Reaction mass ofDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN1}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-) andDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

EC Number:

271-351-7

EC Name:

Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Cas Number:

68541-71-9

Molecular formula:

C32H17CrN6O11S.2Na

IUPAC Name:

disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Test substance: FAT 20011/E
Test substance identity (including alternative names): Acid Blue 317
Intended use: Textile dyeing.
Appearance: A solid blue odourless powder.
Storage conditions: At ambient temperature, in the dark.
Supplier: Sponsor
Batch number: 1309023 (China)
Expiry date: 30 September 2018.
Purity: 61.2 % (a weighing factor of 1.63 was applied)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™:WIST 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:

Animal supply, acclimatisation and allocation
Strain/Species: RccHan™;WIST rat.
Supplier: Harlan (UK) Ltd.
Number of animals ordered: 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: 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: 263 to 322 g
Females: 172 to 209 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 colour-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 minimise entry of external biological and chemical agents and to minimise 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 20-24 ºC 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 acclimatisation, pre-pairing, gestation, littering and lactation periods.
Grid bottomed polypropylene 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 equalise, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fibre 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 haematology 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Identity of treatment groups
Some serial observations needed to be performed without the knowledge of the treatment group; therefore the animal numbering system was such that it was not easy to identify a treatment group from the animal number.
The F1 generation received no direct administration of the test substance, FAT 20011/E. Any exposure to the test substance or metabolites was through the mother to the offspring in utero and/or through the milk.

Formulation
Correction factor: A correction factor (including purity and ratio salt/base) of 1.63 was used when calculating quantities of test substance used during dose preparation.
Vehicle: Purified water
Method of preparation: Sufficient vehicle was added to the required amount of test substance to obtain a paste. The paste was initially mixed, crushing any large particles with a spatula. Further, vehicle was added to make approximately 98 % of the final volume, which was then magnetically stirred. Once the material was fully dissolved it was transferred to a measuring cylinder and made up to final volume and magnetically stirred. Full dissolution was confirmed by placing the formulation in front of a strong light source and inspecting the formulation. The start and end times of the magnetic stirring were recorded in the raw data.
Frequency of preparation: Weekly.
Storage of formulation: Store refrigerated (nominally +4 °C).
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.

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
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 treated groups.
Frequency: Once daily, at approximately the same time each day.
Formulation: 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.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Dosing catheters were dipped in water and wiped clean before administration to minimise dose residue on the external surface.
Storage of formulation: Refrigerated.

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis
Stability and homogeneity: Stability of the test material in the vehicle was determined as part of this programme of work in HLS Study No. TMR0050. Stability confirmed two days at ambient and 15 days refrigerated. Achieved concentration: Samples of each formulation prepared weekly were analysed for achieved concentration of the test substance. The weekly achieved concentration results were provided to the Study Director before formulations were issued to the animal facility. The samples were analysed in accordance with the validated Envigo Analytical Procedure FIA/M036/15. The analytical method involved extraction and dilution in N,N-Dimethylformamide/water 20/80 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 254 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 5 μg/mL to 25 μg/mL.

Concentration in test formulations
For each 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 samples were retained for contingency. These were discarded following acceptable results.

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

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 allowed to litter, rear their offspring and were killed on Day 7 of lactation.
The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

Details on study schedule:

Minimum age at mating of the mated animals in the study:
Males: 83 to 89 days old.
Females: 76 to 82 days old.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group - 1

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

Group - 2

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group - 3

No. of animals per sex per dose:

10 male and 10 female rats per dose

Control animals:

yes, concurrent no treatment

Details on study design:

