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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 10 May 2017 Experimental Completion Date: 13 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

275-602-1

EC Name:

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

Cas Number:

71550-21-5

Molecular formula:

C27H24N6O9S2.2C4H11NO2

IUPAC Name:

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

Test material form:

liquid

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Test item identity (including alternative names): Bayscript Gelb GGN
Benzenesulfonic acid, 3,3’-(carbonylbis(imino(3-methoxy- 4,1-phenylene)-2,1-diazenediyl))bis-, compd. with 2,2’-iminobis(ethanol) (1:2)
Appearance: Russet colored crystalline solid.
Storage conditions: At ambient temperature (15 to 25ºC).
Expiry date: 22 June 2018

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River (UK) Ltd.
Number of animals ordered: 44 males and 48 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: 8 days prior to the commencement of treatment.
Females: 22 days prior to the commencement of treatment.
Age of the animals at the start of the study:
Males: approximately 73 days old.
Females: approximately 87 days old.
Weight range of the animals at the start of the study:
Males: 329 to 408 g.
Females: 245 to 315 g.

Allocation and Identification
Allocation: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed ± 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a microchip 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 Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment: Irregular or abnormal estrous cycle - Four females

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

Animal Accommodation
Cages: 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, pre-pairing, 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.
Polycarbonate shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

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:

PREPARATION OF DOSING SOLUTIONS:
Correction factor: A correction factor (including purity) of 1.12 was used when calculating quantities of test item used during dose preparation.
Method of preparation: The test item was dry ground before weighing. The required amount of test item was weighed and vehicle was added. It was magnetically stirred until a uniform suspension was achieved. The formulation was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8ºC).
.

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:

Stability and homogeneity: The homogeneity and stability of formulations in the concentration range of 1 and 200 mg/mL during ambient and refrigerated storage were determined in Envigo Study No. LR73TX. These investigations demonstrated that formulations in the concentration range 1 to 200 mg/mL are stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analyzed for achieved concentration of the test item.

The mean concentrations were within 9% of the nominal concentration, confirming the accuracy of formulation. The difference from mean values were within 3%, confirming precise analysis.

Duration of treatment / exposure:

Males Two weeks before pairing up to necropsy after minimum of four weeks.
Females Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

The study consisted of one control and three treated groups.
The F1 generation received no direct administration of the test item, Bayscript Yellow GGN. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

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: 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

70 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels selected for investigation in this OECD 421 reproductive/developmental toxicity screening study (0, 10, 30 and 70 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a 14-day preliminary study and an OECD 407 study conducted at these laboratories (Envigo Study No’s. KX37FJ and LR73TX, respectively).
In the 14-day preliminary study dose levels of 25, 50, 100 and 500 mg/kg/day were investigated, and the kidney was identified as a target organ at all dose levels. The dose levels of 100 or 500 mg/kg/day exceeded the maximum tolerated dose. At 500 mg/kg/day, one animal was found dead and the remaining animals required premature termination on Day 4 or 5 of study due to a marked decline in clinical condition, weight loss and low food intake; the kidneys of all animals were markedly increased in weight and pale. Although there were no premature deaths at 100 mg/kg/day, sporadic incidences of hunched posture, piloerection and partially closed eyes were apparent, body weight performance was poor and food consumption was persistently low. At scheduled termination, all animals showed markedly increased kidney weights and all kidneys were pale. At 25 or 50 mg/kg/day, there were no test item-related changes in clinical condition or post-dosing signs. Some short periods of minor weight loss were apparent in individual animals but with the exception of one female given 50 mg/kg/day, all animals recorded overall body weight gain during the course of the study. Food consumption was slightly low at 50 mg/kg/day but was not clearly affected by treatment at 25 mg/kg/day. Increased kidneys weights were evident at
50 mg/kg/day, and all animals at 50 mg/kg/day and one female at 25 mg/kg/day showed pale kidneys at scheduled termination.
In the OECD 407 study, dose levels of 0, 10, 30 and 70 mg/kg/day were employed. There were no changes in clinical condition or effects on body weight performance or food intake at any dose level investigated. Slight/moderate histopathological changes were evident in the kidneys, which were associated with some minor biochemical changes in the plasma, changes in urine output/composition and slight increases in kidney weights, however the magnitude of these changes were considered not to preclude the use of 70 mg/kg/day as the high dose level in the current OECD 421 study.
The high dose level was therefore set at 70 mg/kg/day. The intermediate and low dose levels of 30 and 10 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Parental animals: Observations and examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

Clinical 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
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males:
Week 1 - daily
Week 2 onwards - once each week

F0 females:
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Before dose administration
One to two hours after completion of dosing
As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males:
Once each week

F0 females:
Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13

Body Weight
The weight of animals was recorded as follows:
F0 males:
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.

