Registration Dossier

Administrative data

Description of key information

The main structure of the read-across substance is the same of the CJ304, only the salt form is different. However, this difference does not influence the toxicity of the CJ304. CJ304 contains Li/Na salt form and the read-across substances contains only Na salt. This will not lead to difference on the toxicity results. Based on the results obtained from a  OECD  guideline 422 study, performed  in accordance to GLP, NOAEL (No Observed Adverse Effect Level) for males and females was established at 1000 mg/kg body weight/day.

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The main structure of the read-across substance is the same of the CJ304, only the salt form is different. However, this difference does not influence the toxicity of the CJ304. CJ304 contains Li/Na salt form and the read-across substances contains only Na salt. This will not lead to difference on the toxicity results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test substance: FAT 41001-H TE
Test substance identity: NOVACRON BLUE P-3R (Reactive Blue 049)
CAS number: 72214-18-7
EC number: 276-481-8
Intended use: Textile dye
Appearance: A solid powder
Storage conditions: Frozen (nominally -20 degree C)
Batch number: BOP 01-12 (Lot: BS-1106872)
Expiry date: 31 January 2017
Purity: 77.3%
Purity/weighing factor: 1.29
pH: 7.2
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Animal supply, acclimatization and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Harlan (UK) Ltd.
Number of animals: 44 males and 44 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Five days before commencement of treatment.

Age of animals at the start of the study
Males: 69 to 75 days old
Females: 62 to 68 days old

Weight range of animals at the start of the study:
Males: 257 to 289 g
Females: 159 to 189 g

Allocation and identification
Allocation: On arrival and non-selective allocation to cages.
On Day 1 of study all animals were weighed, and body weights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed ± 20% of the mean for each sex.

Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

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 occupant(s).

Animal housing, diet and water supply
Environmental control
Rodent facility: Full barrier - 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 19 - 23ºC 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:
Cages comprised of a polycarbonate body with a stainless-steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding: Solid bottom cages contained softwood-based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

Diet supply
Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.
Route of administration:
oral: gavage
Details on route of administration:
The oral gavage route of administration was chosen to simulate a condition of potential human exposure.
Vehicle:
water
Remarks:
Purified water
Details on oral exposure:
Correction factor: A correction factor of 1.29 was used when calculating quantities of test substance used during dose preparation (including purity and ratio salt/base).
Vehicle: Purified water.
Method of preparation: Sufficient vehicle was added to the test material to obtain a solution and it was mixed by hand using a spatula. It was mixed using a magnetic stirrer to dissolve the test material. It was made up to volume with the remaining vehicle and mixed with a magnetic stirrer.
A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Ambient temperature (nominally +21ºC) for two days or refrigerated (nominally +4ºC) for 15 days.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation analysis
Stability and homogeneity: The stability of a solution of the test substance in the vehicle at concentrations of 10 mg/mL and 100 mg/mL was demonstrated over a period of 15 days at nominally +4ºC and two days at nominally +21ºC.
Achieved concentration: Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Administration
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
Volume dose: 10 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.
Sequence: Groups dosed in ascending order.
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.
Storage of formulation: The stability was confirmed following storage at ambient temperature (nominally 21ºC) for up to two days, and refrigeration (nominally 2-8ºC) for up to 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical procedure
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places.
General laboratory apparatus and glassware.

Reagents
Control vehicle: Purified water
Acetonitrile (ACN): Gradient HPLC grade
N,N-Dimethylformamide (DMF): HPLC grade
Tetra-n-Butylammonium bromide: Reagent plus, >99.0%
Water: Reverse osmosis
Diluent: DMF/water 20/80 v/v

Preparation of standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of FAT 41001-H TE in diluent (50 mL). A secondary standard solution (250 μg/mL) was prepared by diluting primary standard solution with diluent (20 mL). Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard solution using diluent and contained FAT 41001-H TE at nominal concentrations of 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL and 25 μg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Sample process
A representative sample of test formulation (1 mL, accurately weighed (accurate volume for stability trial)) was diluted using diluent to provide a solution containing FAT 41001-H TE at an expected concentration within the range 10 μg/mL to 20 μg/mL.
The concentration of FAT 41001-H TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: Hypersil BDS, C18, 5 μm 4.6 × 250 mm
Column temperature: 40°C
Sample temperature: 10°C
Mobile Phase A: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 35/65 v/v
Mobile Phase B: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 90/10 v/v

