Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 17 May 2017 Experimental Completion Date: 26 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)
Deviations:
no
Remarks:
No deviations occured that were considered to have affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
12H-phthaloperin-12-one
EC Number:
230-049-5
EC Name:
12H-phthaloperin-12-one
Cas Number:
6925-69-5
Molecular formula:
C18H10N2O
IUPAC Name:
12H-isoindolo[2,1-a]perimidin-12-one
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test item: Macrolex Orange 3G
Test item identity (including alternative names): Macrolex Orange 3G FG
Appearance: Orange powder (with a yellow cast).
Storage conditions: At ambient temperature (15 to 25ºC).
Expiry date: 30 June 2021

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: 6 days prior to the commencement of treatment.
Females: 20 days prior to the commencement of treatment.
Age of the animals at the start of treatment
Males: approximately 71 days old.
Females: approximately 85 days old.
Weight range of the animals at the start of treatment
Males: 324 to 393 g
Females: 240 to 293 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.

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 estrous cycle - One female

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%.
On one occasion conditions were outside the indicated ranges, this deviation was minor and of short duration and was not considered to have influenced the health of the animals or the outcome of the study.
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, before pairing, treatment, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

iet Supply
Diet SDS VRF1 Certified pelleted.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
A sample (100 g) of each batch of diet used was retained within the Pharmacy department (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after finalization of the report.
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:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Correction factor: None.
Method of preparation: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension.
The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of suspensions 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 100 mg/mL during ambient and refrigerated storage were determined in Envigo Study No. CD10HX. These investigations demonstrated that formulations in the concentration range 1 to 100 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 6% of the nominal concentration, confirming the accuracy of formulation. The difference from mean values were within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming continued robustness of the method.
Duration of treatment / exposure:
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.
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, Macrolex Orange 3G. 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 / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
500 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, 100, 300 and 500 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. BX18NR and CD10HX, respectively).
In each of those studies dose levels of 100, 300 and 500 mg/kg/day were investigated. As part of the programme of work with this test item it was demonstrated in a vehicle trial prior to formulation homogeneity and stability investigations (Envigo Study No. CD10HX) that, due to the properties of the test item, it was not possible to prepare a formulation suitable for dose administration above a concentration of 100 mg/mL in corn oil. Since the maximum dose volume for formulations using corn oil is 5 mL/kg body weight, the high dose level for investigation by oral gavage administration was limited to the maximum feasible dose of 500 mg/kg/day. In the 14-day preliminary study there were no premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.
In the OECD 407 study, test item-related effects were limited to the following: slightly low body weight gain during Days 1-15 for males given 300 or 500 mg/kg/day associated with slightly low food consumption during this period for males given 500 mg/kg/day; slight grade hyaline droplets in the kidneys of males given 300 or 500 mg/kg/day and minimal grade yellow/brown pigment in the cortical tubules of the kidneys of females given 500 mg/kg/day (correlating with the kidneys of these females being macroscopically dark), and associated with high kidney weights in all groups of treated males and females with correlated minor
biochemical changes in the plasma; high liver weights in all groups of treated males and females which were associated with some minor biochemical changes in the plasma but had no microscopic correlates. The magnitude of these changes were considered not to preclude the use of 500 mg/kg/day as the high dose level in the current OECD 421 study.
The high dose level was therefore set at 500 mg/kg/day. The intermediate and low dose levels of 300 and 100 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.

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 of all groups
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 mating.
For four days before scheduled termination. Some females in each group were only smeared for three days

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
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: Day 13 of lactation (following terminal blood sampling).
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

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.

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.

Special staining
The kidneys of a single female in Group 1 (No. 51) and two females with tubular pigment present in Group 4 (No’s. 89 and 90) stained for lipofuscin (Schmorls) and for hemosiderin (Prussian Blue/Perls).

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.
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):
Among animals surviving to scheduled termination, the administration of Macrolex Orange 3G at doses up to and including 500 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. Orange staining of the coat was apparent in all treated groups, representative of the colour of the treated formulations which was clearly excreted in the faeces (as demonstrated by the colour of the faeces recorded during the cage bedding observation procedures).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were two premature deaths among Control females during the course of the study both of which were caused by injury by mis-dosing.