F0 females:
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals:
Weekly before pairing, 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 recorded as follows:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

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.

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination - All adults

Sequence of blood sampling on each occasion
In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled as far as possible to allow satisfactory inter-group comparisons.
Conditions - No overnight deprivation of food.
Anesthetic - Isoflurane.
Blood sample site - Sublingual vein.

Parameter:

Thyroid stimulating hormone (TSH):
Anticoagulant - K2 EDTA with no separator gel.
Tubes - Standard Envigo.
Blood volume - 0.5 mL.
Processing - Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.

Thyroxine (T4):
Anticoagulant - None.
Tubes - Greiner Minicollect - with clot activator.
Blood volume: 0.5 mL.
Processing - Samples were kept at ambient temperature (15 to 25ºC) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions: At 2000g for ten minutes at 4°C.
Final storage conditions: Deep frozen (approximately -60°C to -90ºC)
Fate of samples: Dispatched to the Department of Bioanalysis Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears - For 15 days before pairing using cotton swabs
Wet smears - Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until positive evidence of mating detected.
For four days before scheduled termination.

Sperm parameters (parental animals):

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.

Litter observations:

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 maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring

Thyroid Hormone Analysis
Blood samples were collected as follows:

Day 4 of age:
F1 offspring, two females per litter (where possible).
No offspring were allocated to these procedures if the resultant live litter size would fall below ten offspring or if it would leave less than three females.
- one for T4 (serum)#
- one for TSH (plasma)
# priority was given to serum sample

Day 13 of age:
F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample

Sequence of blood sampling on each occasion - In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled as far as possible to allow satisfactory inter-group comparisons.
Conditions - No overnight deprivation of food.
Anesthetic - None.
Blood sample site - Decapitation.

Parameter:

Thyroid stimulating hormone (TSH):
Anticoagulant - K2 EDTA with no separator gel.
Tubes - Standard Envigo.
Blood volume - max possible.
Processing - Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.

Thyroxine (T4):
Anticoagulant - None.
Tubes - Greiner Minicollect - with clot activator.
Blood volume: max possible.
Processing - Samples were kept at ambient temperature (15 to 25ºC) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions: At 2000g for ten minutes at 4°C.
Final storage conditions: Deep frozen (approximately -60°C to -90ºC)
Fate of samples: Dispatched to the Department of Bioanalysis Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.

Postmortem examinations (parental animals):

Method of Kill
All adult animals: Carbon dioxide asphyxiation. Each animal was subsequently exsanguinated.
Sequence: To allow satisfactory inter-group comparison.

Necropsy
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. Examination included a detailed assessment and documentation of any colour changes in the internal organs, adipose tissue or skin. If colour changes were observed, representative photographs were taken before retained tissues were placed in fixative.

Time of Necropsy
F0 males: After at least four weeks of treatment.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 13 of lactation (following terminal blood sampling).

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

Females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.
Female whose litter died before Day 13 of lactation: Mammary tissue appearance.

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

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - Initially in modified Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List:
All F0 animals killed prematurely.
All F0 animals in Groups 1 and 4 at scheduled termination.

Kidneys and Abnormalities :
All F0 animals.

Routine staining:
Sections were stained with hematoxylin and eosin; in addition sections of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined as follows:

Premature deaths:
All F0 animals from all groups - All specified in the table in section 'any other information on materials and methods'
Scheduled kill:
All F0 animals in Groups 1 and 4 - All specified in the table in section 'any other information on materials and methods'
All F0 animals in Groups 2 and 3 - Kidneys and Abnormalities only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

Method of Kill
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone
Sequence: To allow satisfactory inter-group comparison.

Time of Necropsy
F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.

F1 offspring on Day 4 of age: For blood sampling requirements.
Externally normal offspring discarded without examination. Externally abnormal offspring identified on despatch to necropsy, an external macroscopic examination was performed and offspring retained pending possible future examination.

