Hydrocarbon waxes (petroleum), oxidized, Me esters

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2018 to 22 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

various with no impact on results or integrity of study results (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Chinese regulatory test guidelines

Version / remarks:

Inclusion of recovery groups approved by UK Home Office

Deviations:

yes

Remarks:

various with no impact on results or integrity of study results (see below)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

PURPOSE OF STUDY
- The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, by oral gavage administration of the test item, to Han Wistar rats for at least six weeks.
- An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for at least four weeks post treatment. Males and females within the recovery subgroups were not paired.

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

solid

Details on test material:

- Appearance / physical state: Dark brown paste
- Storage conditions: Room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan

Details on species / strain selection:

ANIMAL MODEL
- The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan;WIST was used because of the historical control data available at the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- Supplier: Envigo RMS (UK) Ltd
- Number of animals ordered: 54 males and 58 females. Spare animals were removed from the study room after treatment commenced.
- Duration of acclimatization: Six days prior to the commencement of treatment (males) or 20 days prior to the commencement of treatment (females).
- Age of the animals at the start of the treatment: 84 to 90 days (males) or 98 to 104 days (females).
- Weight range of the animals at the start of treatment: 306 to 354 g (males) or 183 to 244 g (females).

ALLOCATION AND IDENTIFICATION
- Allocation: Non-selective allocation to cages on arrival. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed  20% of the mean for each sex.
- Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
- Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

ANIMAL REPLACEMENT
- Before the commencement of treatment, two females were rejected during the acclimatization period because they had irregular estrous cycles and were replaced with spare animals of suitable weight from the same batch.
ANIMAL CARE AND HUSBANDRY
- Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. On one occasion, the temperature was outside the indicated ranges, this deviation was minor and/or of short duration and was not considered to have influenced the health of the animals and/or the outcome of the study.
- Lighting: Artificial lighting, 12 hours light : 12 hours dark.
- Electricity supply: Public supply with automatic stand-by generators.

ANIMAL ACCOMMODATION
- Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, recovery, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
- Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Number of animals per cage: Up to four animals of one sex (pre-pairing, treatment and recovery); one male and one female (pairing); up to four animals (males after mating); one female (gestation); one female + litter (lactation).

ENVIRONMENTAL ENRICHMENT
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
- Nesting material: From Day 20 after mating and throughout lactation, paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

DIET SUPPLY
- Diet: SDS VRF1 Certified pelleted diet. A sample (250 g) of each batch of diet used was retained within Pharmacy (frozen, -10 to -30°C) 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.

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, the Aspen chew blocks and the polycarbonate shelters.
- No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

IDENTITY OF TREATMENT GROUPS
- The study consisted of one control and three treated groups.
- The F0 generation of the study was identified as shown in the table below.
- 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 / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

ROUTE OF ADMINISTRATION
- The oral route (gavage) was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

RATIONALE FOR DOSE LEVEL SELECTION
- The dose levels for investigation in this study (0, 350, 700 or 1000 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a 28 day toxicity study (with recovery groups) conducted at Covance Shardlow. In that study dose levels of 350, 700 or 1000 mg/kg/day were investigated. The study concluded that the oral administration of test item to Han Wistar rats for a period of 28 consecutive days followed by a 14 day recovery period at dose levels up to 1000 mg/kg/day did not result in any treatment-related changes in animals of either sex at any dose level.
- Given that no effects were reported in the reproductive organs in the 28-day study the high dose level for this screening study could be 1000 mg/kg/day. The intermediate and low doses selected, 700 and 350 mg/kg/day, respectively, provided a geometric spacing of the dose levels.

TEST ITEM PREPARATION AND ANALYSIS
- Formulation: Details are shown in the table below.
- Method of preparation: The test item was heated at a maximum temperature of 70ºC before use. The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred, whilst being heated in a water bath at 50ºC, until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order by dilution of individual weighings of the test item.
- Frequency of preparation: Weekly
- Storage of formulation: Refrigerated (2 to 8 °C)
- 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.

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.
- Dose volume: 4 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.

