Pentyl 2-cyano-(3-(6-methoxybenzothiazol-2-ylimino)-2,3-dihydro-1H-isoindol-1-ylidene)acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Animal arrival) 21 April 2021
Experimental completion date (T4 Bioanalysis) 09 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

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 Sprague-Dawley [Crl:CD(SD)] 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.

Duration of acclimatization
Males: Six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment
Males 69 to 75 days old.
Females 84 to 90 days old.

Weight range of the animals at the start of treatment
Males 337 to 410 g.
Females 239 to 303 g.

Animal facility
Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated. Minimum of 15 supply air changes per hour.

Temperature and relative humidity
Monitored and maintained within the range of 20-24°C 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 throughout the study except during pairing.

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 four animals of one sex
Pairing one male and one female
Males after mating up to four 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 late gestation and littering) and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except during pairing and late gestation and littering) and replaced at the same time as the cages.

Paper shavings Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted.
A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30°C). Samples will be 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.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 0.5% (w/v) methylcellulose.

Details on exposure:

Method of preparation
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder, which had been wetted with vehicle, the mortar was rinsed with vehicle, and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.

Formulated in ascending order of concentration.

Frequency of preparation
Weekly, subdivided into daily aliquots and was prepared up to eight days in advance of the first day of dosing.

Storage of formulation Refrigerated (2 to 8°C) for up to 15 days.

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.


Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined, and specimen formulations at 1 to 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Labcorp Study number 8451440).
The stability of a homogenous suspension of the test item in the vehicle were demonstrated at ambient temperature (15 to 25°C) for four hours and refrigerated (2 to 8°C) for 15 days.

Details on mating procedure:

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:

The analytical method involved dilution with tetrahydrofuran followed by final dilution with water/methanol/formic acid solution prior to quantitation performed using liquid chromatography with tandem mass spectrometric detection (LC MS/MS).

Sample concentrations were determined with reference to external calibration standards freshly prepared in the concentration range 2.5 ng/mL to 25 ng/mL.

LC-MS/MS conditions
Instrument: Sciex API4000 (Analyst version 1.4.2 software) coupled to Waters Acquity UPLC system

Ionisation mode: Ionspray positive

Ion monitoring details: Primary transition m/z 447>359

Parameters: Curtain gas flow: 30 Collision gas pressure: 6
Nebulizer gas 1: 40 Ionspray voltage: 5000
Nebulizer gas 2: 40 Declustering potential: 66
Desolvation temperature: 400 Collision energy m/z 447>359: 33

Column: Acquity UPLC® BEH C18 (2.1 mm x 50 mm, 1.7μm), or equivalent

Column temperature: 45°C

Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M ammonium formate
Mobile phase B: Methanol:formic acid (100:0.1 v:v)

Gradient: Time (mins) %A %B
0 10 90
0.2 10 90
2.0 2 98
4.5 2 98
5.0 10 90
6.0 10 90

Cycle time: 6 minutes

Weak needle wash: Methanol:water:formic acid (90:10:0.1 v:v:v)

Strong needle wash: Propan-2-ol:dimethyl sulfoxide:water:methanol:formic acid (25:25:25:25:1 v:v)

Injection volume: 5 μL

Flow rate: 0.5 mL/min

Retention time: Approximately 1.0 minute

Concentration of Dose Formulations
The first and last week formulations were sampled. For groups 1 to 4, 4 x 1 mL were sampled from the middle of the formulation by Pharmacy personnel.

Duplicate aliquots from all groups were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency and stored refrigerated at approximately +4°C.
Re-dilution analysis was performed for Group 3 last week formulations due to an outlier result for one replicate which suggested a dilution error during the analytical procedure. Re dilution of the sample from initial extract confirmed the analytical error.

Samples were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
The mean concentrations of Disperse Orange FC84508in test formulations analyzed during the study and the deviation of the mean result from the nominal value are detailed in Table 1.
Re-dilution analysis was performed for Group 3 last week formulations due to a dilution error in the original analysis resulting in an outlier result (less than the limit of detection) for one replicate.