Offspring at scheduled termination : For blood sampling requirements.
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

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

Reproductive indices:

Mating Performance and Fertility
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 pairing) x 100


Gestation Length and Index
Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

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 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) 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, 4 (before and after blood sampling) and 13 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):

Administration with Bayscript Yellow GGN at doses up to and including 70 mg/kg/day was considered to be well tolerated throughout the treatment period, with no test item-related changes in general clinical condition or post-dosing signs observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two premature deaths during the course of the study which were unrelated to Bayscript Yellow GGN administration.
One female assigned to the 30 mg/kg/day group (Group 3F No. 76) was killed for welfare reasons during the pairing period (Day 18 of treatment) due to physical injury to the vaginal region most likely caused by the male mating partner.
A further female assigned to the 30 mg/kg/day group (Group 3F No. 79) was killed on Day 3 of lactation following the premature sacrifice of the litter for reasons of animal welfare. Having shown no previous signs following birth, on Day 3 of lactation the 15 pups were found to be cold to touch with little/no milk in stomach; due to their poor survival prognosis the litter was killed. Macroscopic examination of the dam revealed pale and inactive mammary tissue and microscopic examination confirmed decreased/minimal lactation; abnormalities in the offspring were limited to an absence of milk in the stomach. Since there were no similar premature deaths in the 70 mg/kg/day group, the demise of this litter and subsequent sacrifice of the dam were considered fortuitous and unrelated to Bayscript Yellow GGN.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the two-week pre-pairing treatment period, males and females given 70 mg/kg/day showed statistically significant mean body weight loss during Week 1 (9 grams in males and 8 grams in females) compared with mean weight gain in the Control animals of 25 grams in males and 9 grams in females. In Week 2, mean body weight gain was evident in these males and females, however among the males this remained lower than Control (although not attaining statistical significance).
The mean body weight gain of males in the 70 mg/kg/day group remained lower than Control with some differences attaining statistical significance, such that overall mean body weight gain from Day 1-29 of study was 76% lower than Control.
After mating, the mean body weight gain of females in the 70 mg/kg/day group was lower than Control, with statistical significance attained during Days 0-7 and 14-20 after mating; overall mean body weight gain from Day 0-20 of gestation was 27% lower than Control.
Following parturition, the mean body weight performance of females given 70 mg/kg/day during Days 1-7 of lactation was poor compared to Control, with overall mean weight loss of 5 grams compared to mean weight gain of 16 grams among Controls. Thereafter, the mean body weight gain of these females was essentially similar to Control, such that overall mean weight gain during Days 1-13 of lactation was 50% lower than Control, with statistical significance attained.
The body weight performance of males and females given Bayscript Yellow GGN at dose levels of 10 or 30 mg/kg/day was considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Throughout the study, mean food consumption for males and females receiving 70 mg/kg/day was lower than Control, correlating with the reduced mean body weight gain previously discussed.
Mean food consumption for males and females given 10 or 30 mg/kg/day was unaffected by Bayscript Yellow GGN administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment Related Findings
Changes related to treatment with Bayscript Yellow CGN were seen in the kidneys.
There was an increase in the incidence and severity of tubular/collecting duct basophilia, tubular dilatation and intratubular crystals in the kidneys of males and females given 70 or 30 mg/kg/day. This was associated with inflammation in several animals. A summary of the findings in the kidneys is given in a table in section 'any other information on results'

Incidental Findings
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

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males. There was therefore no requirement to measure T4 in the samples obtained from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis. See table in section 'any other information on results'.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatisation period

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Males given 70 mg/kg/day showed low absolute epididymis and testis weights, along with low adjusted prostate weights, with statistical significance attained for the differences in absolute epididymal weight. Among males given 30 or 70 mg/kg/day there was a trend towards low seminal vesicle/coagulating gland weights.

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and index were unaffected by treatment. One female receiving 70 mg/kg/day (No. 82) was found to be not pregnant.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Details on results (P0)

With the exception of one female in the 70 mg/kg/day group which was not pregnant (Group 4F No. 82), all other females which showed positive evidence of mating gave birth to live young.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: reproductive performance

Remarks:

The reduction in the number of uterine implantations at 70 mg/kg/day (and as a consequence litter size) was considered likely secondary to lower maternal body weight at mating, it is considered to be of uncertain aetiology and therefore potentially adverse.

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with Bayscript Yellow GGN.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 70 mg/kg/day, the mean numbers of implantation sites were lower than Control, resulting in lower mean litter size; the mean number of implantation sites and litter size were unaffected at 10 or 30 mg/kg/day.
There was no effect of parental treatment with Bayscript Yellow GGN at any dose level investigated on sex ratio or offspring survival to Day 13 of age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male and female offspring on Day 1 of age were slightly lower than Control in all treated groups although in the absence of a dose response. Subsequent body weight gain of the offspring to Day 13 of age in the 70 mg/kg/day group was reduced by 16% in both sexes compared to Control, with statistical significance attained; the mean body weight gain of offspring in the 10 or 30 mg/kg/day groups was 4-5% lower than Control and not clearly affected by parental treatment.

