4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 May 2017 and 10 November 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

EC Number:

500-429-8

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Cas Number:

159034-96-5

Molecular formula:

(C15 H16 O2 . C9 H13 N O . C4 H10 N2 . C3 H5 Cl O)x

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine Amine adduct with epoxy resin~

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test item: THEMIS (EC 500-429-8)
CAS number: 159034-96-5
Intended use: Industrial chemical.
Appearance: Pale brown powder.
Storage conditions: At room temperature (10 to 30oC) in the dark.
Batch number: 161129
Expiry date: 28 November 2017
Purity: 100%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from the batch of test item, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Strain/Species Crl:CD(SD) rat.
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: six days before commencement of treatment. Females: 20 days before commencement of treatment.
Age of the animals at the start of treatment Males: approximately 69 to 76 days old. Females: approximately 83 to 90 days old.
Weight range of the animals at the start of treatment Males: 307 to 396 g. Females: 251 to 316 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages. Estrous cycles were evaluated prior to treatment commencing. 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 before dosing commenced to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal 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 the start of treatment Body weight range extremes: One male; Irregular estrous cycles: Three females

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

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages were distributed on the racking to 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.
Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after finalization of the report. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% w/v methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The required amount of test item was weighed and ground in an agate mortar. The vehicle was gradually added and mixed with a pestle. The suspension in the mortar was transferred to a measuring cylinder. Any agglomerates were broken down. The mortar and pestle was washed with the vehicle and the washing was transferred to the measuring cylinder. The final volume was adjusted by adding the correct quantity of the vehicle to achieve the required concentrations of 10.6, 31.4, and 94.3 mg/mL. After preparation the dosing formulations were mixed several times by end-over-end rotation and then transferred into brown glass containers.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
- Frequency of preparation: Daily, up to and including Day 10 of treatment. Weekly thereafter.
- Storage of formulation: Ambient temperature (15 to 25°C) for up to 24 hours. Refrigerated storage (2 to 8°C) for up to eight days.
- Test item accounting: 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.

VEHICLE
- Concentration in vehicle: 10.6, 31.4, and 94.3 mg/mL.
- Amount of vehicle (if gavage): 3.5 mL/kg

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:

Preparation of Test Samples
Test samples were prepared by dispensing 1 mL of formulation into a pre-weighed (weight recorded) centrifuge tube. The sample and tube were weighed an accuracy of 0.01 mg. The samples were dried overnight in a drying oven set to 105°C overnight. Five control vehicle samples were prepared with each analysis to calculate the residue weight of controls.
Samples were removed from the oven the next day, allowed to equilibrate to ambient temperature and re-weighed to an accuracy of 0.01 mg.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (0.5%MC) with known amounts of test item. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Validation of the Analytical Procedure The analytical procedure was validated by determining the following parameters:
The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of5 mg/mL, 10 mg/mLand 100mg/mL during the method validation.

Homogeneity and Stability in 0.5% Methylcellulose Formulations
The homogeneity and stability of test item in 0.5% MCformulations was assessed at nominal concentrations of 5 mg/mL, 10 mg/mL and 100 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into 4amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15-25ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) 1 hour and 2 hours (10 mg/mL only), duplicatesamples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 24 hoursstorage the contents were remixed and sampled as detailed above.
On Day 1 of Trial 1 (5 mg/mL and 100 mg/mL), Bottle 1 was left to stir for 1 hour and 2hours to assess the ambient stability, continuously stirred.

Refrigerated Storage (2-8ºC)
The remaining bottleswererefrigerated on receipt and on Days1, 8 sand 15;the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 20minutes and duplicate samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
The low level Trial 1 (5 mg/mL) was not analyzed at Day 15 due to low results obtained throughout the trial.