Linear Gradient:
Time (min.) % A % B
0 100 0
1.0 100 0
20.0 40 60
20.5 100 0
23.0 100 0

Flow rate: 1.2 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 10 μL
Run time: 23 minutes
Approximate retention time:
FAT40061-F TE peak 1: 15.1 minutes
FAT40061-F TE peak 2: 15.3 minutes

Calculation
The peak area response for FAT 41001-H TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The concentration of FAT 41001-H TE was determined using the following equation:

Analysed concentration (mg/mL) = ((Y – I) / S) x (V / 1000)

Where
Y = Peak area response for FAT 41001-H TE in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution factor of sample

Validation of the analytical procedure
The analytical procedure was validated by determining the following parameters: The specificity of the chromatographic analysis in control sample chromatograms. The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range. The repeatability of the lowest and highest concentration calibration standards. The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 10 mg/mL and 100 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (purified water) with known amounts of FAT 41001-H TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Stability in purified water formulations
The homogeneity and stability of FAT 41001-H TE in purified water formulations was assessed at nominal concentrations of 10 mg/mL and 100 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (100 mL) were equally sub-divided (2 × 50 mL) into two amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21ºC)
On receipt, the contents of one bottle of each formulation was mixed by 20-fold inversion (representing 0 hour) and 2 hours, duplicate samples (1 mL accurate volume) were removed for analysis from the middle of the formulation.
The remainder of the bottle was stored at ambient temperature and after 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4ºC)
The remaining bottle was refrigerated on receipt and on Day 2 and Day 15, the bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion and single samples (1 mL accurate volume) were removed for analysis from the middle of the formulation.

Concentration in test formulations
For the First and Last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, 2 × 3 mL for controls accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Results
Method validation
The analytical procedure was successfully validated for FAT 41001-H TE in purified water with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. Results are summarised below:

The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for FAT 41001-H TE in the control sample
chromatogram.
The limit of detection and quantification was estimated as 0.0225 μg/mL and 0.0751 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 5 μg/mL to 25 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 41001-H TE at nominal concentrations of 5 μg/mL and 25 μg/mL
;
Method accuracy and precision were confirmed: a mean procedural recovery value of 101.2% (CV=0.82%, n=6) was obtained for 10 mg/mL and 99.9% (CV=1.31%, n=6) was obtained for 100 mg/mL.

Formulation trial
The stability of FAT 41001-H TE in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 100 mg/mL. Stability was confirmed following storage at ambient temperature for 2 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the duplicate samples remained within 3% of the initial time zero value and difference from mean values were less than 2%.

Concentration in dose formulations
The mean concentrations of FAT 41001-H TE in test formulations analysed during the study and the deviation of the mean result from the nominal value are presented. The mean concentrations were within applied limits ±10%, confirming the accuracy of formulation. Difference from mean values were within 5% confirming precise analysis.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The stability was confirmed for FAT 41001-H TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 41001-H TE in test formulations analysed for the study were within ±10% of nominal concentrations, confirming accurate formulation. Difference from mean values were within 5% confirming precise analysis.
Duration of treatment / exposure:
At least four weeks.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group I
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group II
Dose / conc.:
330 mg/kg bw/day (actual dose received)
Remarks:
Group III
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group IV
No. of animals per sex per dose:
10 / sex / dose
Control animals:
yes
Details on study design:
Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0055).