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION ANALYSIS
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 3.75 and 250 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix.
- Achieved concentration: Samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item.
- Analysis: The method of analysis and results are presented in Annex 2 (attached).

Duration of treatment / exposure:

- Main phase males: Two weeks pre-pairing up to necropsy after minimum of six weeks.
- Main phase females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
- Recovery phase animals: At least six weeks followed by a minimum 28 days recovery period.
- The offspring (F1) received no direct administration of the test item; any exposure was in utero or via the milk.

Frequency of treatment:

Daily

Details on study schedule:

See details of study schedule (attached)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

700 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:

- Control: 15 males and 15 females
- Test item 350 mg/kg bw/day: 10 males and 10 females
- Test item 700 mg/kg bw/day: 10 males and 10 females
- Test item 1000 mg/kg bw/day: 15 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

See details of study design (attached)

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. The final animal check was not performed on one day. 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.

SIGNS ASSOCIATED WITH DOSING
- Detailed observations were recorded at the following times in relation to dose administration and according to the following schedule: F0 males and recovery phase females (Week 1 – daily; Week 2 to 4 - twice each week; Week 5 onwards – weekly) and F0 main phase females (Week 1 – daily; Week 2 – twice; 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 of all groups and as late as possible in the working day (not recorded on one occasion)).

CLINICAL SIGNS
- A detailed physical examination was performed on each animal to monitor general health according to the following schedule: F0 males and recovery phase females (weekly); F0 main phase females (Week 1 and 2 – weekly; Gestation phase - Days 0, 7, 14 and 20; Lactation phase - Days 1, 6 and 13).

BODY WEIGHT
- Body weight of F0 males and recovery phase females was recorded weekly during acclimatisation (not reported); before dosing on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase where applicable; on the day of necropsy.
- Body weight of main phase females was recorded weekly during acclimatisation (not reported); 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 for F0 animals:
- Weekly, from the day that treatment commenced, including the recovery phase where applicable.
- Food consumption was not recorded for Main phase males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
- For Main phase females after mating food consumption was performed to match the body weight schedule (where the end feeder recording matched a body weight day): Days 0-7, 7-14 and 14-20 after mating and Days 1-4, 4-7 and 7-13 of lactation.
- From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

WATER CONSUMPTION
- Water consumption was recorded by weight (over a 3-day period on each occasion) in each of the pre-pairing weeks for each cage of animals, using water bottles fitted with sipper tubes.
- Fluid intake was assessed by daily visual observation in subsequent weeks. No effect was observed, and consequently quantitative measurements were not performed.

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 CYCLE
- Dry smears using cotton swabs:
(i) For 15 days before pairing (main phase females)
(ii) For 15 days from the start of treatment (recovery phase females)
- Wet smears using pipette lavage:
(i) For 14 days before treatment (all females including spares and recovery phase females); animals that failed to exhibit 4-5 day cycles were not allocated to study.
(ii) After pairing until mating for Main phase females.
(iii) For four days before scheduled termination (all females, including the recovery phase females).

Sperm parameters (parental animals):

Not examined

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 of age - all F1 offspring.
- Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

TERMINAL INVESTIGATIONS
- Method of kill: Carbon dioxide asphyxiation with subsequent exsanguination (all adult animals); Decapitation (offspring - selected for thyroid hormone sampling on Day 4 and 13 of age); intraperitoneal injection of sodium pentobarbitone (Offspring - not selected for thyroid hormone sampling).
- 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: After six weeks of treatment (F0 main phase males); Day 25 after last day of pairing (F0 Main phase females failing to litter); Day 13 of lactation (F0 Main phase females); Selected offspring for thyroid hormone analysis - Day 4 of age (F1 offspring); Terminal kill - Day 13 of age (F1 offspring); After at least 28 days without treatment (F0 recovery phase males and females).

PATHOLOGY PROCEDURES FOR ALL ADULT ANIMALS
- The organs weighed, tissue samples fixed, and sections examined microscopically are detailed for F0 animals in the table below.
- Animal ID was retained.
- Each uterine horn (main phase females): Number of implantation sites was counted and confirmed if none were visible at visual inspection.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes, where modified Davidson's fluid was used.

