observed at functional tests (sensory reactivity, landing footsplay, grip strength) and motor activity measurements performed at the end of treatment and recovery periods.

Very slight reductions in body weight were noted in animals of the treated groups when compared to controls. These decrements were statistically significant on Day 22 for males of Group 4, on Days 22 and 29 for females of Groups 2 and 3 and on Day 8 up to the end of the treatment and recovery periods for females of Group 4. No changes in food consumption were noted.

No treatment-related changes were recorded in haematology parameters. Alterations of some biochemical parameters observed during the treatment or recovery phase were considered unrelated to the treatment or, due to their slight entity, they were not deemed to be suggestive of tissue/organ injury or adverse.

No alterations of toxicological relevance were observed on terminal body weights. Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls. No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase.

No treatment-related changes were noted during necropsy. Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition, multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.

The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery period.

On the basis of the above results, it can be concluded that the Basic Blue 159 histopathological alterations observed in kidneys and liver of male animals treated at the high dose level (40 mg/kg body weight/day) were adaptive changes and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day could be considered the No Observed Adverse Effect Level (NOAEL) in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

The toxicity of the test item Basic Blue 159 in rats, following daily oral administration for 4 consecutive weeks and recovery from any treatment-related effects during a period of 2 weeks, were investigated in this study.

Three groups, each of 5 male and 5 female Sprague Dawley rats, received the test item by gavage at dosages of 10, 20 and 40 mg/kg body weight/day. A fourth similarly constituted group received the vehicle alone (water) and acted as a control. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli, assessment of grip strength and motor activity, body weight, food consumption,

clinical pathology investigations, terminal body weight, organ weights, macroscopic observations and histopathological examination.

One male animal of the control group was found dead on Day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.

No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period. No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods. No differences between treated animals and controls which could be considered treatment related were observed at functional tests (sensory reactivity, landing footsplay, grip strength) and motor activity measurements performed at the end of treatment and recovery periods.

Very slight reductions in body weight were noted in animals of the treated groups when compared to controls. These decrements were statistically significant on Day 22 for males of Group 4, on Days 22 and 29 for females of Groups 2 and 3 and on Day 8 up to the end of the treatment and recovery periods for females of Group 4. No changes in food consumption were noted.

No treatment-related changes were recorded in haematology parameters. Alterations of some biochemical parameters observed during the treatment or recovery phase were considered unrelated to the treatment or, due to their slight entity, they were not deemed to be suggestive of tissue/organ injury or adverse.

No alterations of toxicological relevance were observed on terminal body weights. Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls. No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase.

No treatment-related changes were noted during necropsy. Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition, multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.

The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery period.

On the basis of the above results, it can be concluded that the Basic Blue 159 alterations observed in kidneys and liver of animals treated at the high dose level (40 mg/kg body weight/day) were adaptive changes and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day is considered to be the No Observed Adverse Effect Level (NOAEL) in this study.

Justification for classification or non-classification

No alterations of toxicological relevance were observed in this study.

Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls. No remarkable changes in terminal body weight and in absolute/relative organs weight were noted in animals of the recovery phase. Histopathologically, in male animals of Group 4, moderate to marked hyaline droplets accumulation in kidneys and multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed, which showed complete reversibility at the end of the recovery period.

These findings were considered to be adaptive changes due to and increased metabolisation of the dye and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day is considered to be the No Observed Adverse Effect Level (NOAEL) in this study. Based on this NOAEL, the test substance has to be classified as STOT RE 2 (H373) in accordance with CLP regulation (EC) No 1272/2008.