In this preliminary study, FAT 41001 H TE was well tolerated for 14 days at doses up to and including 1000 mg/kg/day. Overall body weight gain and food consumption were satisfactory and there was no evidence of toxicity during routine physical examinations or following dose administration. Kidney weights were marginally high in males given 500 or 1000 mg/kg/day and spleen weights were low amongst males given 1000 mg/kg/day. Macroscopic examination revealed blue or dark colouration in the kidneys, caecum, colon, ileum, jejunum, mesenteric lymph nodes, rectum, stomach and on the fur, tail or paws from the majority of animals which was attributed to the properties of FAT 41001-H TE, which is a blue dye.

Based on these results 1000 mg/kg/day was considered to be an appropriate high dose for this main OECD 422 study. A low and intermediate dose of 100 and 330 mg/kg/day, respectively, have been selected to investigate a dose response and with reference to the Globally Harmonized System of Classification and Labelling of Chemicals (2013).
Positive control:
None.
Sacrifice and pathology:
Terminal procedures
All adult 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. 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.

Time of necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 7 of lactation.
F1 offspring: Day 7 of age.

Method of kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring : Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows for F0 animals:

Females
The following were recorded:
Each uterine horn: Number of implantation sites.
Offspring: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

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: The five lowest numbered surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Kidneys, mesenteric and left axillary lymph nodes, stomach and epididymis: The five lowest numbered surviving F0 males and females in Groups 2 and 3 at scheduled termination.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
The epididymis of two Control males and two Group 4 males were also be stained with Periodic Acid Schiffs (PAS) in order to confirm results observed at the initial histological examination.

Light microscopy
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.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Clinical signs:
no effects observed
Description (incidence and severity):
Signs seen in association with the administration of FAT 41001-H TE were restricted to rales in one male receiving 330 mg/kg/day on Day 3 of treatment. This was observed from 1-2 hours after dosing up to the end of the working day but was no longer evident the following day.
At the detailed physical examination and arena observations, signs seen which were considered to be related to the administration of FAT 41001-H TE included blue staining of various body parts of all males receiving FAT 41001-H TE at 330 or 1000 mg/kg/day, all females receiving 1000 mg/kg/day, nine females receiving 330 mg/kg/day and four males receiving 100 mg/kg/day.
Irritable behaviour was observed in two females receiving 100 mg/kg/day. Vocalisation was evident in females across all groups (inclusive of one control female) and for one control males, however, vocalisation had been observed in a small number of females prior to the commencement of treatment.
Salivation was seen in one male and one female receiving 1000 mg/kg/day, however, this is a common sign observed with oral gavage dosing and is not considered to be a direct response to treatment with FAT 41001-H TE.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean body weight gain for males and females receiving FAT 41001-H TE at 100 mg/kg/day were similar to controls and were considered to be unaffected by treatment. The group mean weight gain of males receiving FAT 41001-H TE at 1000 mg/kg/day was low during Week 1 of treatment when compared with controls; thereafter until Week 4, the mean weight gains of these animals improved and were similar to the gains of the controls.
During Weeks 4-5, group mean weight gain was significantly lower in males receiving FAT 41001-H TE at 330 or 1000 mg/kg/day when compared with controls and subsequently overall weight gain (Weeks 0-5) appeared low in these animals. For females receiving FAT 41001-H TE at 300 or 1000 mg/kg/day, body weight gain during the two weeks of treatment prior to pairing was slightly low when compared with the control animals however these differences were minor and did not demonstrate a clear dose response.
Body weight gain during the gestation period (Days 0-20), for females receiving FAT 41001-H TE at 100, 330 or 1000 mg/kg/day, were similar to that of the Control and showed no effect of treatment.
For the period Days 1-4 of lactation, group mean body weight gain was low in females receiving 100 mg/kg/day, however, this was attributed to a large bodyweight loss (32g) in female No. 56 for this period, resulting in overall lower gains for the period Days 1-7 of lactation. Body weight gain during Days 1-4 of lactation was high at 330 or 1000 mg/kg/day but differences did not attain statistical significance, and were not dose-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment with FAT 41001-H TE on food consumption during the first two weeks of treatment (prior to pairing) in either sex. During treatment Week 4 males treated with FAT 41001-H TE at 100, 330, or 1000 mg/kg/day appeared to consume more diet than controls however these differences were minor and lacked dose relationship; food consumption was not recorded whilst animals were paired for mating (Week 3).