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:

not examined

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 no findings in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Ano-Genital Distance
The mean ano-genital distance of male and female offspring was essentially similar to Control in all treated groups and no effect of parental treatment with Bayscript Yellow GGN was inferred.

Thyroid hormone analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in offspring on Day 13 of age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: See remark

Remarks:

The reduction in the number of uterine implantations at 70 mg/kg/day (and as a consequence litter size) was considered likely secondary to lower maternal body weight at mating, it is considered to be of uncertain aetiology and therefore potentially adverse.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

70 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Thyroid hormone analysis

Mean serum T4 concentrations (pg/mL)

Group	Treatment	Dose		Adult Male at Termination	Day 13 of age Offspring
		(mg/kg/day)			Male	Female
1	Vehicle (Purified water)	0	Mean	39700	42300	43000
			SD	6340	6790	9070
			CV	16.0	16.1	21.1
			N	10	10	10
2	Bayscript Yellow GGN	10	Mean	42400	40400	37500
			SD	5390	4030	10600
			CV	12.7	10.0	28.3
			N	10	9	9
3	Bayscript Yellow GGN	30	Mean	39900	35800	44300
			SD	6050	10400	8110
			CV	15.2	29.1	18.3
			N	10	8	8
4	Bayscript Yellow GGN	70	Mean	37600	37500	44700
			SD	3510	8460	8540
			CV	9.3	22.6	19.1
			N	10	9	9

 

Summary of treatment related findings in the kidneys

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	10	30	70	0	10	30	70
Basophilia, tubules/collecting ducts
Minimal	0	0	9	0	0	0	2	0
Slight	0	0	0	4	0	0	0	4
Moderate	0	0	0	5	0	0	0	6
Marked	0	0	0	1	0	0	0	0
Total	0	0	9	10	0	0	2	10
Crystals, intratubular
Minimal	0	0	7	5	0	0	2	6
Slight	0	0	0	5	0	0	0	4
Total	0	0	7	10	0	0	2	10
Dilatation, tubular
Minimal	0	0	4	5	0	0	2	7
Slight	0	0	0	5	0	0	0	3
Total	0	0	4	10	0	0	2	10
Inflammation
Minimal	0	0	6	6	0	0	0	3
Slight	0	0	0	1	0	0	0	1
Total	0	0	6	7	0	0	0	4
Number of tissues examined	10	10	10	10	10	10	10	10

 

Applicant's summary and conclusion

Conclusions:

In conclusion, oral administration of Bayscript Yellow GGN up to and including
70 mg/kg/day for four weeks in males and for two weeks prior to pairing, throughout gestation and up to Day 13 of lactation in females, was generally well tolerated. Clear adverse reductions in body weight performance and food consumption were evident in the parental animals given 70 mg/kg/day throughout the study, although in the absence of any change in general clinical condition. Test item-related histopathological changes were evident in the kidneys, manifest as an increase in both incidence and severity of tubular collecting duct basophilia, tubular dilatation and intratubular crystals in the kidneys of animals receiving 30 or 70 mg/kg/day, associated with increases in kidney weights in both sexes and with gross changes of pale and granular kidneys seen at necropsy; these kidney changes were considered to be adverse. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level for systemic toxicity was 10 mg/kg/day.
There was no evidence of any adverse effects on mating performance or fertility of the adult animals or on the survival or development of the F1 offspring. The reduction in the number of uterine implantations at 70 mg/kg/day (and as a consequence litter size) was not related to any other changes in reproductive parameters, such as post implantation loss. This effect, however, was statistically significant and outside of the historical control values (representing 22 OECD TG 421 and 422 studies). Although this effect is considered likely to be secondary to the lower maternal body weight at the time of mating, it is considered to be of uncertain aetiology and therefore potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 30 mg/kg/day.
Bayscript Yellow GGN showed no evidence of being an endocrine disruptor.

Executive summary:

Summary

The purpose of this screening test was to assess the potential for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, following administration of Bayscript Yellow GGN by oral gavage administration forat least four weeks. The study was conducted to fulfil the data requirement according to REACH regulation Annex VIII chapter 8.6.1.

Three groups of ten male and ten female CD rats received Bayscript Yellow GGN at doses of 10, 30 or 70 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. 

During the study, for adult animals assessments of clinical condition, body weight, food consumption, estrous cycles, pre‑coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

For the offspring,clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance and nipple counts (males only), and macropathology were also assessed.