The final mean concentrations were within the ideal nominal accuracy limits of +10/ 15% for all formulation occasions tested.

Duration of treatment / exposure:

Males Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

Study initiation (Study Plan signed by Study Director) 13 April 2021

Experimental start date (Animal arrival) 21 April 2021

Animal arrival
Females 21 April 2021
Males 05 May 021

Estrous cycle evaluation commenced 27 April 2021

Treatment commenced 11 May 2021

F0 pairing commenced 25 May 2021

F0 necropsy
Males 10 to 11 June 2021
Females 29 June 2021 to 04 July 2021

Experimental completion date (T4 Bioanalysis) 09 October 2021

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 males, 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
In an acute oral (gavage) toxicity study, a limit dose of 2000 mg/kg body weight test substance as a 20% suspension in Tylose H 4000 G4 PHA (0.5 % in deionized water) was tested in five male and five female Sprague-Dawley rats according to OECD guideline 401. With exception of discolored orange feces, between four hours and one day after administration, no clinical signs were observed until the end of the study. Development of body weight was not impaired, except one female, which showed body weight loss between Day 8 and Day 15. No abnormalities were noted at necropsy.

The sub-acute toxicity of the test substance was examined in an oral (gavage) 28-day repeat dose toxicity study in Sprague-Dawley rats according to OECD guideline 407 followed by a 14 day recovery period. Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 days. On Day 29, five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days. No deaths occurred throughout the study. Behavior, state of health, neurotoxicological, hematological and blood serum parameter analysis remained unaffected by the administration of the test substance in all dose groups. Body weight gain, organ weights and food and water consumption were comparable in control and dose groups. No test substance related changes were detected by urine analysis or at necropsy or histopathology. In conclusion, repeated administration of test substance did not cause any substance related changes at the doses administered. Hence, under the test conditions, NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/day in rats.

Based on these data, dose levels of 100, 300, and 1000 mg/kg bw/day were selected for this study in order to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.

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 day cycles were not allocated to the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management. The 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 allocation Atypical estrous cycles Four females
Body weight range extremes Two males


-

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
Weeks 2 to 4 - twice each week (middle and end of week)
Week 5 onwards - once each week

F0 females
Week 1 - daily
Week 2 - twice (middle and end of week)
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
• Pre-dose observation
• One to two hours after completion of dosing (See Section 4)
• 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
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.

F0 females
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, 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 measured to match the body weight recording:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-7 and 7-13 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.


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.

Oestrous cyclicity (parental animals):

Dry smears For 15 days before pairing using cotton swabs

Wet smears
Using pipette lavage during the following phases:

• For 14 days before treatment; animals that failed to exhibit 4-5 day cycles were not allocated to the study.

• After pairing until mating (for a maximum of 14 days).

• Females showing no evidence of mating: following completion of the pairing period females were separated from the males and vaginal smearing continued for up to five days or until the first estrus smear was seen. If a female showed an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

• For four days before scheduled termination (nominally Days 10 to 13 of lactation).


Estrous Cycle
The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced are presented:
Regular: All observed cycles of 4 or 5 days
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

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:

Records Made During Littering Phase
Clinical observations of offspring 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, 11 and 13 of age.

Ano-genital distance Day 1 - all F1 offspring.

Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy
F0 males After at least 4 weeks of treatment.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 13 of lactation.


Pathology procedures for all F0 parental animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Epididymides (caput, corpus and cauda)** * *
Ovaries * * *
Pituitary *
Prostate * *
Seminal vesicles (with coagulation gland)* *
Testes * * * *
Thyroid *
Uterus with cervix and oviducts *c
*
Seminal vesicles (with coagulation gland)* *
Testes * * * *
Thyroid *
Uterus with cervix and oviducts *c
* Organs weighed, samples fixed or sections examined microscopically.
c Excluding females which were not pregnant, or fail to litter.
Animal ID retained

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.
All adult animals killed prematurely.
All adult animals in Groups 1 and 4 at scheduled termination.
Abnormalities only All adult animals.
Routine staining Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.


Females
The following were recorded (including premature decedents):
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection after modified Salewski staining.