No effect of treatment on food consumption was observed in treated females, during the period of gestation (Days 0-19). Between Day 6 and Day 19 of gestation, the mean food consumption of all groups of treated females was slightly higher than in controls and all differences attained statistical significance except for food intake at 100 mg/kg/day during Days 13-19 of gestation.
No effect of treatment on food consumption was observed in females receiving 100 mg/kg/day, during the period of lactation (Days 1-7). In females receiving 330 or 1000 mg/kg/day food consumption was slightly higher throughout lactation, with the slight increase being dose dependent.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
No formal water consumption measurements/assessments were performed on this study however, on two occasions (at the water bottle change) a marked increase in water consumption was observed amongst animals receiving 1000 mg/kg/day when compared with Controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination during Week 2 of treatment, prior to pairing, and the examination of the clotting parameters in Week 4 of treatment for the males and on gestation Day 17 for the females, revealed no significant response to treatment with FAT 41001-H TE.
A small, but statistically significant increase in mean cell haemoglobin concentration was apparent in males at all dose levels when compared with Controls. A small but statistically significant shortening of the prothrombin time was apparent in males receiving 1000 mg/kg/day.
All other differences from control were minor, lacked dose relationship and were found to be within background control data ranges; therefore these changes were attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical examination of the blood plasma in Week 2 of treatment, prior to pairing revealed low bile acid concentrations amongst all treated groups of males and females when compared with the control animals. There was no dose response evident in the females with
the most pronounced effects being apparent in females receiving 100 or 330 mg/kg/day.
Cholesterol concentration was high in animals receiving 330 or 1000 mg/kg/day and creatinine concentration was high in females at all dose levels. Glucose and urea concentrations were low in males receiving 1000 mg/kg/day when compared with control animals, however, in the case of the urea concentration this was partially attributed to both a high value in one control male (Animal No. 23; plasma urea = 11.62 mmol/L) and a low value in one male receiving 1000 mg/kg/day (Animal No. 1; plasma urea = 5.66 mmol/L); as such these differences in urea concentration were not considered to be of toxicological significance.
High calcium concentration was evident in all treated male groups when compared with the control group; there was no similar effect in the females.
Other changes that attained statistical significance included the low plasma alkaline phosphatase activity in males receiving 1000 mg/kg/day and the low alanine aminotransferase activity in females at all dose levels. As low levels of enzyme activity are not considered to be biologically relevant, no toxicological significance is inferred.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity and grip strength assessments did not reveal any response to treatment with FAT 41001-H TE.

Motor activity assessments during Week 5 of treatment, did not reveal any response to treatment in male animals receiving FAT 41001-H TE. For females treated with FAT 41001-H TE, motor activity assessments between Day 4 and Day 6 of lactation revealed small inter- and intra-group differences in the activity scores.
This is not unusual for lactating females that have been separated from their pups and therefore these differences are considered to be a consequence of natural biological variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After five weeks of treatment, absolute and body weight adjusted kidney weights of males receiving 1000 mg/kg/day were marginally higher than Controls. Absolute testes weights were also high in these animals and in males receiving 330 mg/kg/day.