Concentration of Dose Formulations
For Week 1, 5 and 8, freshly prepared test formulations were sampled (10× 1mL control group and 6 × 1 mL for treated groups, accurately weighed into pre-weighed centrifuge tubes) by Pharmacy personnel and submitted for analysis. Five control samples and three treated samples from each groupwere analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for THEMIS (EC 500-429-8)in 0.5% MCwith respect to the method accuracy and precision.
Method accuracy and precision were confirmed : a mean procedural recovery value of 96.6% (CV=5.73%, n=5) was obtained for 5mg/mL, 102.5% (CV=0.1.77%, n=5) was obtained for 10 mg/mLand 98.9% (CV=0.12%, n=5) was obtained for 100mg/mL.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of test item in 0.5% MCformulations was assessed with respect to the level of concentration at nominal concentrations of 5mg/mL, 10mg/mL and 100 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1days and refrigeration for up to 8 days (5 mg/mL) and 15days(10 mg/mL and 100 mg/mL). At each time-point, the mean analyzed concentration for the sixsamples remained within 6% of the initial time zero value and the coefficient of variation was less than 3%. The low level (5mg/mL) on Day 1 + 1 hour was higher than the results seen for the rest of the trial and was close to nominal, but was +10.6% from time zero results. The 2 hour timepoint waswithin ±10% from time zero but the trial was stopped for this level as the results were low from nominal concentration. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method with the exception of 2 recovery samples –1 recovery at 5mg/mL on Day 0 and 1 recovery at 100 mg/mL on Day0. The 5 mg/mL recovery is in line with recoveries observed for this level and will be reported. The 100 mg/mL recovery value is excluded as it is lower than recoveries at this level. There are 4 other recoveries for this analysis and the results are considered to be unaffected.

Concentration of Dose Formulations
The mean concentrations were within applied limits +10/-15%, confirming the accuracy of formulation with the exception of Group 2 in Week 5. This group has a low result from nominal and wide precision. Contingency samples were prepared and the results were confirmed. A mean of 6 samples is reported for this group.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the methodwith the exception of 10 mg/mL recoveries and 30 mg/mL recoveries in Week 5. These are considered to be preparation error. Samples results are not corrected for recovery values and the results are reported.

CONCLUSION
The analytical procedure was successfully validated with respect to methodaccuracy and precision.
The homogeneity and stability was confirmed for test item n 0.5% MC formulations at nominal concentrations of 5mg/mL, 10 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1day and refrigerated storage for up to 8 days(5 mg/mL) and 15 days (10mg/mL and 100 mg/mL).
The mean concentrations of test item in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation with the exception of Group 2 in Week 5. The coefficient of variationbetween the samples was within 10%, confirming precise analysis, with the exception of Group 2 in Week 5.

Duration of treatment / exposure:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.

Frequency of treatment:

Daily

Details on study schedule:

Study Schedule
Duration of Treatment
Males Two weeks before pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Time Schedule
Study initiation (Study Plan signed by Study Director) 24 May 2017
Experimental start date (pre-study chemistry) 11 May 2017
Animal arrival
Males 7 June 2017
Females 24 May 2017

Estrous cycle evaluation commenced 30 May 2017
Treatment commenced 13 June 2017
F0 paired for mating 27 June 2017
F0 necropsy
Males 18 July 2017
Females 1 to 8 August 2017

Experimental completion date (pathology data locked) 10 November 2017

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

37 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

110 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Study Design and Identity of Treatment Groups
The study consisted of one control and three treated groups. The F0 generation of the study was identified as follows:
Group Treatment Dose (mg/kg/day)# Number of animals Animal numbers
Male Female Male Female
1 Control 0 10 10 1-10 41-50
2 Test item 37 10 10 11-20 51-60
3 Test item 110 10 10 21-30 61-70
4 Test item 330 10 10 31-40 71-80
# Expressed in terms of material as supplied.

The F1 generation received no direct administration of the test item. 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 3.5 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.
Frequency Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
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.

Examinations

Parental animals: Observations and examinations:

Serial Observations
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 or if they exhibited pregnancy loss.
A complete necropsy was performed in all cases as described in "Termnal Investigations Section.

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, 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; 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, 6 and 12

Body Weight
The weight of animals was recorded as follows:
F0 males Weekly during acclimatization. Before dosing on the day treatment commenced (Day 1) and weekly thereafter. On the day of necropsy.
F0 females Weekly during acclimatization. Before dosing on the day 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 first day of treatment until pairing). Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording: Days 0-6, 7-13 and 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant 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):

Estrous Cycles
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 (nominally Days 10-13 of lactation).