Postmortem examinations (offspring):

Offspring
Premature deaths Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained pending possible future examination.

F1 offspring on Day 4 of age Externally normal offspring discarded without examination.

Externally abnormal offspring identified on despatch to necropsy; examined externally, and retained pending possible future examination.

F1 offspring on Day 13 of age All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative.

Thyroid glands were preserved from one male and one female in each litter, where possible.

Statistics:

Please refer to "Any other information on materials and methods"

Reproductive indices:

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100


Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

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

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling), 7, 11 and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

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/ 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) x 100

Group mean values were calculated from individual litter values

Offspring 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 of Disperse Orange FC84508 at dose levels up to and including 1000 mg/kg bw/day was generally well tolerated in adult animals. There were no clear treatment-related changes in clinical condition or signs observed in relation to dose administration at any dose level investigated.

Mortality:

no mortality observed

Description (incidence):

One female (2F 111) was killed for welfare reasons on Gestation Day 18 showing signs of irregular breathing, piloerection, whole body pallor and dull eyes. Microscopic examination revealed marked abscessation of the thoracic cavity and marked epicardial inflammation, which correlated with the necropsy findings of adhesion(s) of the thoracic cavity and thickened pericardium respectively. The thoracic abscessation was considered to have been the major factor contributing to poor clinical condition of this animals and its early death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain for males during treatment and for females before pairing was unaffected by treatment at all dose levels investigated.
During the gestation phase, females receiving 1000 mg/kg bw/day showed a consistent but slightly higher bodyweight gain when compared to Controls, and resulted in a slightly higher overall mean bodyweight gain (GD0-20). There was no effect of treatment on bodyweight gain at 100 or 300 mg/kg bw/day during gestation.
Overall mean body weight gain at 100, 300 or 1000 mg/kg bw/day during lactation was slightly low when compared to Controls, although the differences were considered to be minor and were unrelated to treatment.
PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS- "Body weight and body weight changes - Parental generation"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for the two-week period before pairing for males and females and post-pairing for males, and for females during gestation and lactation was considered to be unaffected by treatment at all dose levels investigated.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

Analysis of serum T4 concentration revealed low group mean T4 concentration in F0 males given 300 or 1000 mg/kg bw/day, when compared to Controls. Mean serum T4 concentration in samples obtained from F0 males given 100 mg/kg bw/day were unaffected by treatment. Assessment of individual T4 values showed that the majority of values in F0 males at 300 or 1000 mg/kg bw/day were within the 95-percentile historical control range. However, four Control values were above this range, resulting in an apparent difference between Control and treated groups. Therefore, the observed differences at 300 or 1000 mg/kg bw/day, when compared to the Control group mean concentration in F0 males, were not attributed to treatment.

When compared to Controls, the mean serum T4 concentration for male and female F1 offspring on Day 13 of age derived from parental animals treated with Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day were unaffected by treatment

PLEASE REFER TO THE ATTACHED TABLES "Thyroid Hormone Analysis"

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), an/or their severity was as expected for Sprague-Dawley rats of this age. Therefore, all findings were considered not test item related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus except one female (3F 123) was confirmed not pregnant.

Please refer to the results tables in "Any other information on results"

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

One female (No. 111) given 100 mg/kg bw /day was killed for welfare reasons, and one female (No. 123) that received 300 mg/kg bw/day was recorded as failed to litter and was dispatched to necropsy on Day 25 after mating confirming non-pregnancy for this female. Therefore, the following litter assessment is based on 10, 9, 9 and 10 litters at 0, 100, 300 and 1000 mg/kg bw/day.

The mean number of implantations and subsequently litter size for females receiving 1000 mg/kg bw/day was slightly high when compared to Controls; with differences attaining statistical significance. Mean number of implantations and litter size for females receiving 100 or 300 mg/kg bw/day was similar to Controls

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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 that were considered to be related to treatment.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, overall mean bodyweight gain for offspring (Day 1-13 of age) was slightly low for male and female offspring when compared to Controls although these differences did not attain statistical significance and was considered to reflect the slightly higher litter size at 1000 mg/kg bw/day.

