m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

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:

Experimental Starting Date: 2 December 2016. Experimental Completion Date: 29 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

EC Number:

275-602-1

EC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Cas Number:

71550-21-5

Molecular formula:

C27H24N6O9S2.2C4H11NO2

IUPAC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Test material form:

liquid

Specific details on test material used for the study:

Identification: Bayscript Yellow GGN
Physical State/Appearance: Russet coloured crystalline solid
CAS Number : 71550-21-5
Storage Conditions: At ambient temperature (15 to 25ºC)
Expiry Date: 22 June 2018

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River (UK) Ltd.
Number of animals: 25 males and 25 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: 12 days before commencement of treatment.
Age of the animals at start of treatment 47 to 54 days.
Weight range of animals at the start of treatment
Males: 240 to 292 g.
Females: 167 to 207 g.

Allocation and Identification
Allocation: Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment commenced: Body weight range extremes - Five males and five females.


Animal Care and Husbandry
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex.
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet: Teklad 2014C Diet.
Availability: Non-restricted (removed overnight before blood sampling for hematology/blood chemistry and during the period of urine collection).

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (removed overnight before blood sampling for hematology/blood chemistry and during the period of urine collection).

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen as an appropriate route to conduct a human risk assessment.

Vehicle:

water

Details on oral exposure:

The dose levels selected for investigation in this OECD 407 28-day repeat dose toxicity study (0, 10, 30 and 70 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a preliminary study conducted at the same laboratories (Envigo Study No. KX37FJ). In that study, dose levels of 25, 50, 100 and 500 mg/kg/day were investigated.
Treatment at 500 mg/kg/day was not tolerated. One female was found dead prior to dose administration on Day 4 of study and the remaining animals in the group were killed for reasons of animal welfare on Day 4 or 5 of study due to a marked decline in clinical condition, weight loss and low food intake. Macroscopic examination revealed increased kidney weights, pale kidneys, yellow discolouration of the majority of internal organs and for 3/6 animals the liver was firm and/or pale.
Although there were no premature deaths, the dose level of 100 mg/kg/day was considered to be slightly above the maximum tolerated dose. General clinical condition was not clearly affected, but sporadic incidences of hunched posture, piloerection and partially closed eyes were observed post-dose. Body weight performance was poor, with 3/6 animals recording overall body weight loss during the 21-day dosing period, and food consumption was persistently low. At scheduled termination, all animals showed markedly increased kidney weights and all kidneys were pale.
At 25 or 50 mg/kg/day, there were no test item-related changes in clinical condition or post dosing signs. Some short periods of minor weight loss were apparent in individual animals during the course of the dosing period, however with the exception of one female in the 50 mg/kg/day group, all animals recorded overall body weight gain during the course of the study. Food consumption was slightly low at 50 mg/kg/day but was not clearly affected by treatment at 25 mg/kg/day. Increased kidneys weights were evident at 50 mg/kg/day, and all animals at 50 mg/kg/day and one female at 25 mg/kg/day showed pale kidneys at scheduled termination.
Based on the results of the preliminary study at 50 and 100 mg/kg/day, the high dose level for the current OECD 407 study was set at 70 mg/kg/day. The intermediate and low dose levels of 30 and 10 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the concentration range 1 to 200 mg/mL were stable following ambient storage (15 to 25°C) for one day and following refrigerated storage (2 to 8°C) for 15 days.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analyzed for achieved concentration of the test item.


Homogeneity and stability was confirmed for Bayscript Yellow GGN in purified water formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. Discrete sampling was confirmed at refrigerated storage for up to 15 days.
The mean concentrations of Bayscript Yellow GGN in test formulations analyzed for the study were within 6% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

70 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females at 10 mg/kg bw/day
5 males and 5 females at 30 mg/kg bw/day
5 males and 5 females at 70 mg/kg be/day

Control animals:

yes, concurrent vehicle

Details on study design:

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose : 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency: Once daily at approximately the same time each day.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Examinations

Observations and examinations performed and frequency:

Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
During the treatment period, detailed observations were recorded at the following times in relation to dose administration:

Daily during Week 1:
Pre-dose observation
At the end of dosing each group
One to two hours after completion of dosing of all groups.
As late as possible in the working day

Twice weekly from Week 2 to termination:
Pre-dose observation
One to two hours after completion of dosing of all groups.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment (mid-late week), detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer who was unaware of the treatment group to which each animal belonged.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Motor Activity
During Week 4 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded twice in the week before treatment commenced, on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded twice weekly for the week before treatment started and for each week throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and water at the following occasion:
Day 29: All animals

Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and water at the following occasion:
Day 29: All animals

Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis
Animals were placed in an individual metabolism cage, without food or water, for overnight urine collection at the following occasion:
Day 29: All animals

The individual samples were examined for the following characteristics:
Using manual methods:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
Ketones (Keto)
Bile pigments (Bili)
Blood pigments (UBld)

Using a Roche P Modular Analyzer:
Protein (T-Prot)
Glucose (T-Gluc)
Creatinine (T-Creat)

Biomarkers
Blood samples were collected after overnight withdrawal of food and water on the following occasion:
Day 29: All animals

Sampling procedures occurred in the morning (AM) and were completed within a period of two hours (for all groups).
Blood sample site: Sublingual vein.
Anesthetic: Isoflurane.
Anticoagulant: EDTA. Microtainers used for collection of samples did not contain separator gel.
Blood volume: 0.5 mL.
Treatment of samples: Samples were kept on wet ice prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at nominally 2 to 8°C within 30 minutes of collection.
Separation and storage of plasma: The resultant plasma was divided into two separate aliquots:
100 µL (0.1 mL) was transferred to an appropriately labeled plastic tube and was retained for potential analysis of T3 and T4.
All remaining plasma was transferred to an appropriately labeled plastic tube and was retained for potential analysis of TSH.
Final storage conditions: All resultant plasma was subsequently frozen on dry ice prior to being transferred to deep frozen storage conditions ( 60 to 80°C).
Fate of plasma samples: All samples (and/or residual samples if analysis was required) will be disposed of upon finalization of the study.

Sacrifice and pathology:

Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy
All study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: Animals were killed following four weeks (28 days) of treatment.
Sequence: To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as displayed in the table in section 'any other information on materials and methods'

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at the scheduled interval.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:

Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Terminal animals of Groups 1 and 4.
Kidneys and abnormalities only: Terminal animals of Groups 2 and 3.
Routine staining: Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:

Scheduled kill:
All animals of Groups 1 and 4 - All specified in the table in section 'any other information on materials and methods'.
All animals of Groups 2 and 3 - Kidneys and Abnormalities only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following sequence of statistical tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The administration of Bayscript Yellow GGN for four weeks at dose levels up to and including 70 mg/kg/day was well tolerated. There no signs observed in relation to dose administration and no test item-related changes in clinical condition.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight performance of males and females given Bayscript Yellow GGN at dose levels up to and including 70 mg/kg/day was considered unaffected by treatment.
All animals recorded overall body weight gain from Day 1 to 25 of treatment with some transient periods of minor weight loss apparent in individual animals during this period. With the exception of a single males it the 10 mg/kg/day group (Number 17) and a single female in the 30 mg/kg/day group (Number 39), body weight loss was apparent between Day 25 and Day 29 of treatment for all animals, including Controls; this was due to the Day 29 bodyweight being recorded after overnight deprivation of food and water for blood/urine collection, and no effect of treatment was inferred.
Overall body weight gain for all groups of treated males was slightly lower than Control but with no dose trend apparent and statistical significance was not attained at any dose level; differences were considered incidental and unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption remained essentially similar to pretreatment and concurrent Control values for males and females in all treated groups throughout the dosing period and was considered unaffected by treatment.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