Treatment groups and doses
The doses used in this study (0, 100, 330 and 1000 mg/kg/day) were selected in conjunction with the Sponsor and were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0049). It was concluded from this study that dose levels of 100, 330 and 1000 mg/kg/day would be suitable for use on this subsequent OECD 422 study. In the preliminary study administration of FAT 20011/E was tolerated for 14 days at doses up to and including 1000 mg/kg/day. Overall bodyweight gain and food consumption were satisfactory and there was no evidence of toxicity during routine physical examinations or following dose administration. Liver weights were marginally high in males and females given 500 or 1000 mg/kg/day. Macroscopic examination did not reveal any lesions related to treatment. However, dark livers and kidneys were apparent among males and females at 1000 or 500 mg/kg/day, and many organs/tissues had a blue colouration at 1000 mg/kg/day, with fewer organs/tissues affected at 500 mg/kg/day, and only a small number of tissues/organs affected at 250 mg/kg/day.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

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 occupant(s). 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
Daily during Week 1 of treatment and once each week thereafter and for females on Days 0, 6, 13 and 20 after mating and Day 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration: One to two hours after completion of dosing as late as possible in the working day.

Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, 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 unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”. 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 the five lowest numbered males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group during Days 4-6 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room, the length of separation of dam from litter was minimized. The cage labels on these individual cages showed only the study and animal number. 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 as far as possible on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 - No reaction or ignores probe/walks past probe
2 - Normal awareness and reaction e.g. approaches and/or sniffs probe
3 - Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 - No response
2 - Normal response e.g. ear twitches/flattens, or animal shakes its head
3 - Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 - No response
2 - Weak response e.g. ear twitch only
3 - Normal response e.g. obvious flinch or startle
4 - Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 - No response
2 - Weak response e.g. turns around slowly or weak vocalization without moving away
3 - Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 - Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength were measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and during Days 4-6 of lactation for females, the motor activity of five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), 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 all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body weight
The weight of animals was recorded as follows:
F0 males:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females:
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Hematology, peripheral blood
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasion:
Occasion: Week 2 prior to pairing
Animals: The five lowest numbered surviving males and females per group

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)

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 an ACL series 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 prior to dosing (where appropriate) at the following occasion:
Occasion: Week 2 prior to pairing
Animals: The five lowest numbered surviving males and females per group

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)
Bile acids (Bi Ac)
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/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration

Mating
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Parturition observations and gestation length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Records made during littering phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter observations:

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

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.

Females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.

Postmortem examinations (offspring):

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Statistics:

See below

Reproductive indices:

Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating = (number of animals mating / animals paired) x 100
Conception rate (%) = (number of animals achieving pregnancy / animals mated) x 100
Fertility index (%) = (number of animals achieving pregnancy / animals paired) x 100

Gestation length and index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (number of live litters born / number pregnant) x 100

Offspring viability indices:

Litter size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1 and 7 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival indices
The following were calculated for each litter:
Post-implantation survival index (%) = (total number of offspring born / total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (number of live offspring on Day 1 after littering / total number of offspring born) x 100
Viability index (%) = (number of live offspring on Day 7 / number of live offspring on Day 1) x 100
Group mean values were calculated from individual litter values.