Offspring bodyweights at 100 or 300 mg/kg bw/day was unaffected 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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance for both male and female offspring was unaffected by parental treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were apparent for male offspring on Day 13 of age.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings observed in offspring killed or that died prior to scheduled termination were predominantly the absence of milk in the stomach.

There were no macroscopic findings for offspring at scheduled termination on Day 13 of age.

Histopathological findings:

not examined

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:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Any other information on results incl. tables

Formulation Analysis

The mean concentrations of formulation samples taken during this study were within +10/-15% of the nominal concentration, confirming accurate formulation and the percentage difference from mean values remained within 7%, confirming precise analysis.

Procedural recovery values were within the validated range confirming the continued accuracy of the analytical method.

 

Thyroid Hormone Analysis

The purpose of this phase of the study was to measure total Thyroxine (T4) concentrations in rat serum using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) using an analytical method validated in Labcorp Study Number FF58YR and documented in Bioanalytical Method Number BM/2016/0632. T4 is an endogenous serum biomarker therefore, concentrations of T4 were measured using a surrogate matrix (4% BSA and 0.2% Tween20 in Phosphate Buffered Saline) with a calibration range of 700 to 70000 pg/mL.

The mean rat serum concentrations of T4 in the samples obtained from F0 male (adult) animals in Groups 1 to 4 in week 5 and the F1 male and female offspring on Day 13 of age following daily oral gavage administration of Disperse Orange FC84508 to adults are summarized the attached "Thyroid Hormone Analysis"

 

	Body weight and body weight change - group mean values (g) for males and for females before pairing (F0)	Request ID: 5447388

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day						Change	Change	Change	Change	Change	Change
/Sex		1	8	15	22	29		1-8	8-15	15-22	22-29	1-15	1-29
Statistics	test	Av	Wi	Wi	Wi	Wi		Wi	Wi	Wi	Wi	Wi	Wi
1M	Mean	361	385	410	432	450		24	24	22	18	49	88
	SD	15.3	15.9	17.3	20.3	21.1		9.8	6.7	7.7	6.2	14.4	16.9
	N	10	10	10	10	10		10	10	10	10	10	10
2M	Mean	359	384	405	422	441		25	21	17	19	46	82
	SD	12.0	14.3	18.5	21.0	23.4		5.5	4.8	5.8	5.5	9.5	14.0
	N	10	10	10	10	10		10	10	10	10	10	10
3M	Mean	368	392	414	431	450		24	22	17	19	46	82
	SD	12.2	13.8	19.0	15.6	19.1		5.7	8.6	8.1	9.1	13.0	17.6
	N	10	10	10	10	10		10	10	10	10	10	10
4M	Mean	367	388	415	433	452		21	27	18	19	48	84
	SD	20.5	24.2	31.0	35.8	37.2		7.5	8.3	8.7	9.0	12.6	22.5
	N	10	10	10	10	10		10	10	10	10	10	10

 

 

(cont)

Body weight and body weight change - group mean values (g) for males and for females before pairing (F0)

Request ID: 5447388

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day				Change	Change	Change
/Sex		1	8	15		1-8	8-15	1-15
Statistics	test	Av	Wi	Wi		Wi	Wi	Wi
1F	Mean	269	274	278		4	4	8
	SD	16.7	19.0	19.7		5.0	4.9	6.5
	N	10	10	10		10	10	10
2F	Mean	267	267	272		1	5	5
	SD	18.4	16.0	19.2		7.6	6.9	4.4
	N	10	10	10		10	10	10
3F	Mean	273	277	282		4	5	9
	SD	11.1	7.1	8.3		6.8	6.1	8.9
	N	10	10	10		10	10	10
4F	Mean	273	279	288		6	9	15
	SD	19.2	17.3	22.0		11.1	8.7	13.1
	N	10	10	10		10	10	10

 

Body weight and body weight change - group mean values (g) for females during gestation (F0)