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 Main phase animals in Groups 1 and 4 at scheduled termination.
- Abnormalities: All adult Main phase and Recovery phase 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.

LIGHT MICROSCOPY
- Tissues preserved for examination were examined as shown in the table below.
- 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.

Postmortem examinations (offspring):

- Premature deaths (offspring): Where possible, a fresh external macroscopic examination with an assessment of stomach for milk content was performed. Abnormal pups were retained.
- F1 offspring on Day 4 of age: Blood sampling required; externally normal offspring discarded without examination; externally abnormal offspring examined and retained pending possible future examination.
- F1 offspring on Day 13 of age: Blood sampling required; all animals were subject to an external macroscopic examination and abnormal tissues were retained; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.

Statistics:

See below

Reproductive indices:

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

Offspring viability indices:

- Litter size: Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.
- Survival indices: The indices shown below were calculated for each litter. Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded. Group mean values were calculated from individual litter values.
(i) Post-implantation survival index (%) = Total number of offspring born / total number of uterine implantation sites * 100
(ii) Live birth index (%) = Number of live offspring on Day 1 after littering / total number of offspring born * 100
(iii) Viability index (%) = Number of live offspring on Day 4 (before blood sampling) / number live offspring on Day 1 after littering * 100
(iii) Lactation index (%) = Number of live offspring on Day 13 after littering / number of live offspring on Day 4 (after blood sampling) * 100
- 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 blood sampling) and 13 of age. Group mean values were calculated from individual litter values. Percentage males = Total number of males in litter / total number of offspring in litter * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

- No animals showed dose signs related to dosing of the test item throughout the treatment period and there were no clinical signs observed during the study that were considered related to treatment.
- One female receiving 350 mg/kg/day test item was killed on Day 25 of gestation because she failed to litter. At necropsy this female was found to be not pregnant and had no macroscopic findings.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weight gains during the treatment period in Main phase males, Recovery phase animals and Main phase females before pairing (F0) were not adversely affected by treatment. During the recovery phase there was no effect on bodyweight gains in the test item previously treated animals.
- Reproductive phase females treated at all dose levels of test item had a body weight gain throughout gestation that was comparable with the Control group. During lactation, females treated with 350, 700 or 1000 mg/kg/day test item had slightly high overall body weight gains during (Day 1-13) when compared with the Control group (115 %, 115 % and 130 % of the Controls, respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Food consumption was unaffected by treatment with the test item throughout treatment, gestation and lactation and also recovery.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- Males and females receiving 1000 mg/kg/day drank slightly more water than the controls at the end of each pre-pairing week (Day 4-5 and Day 11-12) but thereafter no effects were apparent for the remainder of the recorded periods.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no microscopic test item-related lesions seen in the limited list of reproductive tissues examined in animals killed after at least 6 weeks of treatment or after a subsequent 28-day recovery period.
- All histological changes were considered to be incidental and unrelated to treatment with test item.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

ESTROUS CYCLES, PRE-COITAL INTERVAL, MATING PERFORMANCE, FERTILITY, GESTATION LENGTH AND GESTATION INDEX
- All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatisation period.
- Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. One female receiving 350 mg/kg/day test item was killed on Day 25 of gestation because she failed to litter.
- All Main study females were not cycling before termination (Days 10-13 of lactation) and were in diestrous at termination. Recovery phase animals were all cycling before termination.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

Basis for effect level:

other: no adverse effects relevant to humans reported at highest dose tested

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 related to parental treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