Litter observations:

Records Made During Littering Phase
Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to 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 (see Section 4 of this report, ‘Deviation from study plan’ for details of one observation that was missed).

Postmortem examinations (parental animals):

Terminal Investigations
Method of Kill
All adult animals: Carbon dioxide asphyxiation. Each animal was subsequently exsanguinated.
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.

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.
Time of Necropsy
F0 males After at least five weeks of treatment.
F0 females whose litter died before Day 13 On or after day the last offspring died.
F0 females Day 13 of lactation (following terminal blood sampling).
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 as follows for F0 animals:
Pathology procedures for all F0 parental animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Cowpers glands * *
Duodenum * * *
Epididymides (caput, corpus and cauda)* * * *
Glans penis * *
Ileum * * *
Jejunum * * *
LABC (levator ani-bulbocavernosus) muscle * *
Liver * * * *
Lung * * *
Mesenteric lymph node * * *
Ovaries * * * *
Prostate * *
Seminal vesicles (with coagulation gland) * *
Spleen * * * *
Testes * * * *
Thyroids * *
Uterus with cervix and oviducts * *
Vagina *
* Organs weighed, samples fixed or sections examined microscopically

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted.
Female whose litter died before Day 13 of lactation Mammary tissue appearance.

Organ Weights
For bilateral organs, left and right organs were weighed together.

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 adult animals killed prematurely. All adult animals in Groups 1 and 4 at scheduled termination.
Abnormalities and mesenteric lymph nodes All adult animals.
Spleen All adult females in Groups 2 and 3.
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.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All adult animals from all groups. All specified above.
Terminal sacrifice All adult animals in Groups 1 and 4 All specified above.
All adult animals (males and females) Abnormalities and mesenteric lymph nodes.
All adult females in Groups 2 and 3 Spleen.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Photomicrographs were taken of the treatment-related findings reported in the spleen and mesenteric lymph nodes and these are included in the pathology report (refer to Section 4 (Deviations from Study Plan) of this report for further details).

Serial Observations
Clinical Observations
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.
There were no signs associated with dosing and so no Appendix is included in this report.
Body Weight
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (+ SD) was calculated separately for males and females and the group mean values derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period. Offspring body weight change was calculated relative to Day1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.
Food Consumption
Group mean food consumptions and standard deviations were derived from unrounded cage values.
Values presented for the amount of food consumed in each cage in each food consumption period allow for any animal that died or was killed during that period.
Estrous Cycles
The percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 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 show 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.

Postmortem examinations (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 in appropriate fixative.
- F1 offspring on Day 4 of age: Blood sampling was undertaken. Externally normal offspring were discarded without examination. Externally abnormal offspring were examined, and retained pending possible future examination.
- F1 offspring on Day 13 of age: Blood sampling was undertaken. All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormal offspring were retained in appropriate fixative. Thyroid glands were preserved from one male and one female in each litter.

Offspring Examinations
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring of this report for details of an oversight that occurred resulting in nipple counts not being recorded for the offspring of one animal).
There were limited counts and consequently only an appendix is presented in this report.

Terminal Investigations
Organ Weights
The abbreviations used have the following meanings:
LABC Levator ani-bulbocavernosus
Thyroids + Paras Thyroids and parathyroids
Uter+Cerv+Ovid Uterus including cervix and oviducts

Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

Statistics:

See "Any other Information...", below

Reproductive indices:

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = Number of animals mating x 100
Animals paired
Conception rate (%) = Number of animals achieving pregnancy x 100
Animals mated
Fertility index (%) = Number of animals achieving pregnancy x 100
Animals paired
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. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born x 100
Number pregnant
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) 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 x 100
Total number of uterine implantation sites

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 x 100
Total number of offspring born

Viability index (%) = Number of live offspring on Day 4 (before blood sampling) x 100
Number live offspring on Day 1 after littering
Lactation index (%) = Number of live offspring on Day 13 after littering x 100
Number of live offspring on Day 4 (after blood sampling)
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 x 100
Total number of offspring in litter
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 x 100
Total number of offspring in litter