Request ID: 5447389

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day					Change	Change	Change	Change
/Sex		0	7	14	20		0-7	7-14	14-20	0-20
Statistics	test	Wi	Wi	Wi	Wi		Wi	Wi	Wi	Wi
1F	Mean	279	307	338	412		28	31	75	133
	SD	17.5	18.5	23.8	26.3		5.9	9.0	11.2	16.2
	N	10	10	10	10		10	10	10	10
2F	Mean	273	302	333	411		29	31	74	139
	SD	21.0	26.0	25.8	29.0		15.4	4.8	10.4	14.3
	N	10	10	10	9		10	10	9	9
3F	Mean	286	314	348	422		29	34	73	136
	SD	7.9	12.5	17.4	25.8		7.9	8.4	14.8	23.6
	N	9	9	9	9		9	9	9	9
4F	Mean	289	322	358	441		33	36	83	152
	SD	22.0	24.4	28.4	39.9		7.4	9.7	13.7	25.2
	N	10	10	10	10		10	10	10	10

 

Body weight and body weight change - group mean values (g) for females during lactation (F0)

Request ID: 5447390

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day					Change	Change	Change
/Sex		1	4	7	13		1-7	7-13	1-13
Statistics	test	Wi	Wi	Wi	Wi		Wi	Wi	Wi
1F	Mean	310	318	332	347		22	15	36
	SD	20.8	21.7	22.0	17.6		4.8	10.0	9.6
	N	10	10	10	10		10	10	10
2F	Mean	312	315	331	345		19	14	33
	SD	23.5	26.5	26.7	29.4		6.0	13.7	9.9
	N	9	9	9	9		9	9	9
3F	Mean	330	336	347	357		17	10	27
	SD	21.8	22.9	26.2	19.5		10.9	18.1	15.6
	N	9	9	9	9		9	9	9
4F	Mean	333	341	351	363		18	13	31
	SD	33.7	28.0	30.2	25.2		5.2	11.3	14.5
	N	10	10	10	10		10	10	10

 

Estrous cycles - group values before treatment (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

	Number		Regular
	of		4 or 5 day	Irregular
Group	animals		cycles	cycle l	Acyclic y
1	10	N	10	0	0
		(%)	(100)
2	10	N	10	0	0
		(%)	(100)
3	10	N	10	0	0
		(%)	(100)
4	10	N	10	0	0
		(%)	(100)

l              At least one cycle of two, three or six to ten days

y             At least ten days without estrus

 

 

Estrous cycles - group values during treatment (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

	Number		Regular
	of		4 or 5 day	Irregular
Group	animals		cycles	cycle l	Acyclic y
1	10	N	8	2	0
		(%)	(80)	(20)
2	10	N	9	1	0
		(%)	(90)	(10)
3	10	N	10	0	0
		(%)	(100)
4	10	N	8	2	0
		(%)	(80)	(20)

l              At least one cycle of two, three or six to ten days

y             At least ten days without estrus

 

Pre-coital interval - group values (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

	Number
	of		Pre-coital interval (days)
Group	animals		1-4	5-8	9-12	13-14
1	10	N	10	0	0	0
		(%)	(100)
2	10	N	9	1	0	0
		(%)	(90)	(10)
3	10	N	10	0	0	0
		(%)	(100)
4	10	N	10	0	0	0
		(%)	(100)

 

Mating performance and fertility - group values (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group			Number
and	Number	Number	achieving	Percentage	Conception	Fertility
sex	paired	mating	pregnancy	mating	rate (%)	index (%)
1M	10	10	10	100	100	100
2M	10	10	10	100	100	100
3M	10	10	9	100	90	90
4M	10	10	10	100	100	100
1F	10	10	10	100	100	100
2F	10	10	10	100	100	100
3F	10	10	9	100	90	90
4F	10	10	10	100	100	100

 

Gestation length and gestation index - group values (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

	Number of						Number of complete	Gestation
	pregnant		Gestation length (days)				live litters	index
Group	animals		22	22.5	23	23.5	born	(%)
Statistics test			Lt
1	10	N	3	5	2	0	10	100
		(%)	(30)	(50)	(20)
2	10A	N	2	2	5	0	9	90
		(%)	(22)	(22)	(56)
3	9	N	1	2	5	1	9	100
		(%)	(11)	(22)	(56)	(11)
4	10	N	2	1	7	0	10	100
		(%)	(20)	(10)	(70)