On four occasions (Day 12 of study, Group 4 males only; Days 13, 14 and 27 of study,
Group 4 males and females), the visual assessment of water consumption indicated that animals given 70 mg/kg/day had consumed more water than Controls.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of hematological parameters following four weeks of treatment with Bayscript Yellow GGN did not reveal any adverse effects. All inter-group differences from controls, including those which attained statistical significance, were minor and attributed to normal biological variation.
When compared to Controls, a statistically significant dose-dependent decrease in red cell distribution width was apparent for males given 30 mg/kg/day and for both sexes given 70 mg/kg/day. This decrease in red cell distribution width, indicative of less variability in cell size in the treated animals compared to Controls, occurred as a consequence of slight and non-statistically significant reductions in the concentration of reticulocytes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis of plasma after 4 weeks of treatment revealed, when compared to Controls, the following differences which attained statistical significance: increased aspartate amino-transferase activity in both sexes given 70 mg/kg/day and alkaline phosphatase activity in males given 30 or 70 mg/kg/day; increased urea and creatinine concentrations in both sexes at 70 mg/kg/day; low glucose concentrations in males at 70 mg/kg/day; low cholesterol concentrations in all groups of treated males, although in the absence of a dose response relationship; slightly increased triglyceride concentrations in both sexes given 70 mg/kg/day (statistical significance attained for females only).
Assessment of electrolytes indicated a decrease in potassium concentrations in males given 30 mg/kg/day and in both sexes at 70 mg/kg/day, increased phosphorus concentrations for both sexes given 70 mg/kg/day, and slightly low chloride concentrations and slightly increased calcium concentrations for males at 70 mg/kg/day; all of these differences from Control attained statistical significance.
Total protein concentrations were statistically significantly increased among animals given
70 mg/kg/day when compared to Controls, with albumin concentrations also increased in females given 30 or 70 mg/kg/day.
All other inter-group differences from Controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis investigations after 4 weeks of treatment revealed, when compared to Control, markedly high urine output in males and females receiving 70 mg/kg/day, with an associated low urinary pH and specific gravity; these differences from Control attained statistical significance. Males given 70 mg/kg/day showed markedly low total glucose output and females given 70 mg/kg/day showed increased total protein output. In addition, two males in the 70 mg/kg/day group showed a large amount of blood pigments in the urine.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females receiving Bayscript Yellow GGN at 30 or 70 mg/kg/day showed slightly high body weight adjusted mean adrenal and kidney weights compared to Control; with the exception of males given 30 mg/kg/day these differences from Control attained statistical significance, however there was no dose response apparent among females. Females receiving 30 or 70 mg/kg/day showed statistically significantly low body weight adjusted spleen weights compared to Control, although in the absence of a dose response.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 29 of study, pale areas were seen in the kidneys of one male receiving 30 mg/kg/day and one male receiving 70 mg/kg/day. In addition, the liver of two males receiving 70 mg/kg/day was pale.
All other findings were isolated and showed no clear association with treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment Related Findings
Changes related to treatment with Bayscript Yellow GGN were seen in the kidneys.
There was an increase in the incidence and severity of tubular/collecting duct basophilia in the kidneys of males and females given 70 mg/kg/day. In some animals, this was associated with tubular dilatation. In all males given 70 mg/kg/day, intratubular crystals were present predominantly in the renal medulla/papilla. The presence of crystals was associated with neutrophilic inflammation in several animals.
Summary of treatment related findings in the kidneys for animals killed after 4 weeks of treatment is given in the table in section 'any other information on results'

Incidental Findings
Focal hepatic necrosis was present in one male given 70 mg/kg/day. The remaining liver tissue was unaffected. This changed was likely due to strangulation of a portion of the liver and was not considered to be related to treatment.
All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sensory Reactivity and Grip Strength
Sensory reactivity observations and grip strength values of all groups of animals receiving Bayscript Yellow GGN were similar to those for Controls, and considered unaffected by treatment.

Motor Activity
There was considered to be no adverse effect of treatment with Bayscript Yellow GGN on locomotor activity.
Males in the 70 mg/kg/day group showed a trend towards marginally low group mean high and low beam breaks throughout the majority of the 1-hour recording period compared to Controls. Statistical significance was achieved for the difference at the 30-minute interval low beam break score. In addition, the low beam break score at the 12-minute and 30-minute interval, the high beam break score at the 12-minute and 18-minute interval and the total high beam break score were below the Historical Control Data (HCD) range. However, since all other high and low beam break scores were within the HCD range, no statistical significances were attained for any of the high beam scores (the most sensitive to test item-related effects) and as group mean activity scores for all groups of treated females were unaffected, the marginally low scores recorded among males given 70 mg/kg/day were considered unlikely to be of any toxicological significance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

70 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Summary of treatment related findings in the kidneys for animals killed after 4 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	10	30	70	0	10	30	70
Basophilia, Tubules/Collecting Ducts
Minimal	0	0	0	2	0	0	0	1
Slight	0	0	0	2	0	0	0	1
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	5	0	0	0	2
Crystals, Intratubular
Minimal	0	0	0	4	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	5	0	0	0	0
Dilatation, Tubular
Minimal	0	0	0	3	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	4	0	0	0	2
Inflammation
Minimal	0	0	0	1	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	2	0	0	0	0
Number of tissues examined	5	5	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

In conclusion, oral (gavage) administration of Bayscript Yellow GGN at a dose level of 70 mg/kg/day was associated with adverse pathological changes in the kidneys of males and females, consisting of tubular basophilia, tubular dilatation, and in males, additionally tubular crystal formation and inflammation. There were associated changes in kidney weights and in plasma biochemical and urinary markers of renal function. Similar treatment-related changes were not observed in animals given 10 or 30 mg/kg/day, therefore within the context of this study the No Observed Adverse Effect Level was concluded to be 30 mg/kg/day.