- Litter size up to Day 13 of age was unaffected by treatment with test item. The number of females raising their litter to Day 13 of age was 10, 9, 10, 10 for females treated with 0, 350, 700 or 1000 mg/kg/day test item respectively. One female receiving 350 mg/kg/day was killed on Day 25 as she failed to litter.
- Group mean percentage of post implantation survival index, live birth index and viability index were not affected by treatment with the test item.
- The sex ratio of male:female offspring per group for females treated with 700 or 1000 mg/kg/day had a low percentage of males when compared to the sex ratio of the Control offspring. On review of the individual inter-group variation (and if Dam 4F 140 was excluded because she had an atypical litter comprising only four female pups) then the lowest percentage of male offspring was in Group 3F (Dam 125: 15.4%). The second lowest percentage of male offspring was in Group 2F (Dam 111: 25%). The range of ratios for Group 1F and Group 4F were similar (1F, 30.8% to 75% and 4F 33% to 61.5%). In addition, a review of the individual results included in the Historical Control Data set revealed that many females had percentage offspring male ratios on Day 1 that were below the lowest ratio reported in Group 4F on this study (33 %), excluding the atypical litter.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Mean offspring bodyweight and mean bodyweight gain for male and female offspring of parents treated with test item at 350, 700 or 1000 mg/kg/day were not affected by treatment when compared with Controls.

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):

- There was no effect on ano-genital distance in the offspring of parents treated with test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

- No male offspring had nipples.

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

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.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: no adverse effects relevant to humans reported at highest maternal dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

FORMULATION ANALYSIS

- Detailed results are shown in Annex 2 (attached).

-The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

- The homogeneity and stability was confirmed for the test item in Arachis Oil BP formulations at nominal concentrations of 3.75 mg/mL and 250 mg/mL during magnetic stirring for 2 hours and ambient temperature storage (15 to 25 °C) for one day. 

- The mean concentrations of test item in test formulations analysed for the study were within ± 7 % of nominal concentrations (See table below), confirming accurate formulation. The difference from mean remained within 2 %, confirming precise analysis.

- Recovery results during the trial remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method.

- Mean analysed concentrations and difference from mean values were calculated using unrounded figures.

ANALYSED CONCENTRATIONS FOR TEST ITEM IN ARACHIS OIL BP

Occasion	Group	Nominal concentration (mg/mL)	Analysis 1 (mg/mL)	Analysis 2 (mg/mL)	Mean (mg/mL)	RME (%)	Difference from mean (%)	Procedural recovery (%)
Week 1	1	0	ND	ND	–	–	–	–
Week 1	2	87.5	82.8	83.1	83.0	-5.1	±0.19	96.8
Week 1	3	175	166	167	167	-4.6	±0.33	96.5
Week 1	4	250	243	244	243	-2.8	±0.20	98.3
Last week	1	0	ND	ND	–	–	–	–
Last week	2	87.5	82.2	81.6	81.9	-6.4	±0.40	95.1
Last week	3	175	170	173	171	-2.3	±0.84	94.3
Last week	4	250	247	239	243	-2.8	±1.51	94.5

RME = Relative mean error, representing the deviation from nominal

ND = Not detected

 

THYROID HORMONE ANALYSIS

- Detailed results are shown in Annex 3 (attached).

- There was considered to be no effect of treatment on serum Thyroxine (T4) levels in the F0 males or male and female offspring at Day 13 of age.

- Mean serum T4 concentrations (pg/mL) are shown in the table below.

-Slightly high levels of T4 were reported for the test item treated groups of males when compared with the controls (statistical significance attained for all groups).

-However, the difference between the mean T4 data of the control group versus Group 2, 3 and 4 was 23 %, 24 % and 24 %. When this was evaluated against the fact that the method allows for variability in QC samples up to +/- 20 % difference to the nominal concentration these were only very slight increases and it was a very similar response across all the groups. No effect of treatment was apparent in the T4 levels in the Day 13 Offspring and adult male thyroid weights were not affected by treatment. It was therefore considered that this apparent effect on adult male T4 levels was due to normal biological variation and not the result of test item administration.