A       Percentage distribution of gestation lengths calculated from nine animals - one pregnant female euthanized for welfare reasons

 

Stage of estrous cycle at termination - group values (F0)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

	Number			Estrus before		Cycle stage at termination
Group	smeared			termination		M	D	P	E
1	10	N		0		0	10	0	0
		(%)					(100)
2	9	N		0		0	9	0	0
		(%)					(100)
3	9	N		0		0	9	0	0
		(%)					(100)
4	10	N		0		0	10	0	0
		(%)					(100)

D             Diestrus

E             Estrus

M            Metestrus

P              Pro-estrus

 

 

Litter size - group mean values (F1)

Request ID: 5447416

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Implantations	Total @	Live on Day
/Sex				Before bleed		After bleed
				1	4	4	7	11	13
Statistics	test	Wi	Wi	Wi	Wi
1F	Mean	15.9	14.7	14.2	14.1	12.2	12.2	12.2	12.2
	SD	2.18	2.41	2.20	2.08	1.93	1.93	1.93	1.93
	N	10	10	10	10	10	10	10	10
2F	Mean	15.4	14.9	14.6	14.4	12.6	12.6	12.6	12.6
	SD	1.81	2.03	1.88	1.94	2.01	2.01	2.01	2.01
	N	9	9	9	9	9	9	9	9
3F	Mean	15.8	14.3	14.3	14.2	12.4	12.4	12.4	12.4
	SD	2.77	2.45	2.45	2.33	1.81	1.81	1.81	1.81
	N	9	9	9	9	9	9	9	9
4F	Mean	16.8	16.5	16.4*	16.2*	14.2	14.2	14.2	14.2
	SD	2.20	2.37	2.50	2.15	2.15	2.15	2.15	2.15
	N	10	10	10	10	10	10	10	10

 

@ - Includes offspring that died prior to the designated Day 1 of age

 

Offspring survival indices - group mean values (F1)

Request ID: 5447417

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Post implantation	Live birth	Viability index (%)	Lactation index (%)
/Sex		survival index (%)	index (%)	Day 4	Day 13
Statistics	test	Wi	Ch	Ch
1F	Mean	92.2	97.4	98.9	100.0
	SD	4.18	4.45	2.41	0.00
	N	10	10	10	10
	N<100%	9	3	2	0
2F	Mean	95.6	98.0	99.2	100.0
	SD	5.65	4.26	2.38	0.00
	N	9	9	9	9
	N<100%	4	2	1	0
3F	Mean	91.3	100.0	99.3	100.0
	SD	6.09	0.00	1.96	0.00
	N	9	9	9	9
	N<100%	7	0	1	0
4F	Mean	98.1*	99.3	99.0	100.0
	SD	3.06	2.26	3.01	0.00
	N	10	10	10	10
	N<100%	3	1	1	0

 

The following data were used for the statistics tests, animal indices for post implantation survival index and animal indices dichotomized to 1 when 100% and 0 otherwise for live birth and viability indices.

 

Offspring sex ratio - group mean values (F1)

Request ID: 5447418

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Total @			Live (before bleed) on Day						Live (after bleed) on Day
/Sex					1			4			4			13
		M	F	%M	M	F	%M	M	F	%M	M	F	%M	M	F	%M
Statistics	test			Wi			Wi			Wi
1F	Mean	5.6	9.0	38.5	5.5	8.8	38.5	5.4	8.7	38.4	5.4	6.8	44.5	5.4	6.8	44.5
	SD	1.26	1.89	7.17	1.27	1.75	6.89	1.17	1.64	6.90	1.17	1.55	8.44	1.17	1.55	8.44
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
2F	Mean	7.1	7.8	46.9	7.0	7.6	47.5	6.9	7.6	47.0	6.9	5.7	53.9	6.9	5.7	53.9
	SD	2.98	2.39	16.82	2.78	2.51	17.20	2.85	2.51	17.41	2.85	2.35	18.72	2.85	2.35	18.72
	N	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9
3F	Mean	7.1	7.2	48.9	7.1	7.2	48.9	7.0	7.2	48.7	7.0	5.4	55.5	7.0	5.4	55.5
	SD	3.02	2.39	15.94	3.02	2.39	15.94	2.83	2.39	15.64	2.83	2.24	18.27	2.83	2.24	18.27
	N	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9
4F	Mean	8.8	7.7	52.5*	8.7	7.7	52.1*	8.6	7.6	52.2*	8.6	5.6	59.6	8.6	5.6	59.6
	SD	2.57	1.06	9.03	2.71	1.06	9.64	2.55	1.07	9.79	2.55	1.07	10.43	2.55	1.07	10.43
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