On Day 7 of lactation, absolute and body weight adjusted adrenal weights for all treated groups of females were low when compared with Controls, attaining statistical significance for adjusted adrenal weights for all dose levels. Absolute and body weight adjusted kidney weights of females receiving 1000 mg/kg/day, were higher than Controls, attaining statistical significance for adjusted kidney weights only.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination performed in males after five weeks of treatment and in females on Day 7 of lactation, did not reveal any treatment-related findings for animals receiving FAT 41001-H TE at 100 mg/kg/day.
In males receiving 330 or 1000 mg/kg/day, blue colouration (including blue colouration of the contents) of the rectum, ileum, caecum, colon, stomach, kidneys and mesenteric lymph nodes was evident with the incidence higher at 1000 mg/kg/day. Further blue coloration was evident at 1000 mg/kg/day in the skin, jejunum, lymph nodes (mandibular, pancreatic, inguinal, axillary and lumbar), testes and epididymides. In females receiving 330 or 1000 mg/kg/day, blue colouration (including blue coloration of the contents) of the rectum,
caecum, kidneys and colon was evident with the incidence higher at 1000 mg/kg/day.
Further blue colouration was evident at 1000 mg/kg/day in the skin (including pinnae), jejunum, ileum, mesenteric lymph nodes, uterus and uterine cervix, vagina, thymus, stomach and duodenum. These findings were considered to be due to the administration of the test article, FAT 41001-H TE, which is a blue dye. In addition, two females receiving 1000 mg/kg/day were observed to have a dark colon with abnormally dark contents; this is also considered to be in relation to the administration of the test article, FAT 41001-H TE.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For males killed after four weeks of treatment and females killed on Day 7 of lactation, changes related to administration with FAT 41001-H TE were seen in the kidneys, mesenteric and left axillary lymph nodes, stomach and epididymides:

Kidneys:
Vacuolation of the cortical tubules with granular deposits was seen in males and females given 330 or 1000 mg/kg/day. Increased severity of the vacuolation was recorded in the females when compared with the males given 1000 mg/kg/day. Hyaline droplets in the cortical tubules were recorded in the males given 330 or 1000 mg/kg/day.

Lymph nodes
Vacuolated macrophages were recorded in the majority of males and females given 1000 mg/kg/day.

Stomach
Foveolar hyperplasia was recorded in the stomach of three males and the majority of females given 1000 mg/kg/day.

Epididymides
Epithelial vacuolation was recorded in the epididymides of males given 1000 mg/kg/day. A PAS stain was carried out in two control males and two males treated with 1000 mg/kg/day. In the controls all clear cells stained strongly PAS positive, whereas in the treated animals only some of the clear cells stained PAS positive.

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
See results
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development
Critical effects observed:
no

Formulation analysis

The stability was confirmed for FAT 41001-H TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.  The mean concentrations of FAT 41001-H TE in test formulations analysed for the study were within ±10% of nominal concentrations, confirming accurate formulation. Differences from mean values were within 5% confirming precise analysis.

Histopathology

Kidneys

Summary of treatment related findings in the kidneys for animals killed after four weeks of treatment and females killed on Day 7 of lactation
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
                 
Cortical Tubular Vacuolation                        
Minimal 0 0 5 0 0 0 6 0
Slight 0 0 0 4 0 0 1 1
Moderate 0 0 0 1 0 0 0 4
                 
Total 0 0 5 5 0 0 7 5
                 
Cortical Tubular Hyaline Droplets              
Minimal 0 0 3 3 0 0 0 0
Slight 0 0 0 2 0 0 0 0
                 
Total 0 0 3 5 0 0 0 0
                 
Number of tissues examined 5 5 5 5 5 5 7 5

Lymph nodes

Summary of treatment related findings in the mesenteric and axillary lymph nodes for animals killed after four weeks of treatment and females killed on Day 7 of lactation
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
                 

Mesenteric lymph nodes

Vacuolated macrophages                          

Minimal 0 0 0 2 0 0 0 4
Slight 0 0 0 2 0 0 0 0
Moderate 0 0 0 1 0 0 0 0
Total 0 0 0 5 0 0 0 4
Left axillary lymph nodes                          
Vacuolated macrophages                        
Minimal 0 0 0 4 0 0 0 3
Total 0 0 0 4 0 0 0 3
                 
Number of tissues examined 5 5 5 4 5 5 5 5

Stomach

Summary of treatment related findings in the stomach for animals killed after four weeks of treatment and females killed on Day 7 of lactation
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
                 
Foveolar hyperplasia                        
Minimal 0 0 0 3 0 0 0 4
Total 0 0 0 3 0 0 0 4
                 
Number of tissues examined 5 5 5 5 5 5 5 5

Epididymides

Summary of findings in the epididymides for animals killed after four weeks of treatment
Group/sex 1M 2M 3M 4M
Dose (mg/kg/day) 0 100 330 1000
         