Sex ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1 and 7 of age.
Percentage males = (number of males in litter / total number of offspring in litter) x 100
Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical sign of blue staining, of various part of the body, was apparent at 1000 mg/kg/day, at 330 or 100 mg/kg/day staining was predominantly on the tail. Blue bedding and blue/dark faeces were recorded on animals receiving 330 or 1000 mg/kg/day a dose response was noted with the signs appearing more often in the high dose animals. This sign was attributed to the colour of the test material and not considered to be adverse. A higher incidence of vocalisation was observed in females in all treated groups a dose response was not apparent, the significance of this finding was unclear.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Low bodyweight gain was apparent in males receiving 1000 mg/kg/day. There were occasional periods of slightly low body weight gain in males receiving 330 mg/kg/day with overall being marginally lower than in Controls. There was no effect on body weight performance of females before pairing, during gestation or lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects of FAT 20011/E TE on food consumption of males throughout or females before pairing, during gestation or lactation. In females at 330 or 1000 mg/kg/day high food consumption attained statistical significance on Day 6-12 of gestation, the magnitude of these differences was small and was considered not be adverse at the degree observed.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects of FAT 20011/E on haematology parameters assessed. In females receiving 1000 mg/kg/day statistical significance was attained for low haematocrit count and haemoglobin concentration; however these parameters lacked any clear dose related trend and the magnitude of these changes were small.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry assessment during week 2 before pairing revealed the following consistent statistical differences to control in both males and females; high alanine amino-transferase at 1000 mg/kg/day and low bilirubin at 100 (females only), 330 and 1000 mg/kg/day. The following statistical differences to the Control were apparent in males only; low alkaline phosphatase at 1000 mg/kg/day; low bile acid concentration at 330 and 1000 mg/kg/day; low glucose and potassium concentrations at all dose levels. The following statistically significant differences to the control were apparent in females only; low calcium at 1000 mg/kg/day and low total protein at 330 and 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of FAT 20011/E on motor activity. In males statistical significance was attained at 1000 mg/kg/day for low group mean beam breaks during one period at each of the low and high beam heights. In females there were no periods of statistical significance for high beam breaks, group mean low beam break scores for two periods at 1000 mg/kg/day and one period at 330 mg/kg/day were statistically lower than control. The Control male mean total high and low beam scores were slightly above the historical control data range and, in the absence of any block of consecutive periods attaining statistical significance and the absence of any statistical significance of the overall activity scores these differences are considered to be unrelated to treatment and not to be adverse.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment related findings
Changes related to treatment with FAT 200011/E were seen in the kidneys.

Kidneys
In haematoxylin and eosin stained sections, a cytoplasmic blue-grey pigment was observed in the epithelium of cortical tubules in all animals receiving 1000 mg/kg/day. Application of special stains to additional sections of kidney from representative animals indicated that the pigment was Perls negative and Schmorls positive, suggesting that the pigment was lipofuscin.

Incidental findings
Accumulation of pigmented macrophages was seen in the lungs of occasional treated animals, correlating with the reporting of dark areas at necropsy. These were considered to represent a background finding. In one high dose male (Animal No. 2), this change was accompanied by the presence of myxoid material in the bronchioles, alveolar oedema and alveolitis. These changes were considered to be indicative of an incidental inspiration of test item during dosing, and not to represent a systemic effect of administration of FAT 200011/E. All other findings were consistent with the background of microscopic changes commonly seen in Han Wistar rats at these laboratories. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Dosing signs
Signs observed in association with dosing attributed to FAT 20011/E were limited to one male receiving 330 mg/kg/day and one female receiving 100 mg/kg/day displaying noisy breathing/rales and one female receiving 1000 mg/kg/day displaying piloerection. In the absence of any rales in animal receiving 1000 mg/kg/day this sign was not considered to reflect an adverse effect of treatment and the observation of piloerection in a single animal on a single day was considered to be fortuitous.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Evaluation of reproductive parameters concluded there was no effect of FAT 20011/E on pre-coital interval, mating performance, fertility, gestation length or gestation index. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No treatment related adverse effect was observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were considered to be no adverse effects on the clinical condition of offspring. At the high dose level blue staining of the offspring was apparent however as the test material is a blue dye and the external surfaces of the dams, the faeces and bedding were all stained blue the transfer of some of this colour to the offspring was not considered to be adverse.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight was notably low on Day 1 of age in offspring derived from animals receiving 1000 mg/kg/day. Growth in these pups from Day 1 was also slightly lower than concurrent control. Though a relationship to treatment has not been excluded the response was considered not to be adverse at the degree observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were considered to be no adverse effects detected at necropsy for the offspring. At the high dose level blue staining of the offspring skin was apparent. The test material is a blue dye and the external surfaces of the dams, the faeces and bedding were all stained blue the transfer of some of this colour to the offspring was not considered to be adverse.

Histopathological findings:

not examined

Details on results (F1)

Litter size, sex ratio and survival indices: There were no indications of any effects on litter size or survival. Sex ratio was highly variable between groups however in the absence of any clear trends no effect of treatment was inferred.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no treatment related adverse effect was observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Formulation analysis

The mean concentrations of FAT 20011/E in test formulations analysed for the study were within 10 % of nominal concentrations, confirming accurate formulation. Difference from mean values were within 3 % confirming precise analysis.