 

@ - Includes offspring that died prior to the designated Day 1 of age and excludes unsexed offspring

 

 

Ano-genital distance - group mean absolute and adjusted values for offspring (F1)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Body weight (g)	Ano-genital
/Sex		Day 1	distance (mm)
Statistics test		Wi
1M	Mean	7.1	4.2
	SD	0.66	0.28
	N	10	10
2M	Mean	7.0	4.1
	SD	0.58	0.24
	N	9	9
3M	Mean	7.4	4.2
	SD	0.75	0.35
	N	9	9
4M	Mean	7.0	4.2
	SD	0.57	0.25
	N	10	10
Statistics test			Wi
1M	Adjusted Mean		4.2
2M	Adjusted Mean		4.1
3M	Adjusted Mean		4.2
4M	Adjusted Mean		4.2

 

(cont),

Ano-genital distance - group mean absolute and adjusted values for offspring (F1)

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Body weight (g)	Ano-genital
/Sex		Day 1	distance (mm)
Statistics test		Wi
1F	Mean	6.6	2.2
	SD	0.75	0.10
	N	10	10
2F	Mean	6.7	2.1
	SD	0.56	0.07
	N	9	9
3F	Mean	7.1	2.2
	SD	0.73	0.16
	N	9	9
4F	Mean	6.6	2.2
	SD	0.63	0.09
	N	10	10
Statistics test			Wi
1F	Adjusted Mean		2.2
2F	Adjusted Mean		2.1
3F	Adjusted Mean		2.2
4F	Adjusted Mean		2.2

 

Body weight and body weight change - group mean values (g) for offspring (F1)

Request ID: 5447419

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day of age (before bleed)			Day of age (after bleed)					Change	Change	Change	Change
/Sex		1	1 @	4	4	7	11	13		1-4	4-7	11-13	1-13
Statistics	test	Wi	Wi	Wi	Wi	Wi	Wi	Wi		Wi	Wi	Wi	Wi
1M	Mean	7.1	7.1	9.9	9.9	15.0	22.9	27.2		2.8	5.0	4.3	20.1
	SD	0.66	0.66	1.42	1.42	2.51	3.15	3.67		0.88	1.19	0.63	3.19
	N	10	10	10	10	10	10	10		10	10	10	10
2M	Mean	7.0	7.0	9.7	9.7	14.7	22.4	26.4		2.6	5.0	4.0	19.3
	SD	0.58	0.58	0.53	0.53	1.22	2.01	3.02		0.36	0.92	1.18	2.81
	N	9	9	9	9	9	9	9		9	9	9	9
3M	Mean	7.4	7.4	10.5	10.5	15.8	24.2	28.2		3.1	5.3	3.9	20.8
	SD	0.75	0.75	1.39	1.39	2.02	3.39	3.68		0.79	0.78	0.65	3.20
	N	9	9	9	9	9	9	9		9	9	9	9
4M	Mean	7.0	7.0	9.6	9.6	14.1	21.2	25.0		2.6	4.5	3.9	18.0
	SD	0.57	0.58	0.93	0.93	1.77	2.76	2.99		0.45	0.97	0.47	2.63
	N	10	10	10	10	10	10	10		10	10	10	10

 

@ - Includes only those pups surviving after the bleed

 

(cont)