Epithelial vacuolation            
Minimal 0 0 0 1
Slight 0 0 0 4
Total 0 0 0 5
         
Number of tissues examined 5 6 5 5
Conclusions:
In absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day.
Executive summary:

This study was designed to assess the general systemic toxic potential of FAT 41001-H TE (a blue textile dye) in rats, including a screen for reproductive/developmental effects. The study was designed to meet the requirements of OECD 422 guideline for testing of chemicals adopted 22 March 1996: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test. The study was conducted in accordance with the requirements of current, internationally recognised Good Laboratory Practice Standards, and the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

Three groups, each comprising ten male and ten female rats received FAT 41001-H TE at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing up to necropsy, after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were killed on Day 7 of lactation. A similarly constituted Control group received the vehicle (purified water) at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight, macroscopic pathology and histopathology investigations were undertaken.  The clinical condition, litter size, survival, sex ratio, body weight and macropathology for all offspring were also assessed.

Results

Oral administration of FAT 41001-H TE at doses of 100, 330 or 1000 mg/kg/day was generally well tolerated with no mortalities related to treatment. There were no adverse effects attributed to treatment on sensory reaction, grip strength or motor activity; in addition all of the reproductive/developmental endpoints assessed were unaffected by treatment and included, pre-coital interval, mating performance, fertility, gestation length, offspring weights, litter size, sex ratio and offspring survival.

Signs in association with the administration of FAT 41001-H TE were restricted to rales in one male receiving 330 mg/kg/day on Day 3 of treatment. At the detailed physical examination and arena observations, signs seen in relation to treatment were confined to blue staining of various body parts at all dose levels (100, 330 or 1000 mg/kg/day), with the magnitude of incidence increasing as the dose level increased.

Group mean body weight gain for males and females receiving FAT 41001-H TE at 100 mg/kg/day were similar to controls throughout the study and were considered unaffected by treatment.  In males treated at 330 or 1000 mg/kg/day, overall group mean body weight gain was low when compared with Controls however this was predominately a result of low weight gains in Week 0-1 (males treated at 1000 mg/kg/day) and Week 4-5 (males treated at 330 or 1000 mg/kg/day). For females receiving FAT 41001‑H TE there was no conclusive effect of treatment on body weight gain.  

There was no effect of treatment on food consumption in males, or in females prior to pairing.

During Days 6-19 gestation, food consumption of all groups of treated females was slightly high. Throughout lactation, food consumption was slightly higher in females receiving 330 or 1000 mg/kg/day, with the increase being dose dependent.  

On two occasions during treatment, a marked increase in water consumption was observed amongst animalsreceiving 1000 mg/kg/day when compared with Controls.  

Haematological examination during Week 2 of treatment, prior to pairing, and the examination of the clotting parameters in Week 4 of treatment for the males and on gestation Day 17 for the females, revealed no significant response to treatment with FAT 41001-H TE.

Biochemical examination of the blood plasma in Week 2 of treatment revealed lowbile acid concentrations amongst all treated groups of males and females when compared with the control animals. Cholesterol concentration was high in animals receiving 330 or 1000 mg/kg/day and creatinine concentration was high in females at all dose levels. Glucose concentrations were low in males receiving 1000 mg/kg/day. High calcium concentration was evident in all treated male groups when compared with the control group; there was no similar effect in the females.

At routine examination of the offspring, signs that were observed in relation to treatment with FAT 41001 H-TE were limited to dark areas on the lower ventral abdomen at 100, 330 and 1000 mg/kg/day and blue staining was observed in offspring at 330 and 1000 mg/kg/day; this was considered to be related to the colour of the test item.

After five weeks of treatment, absolute and body weight adjusted kidney weights of F0 males and females receiving 1000 mg/kg/day were marginally higher than Controls.  Absolute testes weights were also high in these males and in males receiving 330 mg/kg/day. On Day 7 of lactation, absolute and body weight adjusted adrenal weights for all treated groups of F0 females were low when compared with Controls.