MEAN SERUM T4 CONCENTRATIONS (pg/mL)

Group	Treatment	Dose (mg/kg bw/day)		Adult terminal males	Male offspring on Day 13 of age	Female offspring on Day 13 of age
1	Control	0	Mean	49900	54600	57700
1	Control	0	SD	12075.52	5878.067	6776.307
1	Control	0	N	10	10	10
2	Test Item	0	Mean	61300 *	54400	54600
2	Test Item	0	SD	8641.663	8894.942	8094.511
2	Test Item	0	N	10	9	9
3	Test Item	0	Mean	66500 *	59800	62800
3	Test Item	0	SD	11179.17	4877.203	9437.49
3	Test Item	0	N	10	10	10
4	Test Item	0	Mean	62100 *	59000	61900
4	Test Item	0	SD	16271.68	9500.102	13404.21
4	Test Item	0	N	10	9	10

* Significant differences between the groups compared: p < 0.05

Applicant's summary and conclusion

Conclusions:

Oral administration of test item to parental Han Wistar rats at dose levels of 350, 700 or 1000 mg/kg/day for six 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. No findings related to the previous administration of the test item were apparent in the recovery animals after four weeks post treatment. Mating performance, fertility, litter size and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or growth of the offspring. It was therefore concluded that there was no adverse effect on the reproductive indices measured on this study. 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 effects was considered to be 1000 mg/kg/day.

Executive summary:

GUIDELINE

The study was designed to meet the requirements of Organisation for Economic Co-operation and Development: Testing of Chemicals (Guideline 421; Reproduction/Developmental Toxicity Screening Test; 29 July 2016). The recovery groups were included for compliance with Chinese Regulatory Test guidelines and were approved by the UK home office.

 

METHODS

Test item dose levels were 0, 350, 700 and 1000 mg/kg bw/day.

 

Main phase males (10) were treated daily for two weeks before pairing up to necropsy after a minimum of six consecutive weeks.

 

Main phase females (10) were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. The F1 generation was killed on Day 4 or Day 13 of age but received no direct administration of test item; any exposure was in utero or via the milk.

 

Recovery phase animals (5 males and 5 females from the 1000 mg/kg bw/day group) were treated daily for a minimum of six consecutive weeks followed by a minimum of 28 days recovery.

 

A similarly constituted Control group was assigned to each phase, and received the vehicle, Arachis oil BP, throughout the same relative treatment period.

 

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

 

The clinical condition, litter size and survival, sex ratio, body weight, 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 (not analysed) and Day 13 of age for thyroid hormone analysis. 

 

RESULTS

Parental (F0) responses

There were no treatment-related deaths. There were no signs seen in association with the dosing procedure or at the routine weekly examinations.

The bodyweight performance of the Main phase males, Recovery phase animals and Main phase females before pairing (F0) were not adversely affected by treatment. During the recovery phase there was no effect on bodyweight gains in the test item previously treated animals.

There were no effects on bodyweight gain during the gestation period but during the lactation phase females treated with 350, 700 or 1000 mg/kg/day test item had slightly high overall body weight gains (Day 1-13) when compared with the Control group (115%, 115% and 130% of the Controls, respectively).   

Food consumption was unaffected by treatment.

Water consumption was slightly high, when compared with the Controls, in males and females receiving 1000 mg/kg/day at the end of each pre-pairing week (Day 4-5 and Day 11‑12) but thereafter no effects were apparent for the remainder of the recorded periods.

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. One female receiving 350 mg/kg/day test item was killed on Day 25 of gestation because she failed to litter. All Main study females were in diestrous at termination. Recovery phase animals were all cycling before termination.

There was no effect of treatment on organ weights in animals killed at the end of the treatment period. At the end of the recovery phase the uterus (including cervix and oviducts) absolute and adjusted weights were low in females previously given 1000 mg/kg/day, when compared with Controls (54% of Control, statistical significance attained).

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

CONCLUSION

Oral administration of test item to parental Han Wistar rats at dose levels of 350, 700 or 1000 mg/kg/day for six 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. No findings related to the previous administration of the test item were apparent in the recovery animals after four weeks post treatment.

Mating performance, fertility, litter size and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or growth of the offspring. It was therefore concluded that there was no adverse effect on the reproductive indices measured on this study.

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 effects was considered to be 1000 mg/kg/day.