Female No. 53 was killed on Day 8 of lactation, with antemortem signs including irregular breathing, piloerection, decreased activity and partially closed eyelids. Microscopic examination revealed slight focal pyogranulomatous inflammation of the serosa of the oesophagus, correlated with a macroscopic finding of oesophageal perforation. Marked pyogranulomatous inflammation of the thoracic cavity was seen on microscopic examination, featuring multiple adhesions between the thoracic wall and lung, with pleuritis. These findings correlated with macroscopic findings in the thoracic cavity of adhesions involving multiple organs and abnormal thin clear fluid content, and were considered sequelae to oesophageal perforation.

Female No. 59 was killed on Day 7 of gestation, with antemortem signs including irregular breathing, piloerection, dull eyes, hunched posture, dark skin over the whole body and a thin build. On microscopic examination, purulent debris in the lumen of the oesophagus was correlated with a macroscopic finding of oesophageal perforation. Severe pyogranulomatous inflammation of the thoracic cavity was seen on histopathology, featuring multinucleated giant cells, pleuritis and subpleural inflammation of lung tissue, and multiple adhesions between the thoracic wall, lungs and oesophagus. These findings were correlated with a macroscopic finding of adhesions involving multiple organs in the thoracic cavity.
This animal also had dark pericardial fluid at necropsy. The above findings were all considered sequelae to oesophageal perforation. Additionally on microscopic examination, slight multifocal necrosis was seen randomly distributed throughout the liver, featuring a mixed mononuclear and neutrophilic inflammatory cell infiltrate.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight performance of males and females given Macrolex Orange 3G at dose levels up to and including 500 mg/kg/day was considered to be unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect of Macrolex Orange 3G administration on mean food consumption for males and females at any dose level investigated.
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):
Changes related to treatment with Macrolex Orange 3G were seen in the kidney.

Kidney
An increase in incidence and severity of hyaline droplets in cortical tubules was seen in treated males.
Increased cortical tubular pigment was seen in treated females. Such yellow-brown pigment representing lipofuscin is common in the kidneys of rats, but haemosiderin can appear similar (Frazier et al, 2012). In a previous OECD 407 study with the same test item (Envigo Study number CD10HX), lipofuscin was identified. In the current study, special staining was performed on one Control female (No. 51) and two females treated at 500 mg/kg/day that featured the increased yellow-brown pigment (Nos. 89 and 90). Perls/Prussian Blue staining to detect haemosiderin was negative in all three animals. Schmorls staining for lipofuscin was positive in all three animals, with the intensity and distribution of staining clearly increased in treated females compared to the Control.

Procedural Findings
Two Control males (Nos. 2 and 5) had microscopic findings consistent with having been mis-dosed. On microscopic examination, the animals had moderate or marked pyogranulomatous inflammation and multiple adhesions between the thoracic wall and organs of the thoracic cavity including lungs (and oesophagus in No.5). These findings correlated with macroscopic findings in the thoracic cavity of abnormal content (thick, pale fluid) and adhesions involving multiple organs. In male No. 5, a severe abscess was also found in the thoracic cavity, correlating with a macroscopic soft pale mass, and marked pyogranulomatous inflammation of the pericardium correlated with a macroscopic observation of thickened pericardium.

Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
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.

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.
Estrous cyclicity was unaffected by treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
In the testes, 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-specific abnormalities were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval, mating performance, fertility, gestation length and index were unaffected by treatment.
All females, including the premature decedents were pregnant, and with the exception of Group 1F No. 59 (discussed previously) successfully gave birth to live young.
There was no effect of parental treatment with Macrolex Orange 3G on the mean numbers of implantation sites, litter size, sex ratio or offspring survival to Day 13 of age.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

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 Macrolex Orange 3G.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effect of parental treatment with Macrolex Orange 3G on offspring survival to Day 13 of age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean offspring body weights on Day 1 of age and subsequent body weight gain up to Day 13 of age was similar in all groups and no effect of parental treatment with Macrolex Orange 3G was inferred.
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):
There was no effect of treatment on the circulating levels of thyroxine (T4) in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age and none of the TSH (thyroid stimulating hormone) samples required analysis.