Kidneys

In haematoxylin and eosin stained sections, a cytoplasmic blue-grey pigment was observed in the epithelium of cortical tubules in all animals receiving 1000 mg/kg/day. Application of special stains to additional sections of kidney from representative animals indicated that the pigment was Perls negative and Schmorls positive, suggesting that the pigment was lipofuscin.

Summary of treatment related findings in the kidney for animals killed after 5 weeks of treatment or 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 pigment
Minimal	0	0	0	5	0	0	0	5
Total	0	0	0	5	0	0	0	5
Number of tissues examined	5	5	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level for reproduction and developmental toxicity was 1000 mg/kg/day.

Executive summary:

The objective of this study was the assessment of general systemic toxic potential in rats, including a screen for reproductive/developmental effects, with administration of FAT 20011/E (used in textile dyeing) by oral gavage administration for at least four weeks. 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 recognized Good Laboratory Practice Standards, and the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act). Three groups of 10 male and 10 female rats received FAT 20011/E 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 allowed to litter, rear their offspring and were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water. 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 and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

Results

There were no dosing signs attributed to FAT 20011/E and there were no adverse effects detected for sensory reactivity and grip strength assessments, motor activity, body weight performance (females only), food consumption, haematology or blood chemistry parameters assessed and, organ weights. Changes related to the properties of the test material (a blue dye) included blue staining of various part of the body, blue bedding, blue/dark faeces. The macroscopic examination performed on animals killed after 5 weeks of treatment or on Day 7 of lactation revealed abnormal (blue) colouration of the fur, skin (including tail), skeletal muscle, sciatic nerves, mammary tissue, axillary lymph nodes, brain, eyes, and the majority of the tissues in the abdominal and thoracic cavities, with abnormal colouration of the contents of the gastro‑intestinal (GI) tract. A combination of some or the majority of these changes were evident in all animals receiving 1000 mg/kg/day, and the majority of animals receiving 330 mg/kg/day, with a greater number of males being affected than females in this dose group. Blue colouration in animals receiving 100 mg/kg/day, was generally limited to the mesenteric lymph nodes, contents of the GI tract and staining of the tails in occasional males. At the high dose level external blue staining of the offspring was apparent in-life and confirmed at necropsy. As these are due to an intrinsic property of the test material these changes are considered not to be adverse. A higher incidence of vocalisation was observed in females in all treated groups a dose response was not apparent. Low bodyweight gain was apparent in males receiving 1000 mg/kg/day. There were occasional periods of slightly low body weight gain in males receiving 330 mg/kg/day with overall gain prior to pairing lower than in Controls. Terminal body weights were slightly low in males at necropsy after 5 weeks of treatment. None of the organ weights assessed was statistically different to the Control. In females slightly high adjusted mean kidney weights in animals receiving 1000 mg/kg/day attained statistical significance. Histopathology changes related to treatment with FAT 200011/E were seen in the kidneys of animals treated with 1000 mg/kg/day. A cytoplasmic blue grey pigment was observed in the epithelium of renal cortical tubules in all animals receiving 1000 mg/kg/day. These changes were not present in the animals treated with 330 or 100 mg/kg/day. These changes are considered not to be adverse in the context of this study. Evaluation of reproductive parameters concluded there was no effect of FAT 20011/E on pre-coital interval, mating performance, fertility, gestation length or gestation index. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted. There were considered to be no adverse effects on the clinical condition of offspring, litter size, survival or sex ratio. Body weight was notably low on Day 1 of age in offspring derived from animals receiving 1000 mg/kg/day. Growth in these pups from Day 1 was also slightly lower than concurrent control however, survival and clinical condition were unaffected. There were no adverse effects detected at necropsy of the offspring. 

Conclusion

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.