Executive summary:

Summary

The purpose of this study was to assess the systemic toxic potential of Bayscript Yellow GGN (an industrial colourant) when administered for four weeks by oral gavage to CD rats. The study was conducted to fulfil the data requirement according to REACH regulation Annex VIII chapter 8.6.1.

Three groups, each comprising five male and five female Crl:CD(SD) rats, received Bayscript Yellow GGN at doses of 10, 30 or 70 mg/kg/day at a volume dose of 5 mL/kg body weight. A similarly constituted control group received the vehicle, purified water, at the same volume dose over the same period. 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual assessment), hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

At study termination, blood samples were taken from all animals with the resultant plasma stored at -60°C to ‑80°C pending possible future analysis of the biomarkers T3, T4 and TSH.

Results

Treatment with Bayscript Yellow GGN at doses up to and including 70 mg/kg/day was well tolerated; there were no mortalities and no treatment-related clinical signs observed. Neurobehavioural assessments conducted during Week 4 of treatment did not reveal any adverse effects on locomotor activity, sensory reactivity or grip strength at any dose level investigated.

Mean body weight performance and mean food consumption were unaffected by treatment in all groups of males and females. A daily visual assessment of water consumption revealed occasional periods of increased water intake in animals given 70 mg/kg/day.

The haematological investigation conducted after four weeks of treatment revealed no treatment-related changes. Test article-related biochemical changes in the plasma comprised increased aspartate amino-transferase activity in both sexes given 70 mg/kg/day and alkaline phosphatase activity in males given 30 or 70 mg/kg/day, elevated urea and creatinine concentrations in both sexes at 70 mg/kg/day, reduced glucose concentrations in males at 70 mg/kg/day, low cholesterol concentrations in all groups of treated males, and slightly increased triglyceride concentrations in both sexes given 70 mg/kg/day. In addition, decreased potassium concentrations in males given 30 mg/kg/day and in both sexes given 70 mg/kg/day, increased phosphorus concentrations for both sexes given 70 mg/kg/day, and slightly low chloride concentrations and slightly increased calcium concentrations for males at 70 mg/kg/day were also apparent. Increased total protein concentrations were observed among animals given 70 mg/kg/day, with albumin concentrations also increased in females given 30 or 70 mg/kg/day.

Analysis of urine collected after 4 weeks of treatment revealed, when compared to Control, markedly high urine output in males and females receiving 70 mg/kg/day, with an associated low urinary pH and specific gravity; males given 70 mg/kg/day showed markedly low total glucose output and females given 70 mg/kg/day showed increased total protein output. In addition, two males in the 70 mg/kg/day group showed a large amount of blood pigments in the urine.

The analysis of organ weights following 4 weeks of treatment indicated slightly high body weight-adjusted mean adrenal and kidney weights compared to Control for both sexes given 30 mg/kg/day or above. Females receiving 30 or 70 mg/kg/day showed statistically significantly low body weight adjusted spleen weights compared to Control, although in the absence of a dose response.

Test item-related abnormalities detected at macroscopic examination were limited to pale areas in the kidneys of one male receiving 30 mg/kg/day and of one male receiving 70 mg/kg/day, and pale liver in two males receiving 70 mg/kg/day.

Histopathological changes that were attributable to treatment occurred in the kidneys of animals given 70 mg/kg/day, where an increase in the incidence and severity of tubular/collecting duct basophilia was observed. In some animals, this was associated with tubular dilatation. In all males given 70 mg/kg/day, intratubular crystals were present predominantly in the renal medulla/papilla. The presence of crystals was associated with neutrophilic inflammation in several animals. 

Conclusion

In conclusion, oral (gavage) administration of Bayscript Yellow GGN at a dose level of 70 mg/kg/day was associated with adverse pathological changes in the kidneys of males and females, consisting of tubular basophilia, tubular dilatation, and in males, additionally tubular crystal formation and inflammation. There were associated changes in kidney weights and in plasma biochemical and urinary markers of renal function. Similar treatment-related changes were not observed in animals given 10 or 30 mg/kg/day, therefore within the context of this study the No Observed Adverse Effect Level was concluded to be 30 mg/kg/day.