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):

No signs that were treatment-related were seen in association with the dosing procedure in males or females.
When compared with the controls, the only treatment-related clinical signs recorded at the routine weekly examinations were slightly higher incidences of encrustations which were seen on the lower dorsal thorax and the muzzle of males receiving 330 mg/kg/day and slightly higher incidences of brown staining of the upper dorsal thorax in females in the lactation phase receiving test item at all dose levels (incidences of 1, 5, 4 and 5 in Groups 1, 2, 3 and 4, respectively).
One female receiving 330 mg/kg/day (Animal No. 79) had a swollen area on the upper ventral thorax which was evident during the gestation and lactation periods. Macroscopic examination did not reveal any abnormalities to account for the swollen area. A further female from this group (Animal No. 80) had aqueous red discharge from the vagina on Day 1 of the lactation period but otherwise her health was satisfactory

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male receiving 37 mg/kg/day (Animal No. 19) was killed on Day 22 of treatment for reasons of animal welfare. The animal had a marked decline in general clinical condition, with notable body weight loss (a loss of 52 grams between Day 15 and 22). Macroscopic examination revealed multiple pale areas on the lungs and bronchi, the left lobe of the thyroids was small and scabs were present on the hind limbs. Histopathological examination revealed a moderate focal epidermal ulceration associated with a slight scab in the skin, which correlated with the macroscopic lesion. There were no microscopic correlates for the other lesions. In addition, minimal multifocal vacuolated macrophages were observed in the mesenteric lymph node. This premature death was not considered treatment related. The skin ulceration was considered to be the major factor contributing to the death of this animal.
One female given 110 mg/kg/day (Female No. 66) was killed before Day 1 of lactation due to total litter loss. There were no significant findings on macroscopic or microscopic examination but macroscopic examination did reveal that the mammary tissue appeared inactive and pale.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on the body weights of the males at all dose levels.
Body weight gains for females receiving 330 mg/kg/day were unaffected by treatment during the pre-pairing phase but were slightly low, when compared with controls, throughout the gestation phase but particularly during Day 14 to 20. The overall body weight gain (Day 0 to 20) for these females was 92% of control. Low gains continued to be seen in these females during the lactation phase from Day 4 to 13; the overall gain during this phase was 74% of control.
Mean body weight gains for females receiving 110 mg/kg/day were unaffected by treatment with THEMIS (EC 500-429-8) throughout the pre-pairing and gestation phase, however overall body weight gain during the lactation phase was slightly reduced when compared with the controls (83% of control).
There was no effect of treatment on the body weights of females receiving 37 mg/kg/day. A small group mean body weight loss was observed in this group between Days 1 to 4 of lactation with seven of the ten females losing weight (between 2 and 12 grams) but the subsequent weight gain in these females was satisfactory. This was considered a chance event and not related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on the food consumption of males or females receiving test item at 37, 110 and 330 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or 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 or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis. (Table 1 below)

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment Related Findings
Findings related to treatment were seen in the mesenteric lymph nodes and spleen.
Mesenteric Lymph Node
When compared with the controls, an increase in the number of vacuolated macrophages was recorded in the mesenteric lymph nodes of males given 37, 110 or 330 mg/kg/day, and females given 110 or 330 mg/kg/day, with a dose-related increase in incidence and/or severity.
Text-table 3: Summary of treatment related findings in the mesenteric lymph nodes for animals killed at the end of the treatment periods
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 37 110 330 0 37 110 330
Vacuolated Macrophages, Increased
Minimal 0 5 9 7 0 0 2 5
Slight 0 0 0 3 0 0 0 0
Total 0 5 9 10 0 0 2 5
Number of tissues
examined 10 10 10 10 10 10 10 10

Spleen
When compared with the controls, an increase in the number of vacuolated macrophages was recorded in four females given 330 mg/kg/day.
Text-table 4: Summary of treatment related findings in the spleen for animals killed at the end of the treatment periods
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 37 110 330 0 37 110 330
Vacuolated Macrophages, Increased
Minimal 0 0 0 0 0 0 0 4
Total 0 0 0 0 0 0 0 4
Number of tissues
examined 10 0 0 10 10 10 10 10
These findings were considered not adverse.