Blood samples were collected from all adult animals at termination and from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis. 

Results

Parental (F0) responses

Treatment with Bayscript Yellow GGN at doses up to and including 70 mg/kg/day was generally well tolerated; there were no changes in general clinical condition or post-dosing signs observed. There were two premature deaths during the course of the study in the
30 mg/kg/day group but these were unrelated to treatment.

Body weight loss was observed in males and females given 70 mg/kg/day in Week 1 of treatment (prior to pairing). Subsequent body weight gain was evident in Week 2 for these animals, however remained lower than Control for males. Body weight gain of males receiving 70 mg/kg/day from Day 1-29 of study remained lower than that of Control, such that by termination the overall body weight gain of these animals was 76% lower than Controls. 

After mating, the body weight gain of females receiving 70 mg/kg/day during both gestation and lactation was lower than that of Control; during gestation, overall mean body weight gain was 27% lower than Control and during Days 1-7 of lactation these females recorded mean weight loss of 5 grams compared to a mean body weight gain of 16 grams among Controls. Overall mean body weight gain of females receiving 70 mg/kg/day during Days 1-13 of lactation was 50% lower than that of Control.

Mean food consumption for males and females receiving 70 mg/kg/day was lower than Control throughout the study and analogous with body weight performance. 

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and index were unaffected by treatment with Bayscript Yellow GGN; with the exception of one female receiving 70 mg/kg/day, all females showing positive evidence of mating were pregnant with live young.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

In accordance with the previously described effects on body weight performance at 70 mg/kg/day, low mean terminal body weights were seen in males after 4 weeks of treatment and in females on Day 13 of lactation when compared to Control. Changes in organ weights consisted of low absolute epididymis and testis, along with low adjusted prostate weights in males receiving 70 mg/kg/day. In males receiving 30 or 70 mg/kg/day low seminal vesicle/coagulating gland weights were seen. An increase in kidney weights was evident in both males and females receiving 30 or 70 mg/kg/day.

Macroscopic examination of the adult males and females revealed abnormal colour (pale) and/or granular appearance of the kidneys at 30 or 70 mg/kg/day. One male receiving
70 mg/kg/day was seen to have a 2-9mm firm pale mass on the left epididymis and vas deferens, however this had no impact on the reproductive capacity of this animal. Any additional findings were isolated and showed no clear association with treatment.

Histopathological evaluation of retained tissues revealed an increase in both incidence and severity of tubular/collecting duct basophilia, tubular dilatation and intratubular crystals in the kidneys of males and females given 30 or 70 mg/kg/day; this was associated with inflammation in several animals.

F1 Litter responses

The clinical condition, sex ratio, survival and ano-genital distances of the F1 offspring was unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment.

A reduction in mean implantation sites of females receiving 70 mg/kg/day resulted in lower mean litter size when compared with Control.

On Day 1 of age, all treated groups were seen to have a slightly lower mean body weight when compared to Control (6-9% less), this was however in the absence of a dose response. Mean body weight gain of the offspring up until termination on Day 13 of age was 16% lower than that of Controls in males and females receiving 70 mg/kg/day.

Conclusion

In conclusion, oral administration of Bayscript Yellow GGN up to and including 70 mg/kg/day for four weeks in males and for two weeks prior to pairing, throughout gestation and up to Day 13 of lactation in females, was generally well tolerated. Clear adverse reductions in body weight performance and food consumption were evident in the parental animals given 70 mg/kg/day throughout the study, although in the absence of any change in general clinical condition. Test item-related histopathological changes were evident in the kidneys, manifest as an increase in both incidence and severity of tubular collecting duct basophilia, tubular dilatation and intratubular crystals in the kidneys of animals receiving 30 or 70 mg/kg/day, associated with increases in kidney weights in both sexes and with gross changes of pale and granular kidneys seen at necropsy; these kidney changes were considered to be adverse. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level for systemic toxicity was 10 mg/kg/day.

There was no evidence of any adverse effects on mating performance or fertility of the adult animals or on the survival or development of the F1 offspring. The reduction in the number of uterine implantations at 70 mg/kg/day (and as a consequence litter size) was not related to any other changes in reproductive parameters, such as post implantation loss. This effect, however, was statistically significant and outside of the historical control values (representing 22 OECD TG 421 and 422 studies). Although this effect is considered likely to be secondary to the lower maternal body weight at the time of mating, it is considered to be of uncertain aetiology and therefore potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 30 mg/kg/day.

Bayscript Yellow GGN showed no evidence of being an endocrine disruptor.