Body weight and body weight change - group mean values (g) for offspring (F1)

Request ID: 5447419

 

	Control	Disperse Orange FC84508
Dose Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000

 

Group		Day of age (before bleed)			Day of age (after bleed)					Change	Change	Change	Change
/Sex		1	1 @	4	4	7	11	13		1-4	4-7	11-13	1-13
Statistics	test	Wi	Wi	Wi	Wi	Wi	Wi	Wi		Wi	Wi	Wi	Wi
1F	Mean	6.6	6.6	9.3	9.3	14.2	22.2	26.0		2.7	4.9	3.8	19.5
	SD	0.77	0.73	1.36	1.37	2.33	2.96	3.47		0.74	1.04	0.77	2.96
	N	10	10	10	10	10	10	10		10	10	10	10
2F	Mean	6.7	6.8	9.3	9.3	14.2	21.9	25.8		2.5	4.9	3.9	19.0
	SD	0.56	0.58	0.58	0.58	1.23	2.03	2.64		0.33	0.79	0.79	2.48
	N	9	9	9	9	9	9	9		9	9	9	9
3F	Mean	7.1	7.1	10.1	10.2	15.3	23.5	27.5		3.1	5.2	4.0	20.4
	SD	0.73	0.72	1.12	1.14	1.81	3.08	3.44		0.61	0.83	0.60	3.15
	N	9	9	9	9	9	9	9		9	9	9	9
4F	Mean	6.6	6.5	9.0	8.9	13.3	20.4	24.0		2.4	4.3	3.6	17.5
	SD	0.63	0.64	1.11	1.06	2.04	2.92	3.32		0.57	1.14	0.53	2.93
	N	10	10	10	10	10	10	10		10	10	10	10

 

@ - Includes only those pups surviving after the bleed

 

Applicant's summary and conclusion

Conclusions:

Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity is above 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Disperse Orange FC84508, by oral gavage administration for at least four weeks.

Three groups of ten male and ten female rats (F0 generation) received Disperse Orange FC84508 at doses of 100, 300 or 1000 mg/kg bw/day by oral gavage administration. 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 offspring (F1 generation) received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, aqueous 0.5% (w/v) methylcellulose, at the same volume dose (10 mL/kg bw/day) as treated groups.

During the study, clinical condition, body weight, food consumption, estrous cycles, pre-coital interval, mating performance, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.

Results

Parental responses

Analysis of serum T4 concentration revealed low group mean T4 concentration in F0 males given 300 or 1000 mg/kg bw/day, when compared to Controls. Mean serum T4 concentration in samples obtained from F0 males given 100 mg/kg bw/day were unaffected by treatment. Assessment of individual T4 values showed that the majority of values in F0 males at 300 or 1000 mg/kg bw/day were within the 95-percentile historical control range. However, four Control values were above this range, resulting in an apparent difference between Control and treated groups. Therefore, the observed differences at 300 or 1000 mg/kg bw/day, when compared to the Control group mean concentration in F0 males, were not attributed to treatment with the test substance.

Administration of Disperse Orange FC84508 at dose levels up to and including 1000 mg/kg bw/day was generally well tolerated in adult animals. There were no clear treatment-related changes in clinical condition or signs observed in relation to dose administration at any dose level investigated. One female given 100 mg/kg bw/day was killed for welfare reasons on Gestation Day 18, and the major contributing factor to its poor clinical condition was thoracic abscessation. This finding was considered to be incidental and was not related to treatment with Disperse Orange FC84508.

Body weight gain and food consumption for males during treatment and for females before pairing, during gestation and lactation were unaffected by treatment at all dose levels investigated.

Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment.

Litter responses

The clinical condition and survival of the F1 offspring, with litter size, sex ratio, offspring birth weights, offspring body weight gain and ano-genital distance were not adversely affected by parental treatment of Disperse Orange FC84508. There were no macroscopic findings for offspring and there were no nipples apparent for male offspring on Day 13 of age. Mean serum T4 concentration for male and female F1 offspring on Day 13 of age were unaffected by parental treatment.

Conclusion

Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity is above 1000 mg/kg bw/day.