Incidental findings
One female given 37 mg/kg/day (Female No. 55) had findings suggestive of gavage mis dosing with deposition of test-material and vehicle in the thorax. There were multiple areas surrounding the lungs consisting of severe granulomatous inflammation. These inflammatory changes consisted of large aggregates of foreign body cells encompassing numerous round eosinophilic globules and food material in some areas, with interspersed fibrosis, and pleural hyperplasia and fibrosis. This was considered due to gavage error and therefore procedural.
A single incidence of a hepatodiaphragmatic nodule was observed in one male given
330 mg/kg/day (Male No. 37). This finding correlated macroscopically with a mass located in the median lobe of the liver, which had protruded through the diaphragm. This is a common congenital spontaneous liver abnormality that can occur in rats of any age (Eustis et al., 1990).
The incidence and distribution of all other findings were considered unrelated to treatment.

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 acclimitisation period.
Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. One female on the study was (Group 3 Female No. 66 receiving 110 mg/kg/day) was killed before Day 1 of lactation after showing a total litter loss.
All females were not cycling before termination (Days 10-13 of lactation) and were in diestrous at termination.

Reproductive performance:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimitisation period.
Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. One female on the study was (Group 3 Female No. 66 receiving 110 mg/kg/day) was killed before Day 1 of lactation after showing a total litter loss.
All females were not cycling before termination (Days 10-13 of lactation) and were in diestrous at termination.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive/developmental toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

37 mg/kg bw/day (nominal)

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs related to parental treatment

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on offspring body weight.

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:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance of both male and female offspring on Day 1 of age showed no adverse effects of parental treatment.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

One Control litter had one male offspring with nipples (one pup in Litter 47). Three of the Group 2 litters had male offspring with nipples (one pup in Litter 52, two pups in Litter 54 and one pup in Litter 58). One Group 3 Litter (Litter 62) also had two male offspring with nipples on Day 13 of age. No nipples were observed in all other male offspring. The presence of nipples in these offspring is considered fortuitous and is not related to treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter Size, Sex Ratio and Survival Indices
The number of implantations was marginally lower than control in females receiving 330 mg/kg/day and this resulted in a slightly smaller group mean litter size for these females on Day 1; however values were within the historical control data (HCD) range so no effect of treatment is inferred. The post implantation survival index was also slightly lower than control, but was within the HCD range, and the live birth, viability and lactation indices were all satisfactory. There was also no effect on the sex ratio of the offspring.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

330 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

clinical biochemistry

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

System:

other: various

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

330 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

TABLE 1 : Mean T4 Serum Sample Concentrations (pg/mL)

Group		Treatment		Dose				Adult Males at Termination		Day 13 of age Offspring
			(mg/kg/day)		Male				Female
1		Vehicle (0.5% w/v methyl cellulose)		0		Mean		39400		38500		43600
			SD			10200		6170		7150
			% CV			25.9		16.0		16.4
			N			10		10		10
2		Test item		37		Mean		41000		40700		46200
			SD			5300		5710		4100
			% CV			12.9		14.0		8.9
			N			9		10		10
3		Test item		110		Mean		40400		39100		43900
			SD			4330		5550		6620
			% CV			10.7		14.2		15.1
			N			10		9		9
4		Test item		330		Mean		39900		42100		45500
			SD			3510		8000		6240
			% CV			8.8		19.0		13.7
			N			10		10		10

Applicant's summary and conclusion

Conclusions:

In conclusion, oral administration of test item, to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 37, 110 or 330 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit a reduction in body weight gain in females receiving 330 mg/kg/day, during the gestation and lactation phases of the study. This was not of sufficient magnitude to be considered adverse.
At the histopathological examination increased vacuolated macrophages were recorded in the mesenteric lymph nodes of males given 37, 110 or 330 mg/kg/day and females given 110 or 330 mg/kg/day and there was also an increase in the number of vacuolated macrophages seen in the spleen of four females given 330 mg/kg/day. Although the tissue distribution of the vacuolated macrophages was different from the 28-day study (Ref: LSI Medience Corporation Study No. PI50573) and the severity of the finding was minimal, there is insufficient evidence to demonstrate non-adversity in this study.
Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.
In the context of this study, the test item showed no evidence of being an endocrine disruptor.
The no-observed-adverse-effect-level (NOAEL) for reproductive/developmental toxicity was considered to be 330 mg/kg/day but due to the histopathological finding of vacuolated macrophages a NOAEL was not established for general toxicity in the males but can be given as 37 mg/kg/day for the females.

Executive summary:

This study was a screening test for reproductive/developmental effects, with the administration by oral gavage of test item, an industrial chemical, to Sprague Dawley rats for at least five weeks.

Three groups of ten male and ten female rats received test item at doses of 37, 110 or 330 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 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. A similarly constituted Control group received the vehicle, 0.5% w/v% methylcellulose, at the same volume-dose (3.5 mL/kg/day) as treated groups.

For the adult animals the following assessments were made: clinical condition, body weight, food consumption, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight, macroscopic pathology and histopathology investigations.

For the offspring the following assessments were made: clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance and nipple counts (males only), and macropathology. Blood samples were also collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis (T4).

Results

Parental (F0) responses

There were no treatment-related deaths. There were no signs seen in association with the dosing procedure. At the routine weekly examinations, when compared with the controls, encrustations on the lower dorsal thorax/muzzle were seen at a higher incidence in males receiving 330 mg/kg/day and a higher incidence of brown staining of the body was seen in females receiving test item at all dose levels. 

The body weight performance of the males was unaffected by treatment. Body weight gains for females receiving 330 mg/kg/day were slightly low, when compared with controls, throughout the gestation phase but particularly during Day 14 to 20 (overall body weight gain, Day 0 to 20; 92% of control). Low gains continued to be seen in these females during the lactation phase from Day 4 to 13 (overall body weight gain; 74% of control) and were also evident in females receiving 110 mg/kg/day (83% of control) in this phase.

Food consumption was unaffected by treatment.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

Estrous cycles, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. All females were in diestrous at termination.

There was no effect of treatment on organ weights.

The macropathological examination of the adult males and females did not reveal any findings considered related to the administration of test item.

The histopathological examination revealed treatment-related findings in the mesenteric lymph nodes and spleen. When compared with the controls, increased vacuolated macrophages were recorded in mesenteric lymph nodes of males given 37, 110 or 330 mg/kg/day, and in females given 110 or 330 mg/kg/day, with a dose-related increase in incidence and/or severity. Increased vacuolated macrophages were observed in the spleen of four females given 330 mg/kg/day. These findings were considered not adverse.

F1 Litter responses

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

There was no effect of treatment on litter size or offspring survival indices. There was also no effect on the sex ratio of the offspring. 

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment.

There was no effect of treatment on offspring body weight.

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

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Conclusion

In conclusion, oral administration of test item, to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 37, 110 or 330 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit a reduction in body weight gain in females receiving 330 mg/kg/day, during the gestation and lactation phases of the study. This was not of sufficient magnitude to be considered adverse. 

At the histopathological examination increased vacuolated macrophages were recorded in the mesenteric lymph nodes of males given 37, 110 or 330 mg/kg/day and females given 110 or 330 mg/kg/day and there was also an increase in the number of vacuolated macrophages seen in the spleen of four females given 330 mg/kg/day. Although the tissue distribution of the vacuolated macrophages was different from the 28-day study (Ref: LSI Medience Corporation Study No.PI50573) and the severity of the finding was minimal, there is insufficient evidence to demonstrate non-adversity in this study.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.

In the context of this study, test item showed no evidence of being an endocrine disruptor.

The no-observed-adverse-effect-level (NOAEL) for reproductive/developmental toxicity was considered to be 330 mg/kg/day but due to the histopathological finding of vacuolated macrophages a NOAEL was not established for general toxicity in the males but can be given as 37 mg/kg/day for the females.