Macroscopic examination performed in F0 males after five weeks of treatment and in F0 females on Day 7 of lactation, did not reveal any treatment-related findings for animals receiving FAT 41001-H TE at 100 mg/kg/day. Findings observed at macroscopic examination of the males and females treated at 330 or 1000 mg/kg/day were confined to blue or dark coloration (including some blue or dark colouration of the contents)in a variety of tissues; this was considered to be due to the coloured nature of the compound and was not representative of any pathological change. At macroscopic examination of the offspring, findings observed in relation to treatment were confined to dark contents of the gastrointestinal tract at 1000 mg/kg/day. In addition, one litter at 1000 mg/kg/day were observed to have blue skin.

Microscopic examination revealed treatment related changes within the kidneys, mesenteric and left axillary lymph nodes, stomach of both sexes and epididymides of the males. Renal cortical tubular vacuolation was recorded in males and females treated with 330 or 1000 mg/kg/day, accompanied by hyaline droplets in the males at both dose levels. Vacuolated macrophages were recorded in the mesenteric and left axillary lymph nodes of most males and females given 1000 mg/kg/day. Foveolar hyperplasia was recorded in the stomach of both sexes given 1000 mg/kg/day. Epithelial vacuolation of the epididymides was recorded in all males treated with 1000 mg/kg/day.

Conclusion

It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Key study with reliability rating 1 performed according to OECD guideline 422 and in accordance with GLP.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
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

A study was designed to assess the general systemic toxic potential of FAT 41001 /H TE (a blue textile dye) in rats, including a screen for reproductive/developmental effects. Study was conducted in accordance to OECD guideline 422 and in compliance to GLP.

Three groups, each comprising ten male and ten female rats received FAT 41001 /H TE at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing up to necropsy, after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.  No effect of treatment on the survival, clinical condition or behavioural/reproductive performance of these animals were observed, and no supporting macroscopic or microscopic changes were detected in the full list of tissues examined. It was therefore concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The test substance has very low vapour pressure, so the potential for the generation of inhalable forms is low, also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Furthermore, in the 28 - days repeated dose study via oral gavage administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can be extrapolated to repeated inhalation route administration.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

The test substance has very low vapour pressure, so the potential for the generation of inhalable forms is low, also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Furthermore, in the 28 - days repeated dose study via oral gavage administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can be extrapolated to repeated inhalation route administration.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

The substance FAT 41001 has been tested for acute oral toxicity and has been found to be not toxic to the animals investigated LD50 (males/females) >2000 mg/kg bw. None of the animals died and none of the animals exhibit any notable signs of toxicity. In addition in animal tests on skin irritation/corrosion effects the test substance has not been found to cause severe irritating or corrosive effects to the skin or any other effect resulting in a destruction of an intact skin barrier. In addition old tests on skin irritation performed on abraded skin areas of animals resulting in a partial destruction of the skin barrier, also no signs of toxicity were noted.

The substance itself is characterized as a dry powdery substance which is marketed in a dedusted form or in a liquid mixture only. From basic research on internal and external barriers of humans it is known, that such molecules are almost not able to permeate the skin resulting in an enhanced bioavailability of the substance applied topically.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

The substance FAT 41001 has been tested for acute oral toxicity and has been found to be not toxic to the animals investigated LD50 (males/females) >2000 mg/kg bw. None of the animals died and none of the animals exhibit any notable signs of toxicity. In addition in animal tests on skin irritation/corrosion effects the test substance has not been found to cause severe irritating or corrosive effects to the skin or any other effect resulting in a destruction of an intact skin barrier. In addition old tests on skin irritation performed on abraded skin areas of animals resulting in a partial destruction of the skin barrier, also no signs of toxicity were noted. The substance itself is characterized as a dry powdery substance which is marketed in a dedusted form or in a liquid mixture only. From basic research on internal and external barriers of humans it is known, that such molecules are almost not able to permeate the skin resulting in an enhanced bioavailability of the substance applied topically.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and therefore no classification is needed.