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 Macrolex Orange 3G was inferred.

The low incidence of nipples observed among male offspring on Day 13 of age showed no relationship to parental treatment with Macrolex Orange 3G.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Mean serum T4 concentrations (pg/mL)

 

 

Group

 

Treatment

 

Dose

 

(mg/kg/day)

 

Adult Male at     Termination

Day 13 of age Offspring

Male

Female

 

 

1

 

 

Vehicle (corn oil)

 

 

0

Mean

40500

54800

50800

SD

6350

19600

8320

CV

15.7

35.8

16.4

N

10

8

8

 

 

2

 

 

Macrolex Orange 3G

 

 

100

Mean

42700

51700

53400

SD

11900

9970

6760

CV

27.9

19.3

12.7

N

10

10

10

 

 

3

 

 

Macrolex Orange 3G

 

 

300

Mean

44100

50400

50700

SD

7030

5690

5330

CV

15.9

11.3

10.5

N

10

10

10

 

 

4

 

 

Macrolex Orange 3G

 

 

500

Mean

38900

49700

48600

SD

5400

7060

6440

CV

13.9

14.2

13.3

N

10

10

10

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

 

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

500

0

100

300

500

Hyaline Droplets, Increased

 

 

 

 

 

 

 

 

Minimal

2

4

4

7

0

0

0

0

Slight

0

2

2

1

0

0

0

0

Total

2

6

6

8

0

0

0

0

Tubular Pigment, Increased

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

2

4

7

Total

0

0

0

0

0

2

4

7

Number of tissues examined

10

10

10

10

8

10

10

10

 

Applicant's summary and conclusion

Conclusions:
In conclusion, within the context of this reproductive/developmental toxicity screening study it was concluded that a dose level of 500 mg/kg/day, the maximum feasible dose, represented the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity in the CD rat. Macrolex Orange 3G 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 Macrolex Orange 3G by oral gavage administration for at 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 Macrolex Orange 3G at doses of 100, 300 or 500 mg/kg/day (the maximum feasible dose) by oral gavage administration at a volume dose of 5 mL/kg/day. A similarly constituted Control group received the vehicle, corn oil, 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

Oral administration of Macrolex Orange 3G to parental Sprague Dawley (Crl:CD(SD) rats at dose levels of 100, 300 or 500 mg/kg/day (maximum feasible dose) for two weeks prior to pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was well tolerated. There were no test item-related premature deaths, changes in general clinical condition or post-dosing signs observed, and body weight performance and food consumption among parental animals were unaffected.

Estrous cyclicity, pre-coital interval, mating performance, gestation length and index were unaffected by treatment with Macrolex Orange 3G.

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.

The analysis of organ weights at scheduled termination indicated slightly high body weight-adjusted mean kidney weights compared to Control in all groups of treated males and females.

Test item-related abnormalities detected at macroscopic examination were limited to orange colouration of the lower gastro-intestinal tract contents and urinary bladder among males, indicative of the colour of the test item, and pale areas on the liver. Among treated females, there was a non-dose-dependent increased incidence of dark kidneys when compared to Controls.

Histopathological changes that were attributable to treatment occurred in the kidneys of animals in all treated groups. An increase in the incidence and severity of hyaline droplets in cortical tubules was seen in all groups of treated males. Among all groups of treated females, yellow-brown pigment was seen in cortical tubules of the kidneys. Special staining was used to identify the pigment in a Control female and two females treated at 500 mg/kg/day.

Perls/Prussian Blue staining to detect haemosiderin was negative. Schmorls staining for lipofuscin was positive, with the intensity and distribution of staining clearly increased in treated females compared to Controls.

F1 Litter responses

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

Conclusion

In conclusion, within the context of this reproductive/developmental toxicity screening study it was concluded that a dose level of 500 mg/kg/day, the maximum feasible dose, represented the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity in the CD rat. Macrolex Orange 3G showed no evidence of being an endocrine disruptor.