Reaction mass of 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a GLP-compliant OECD Guideline 443 study with 2-Amylanthraquionone administered at 5, 20 and 75 mg/kg bw/day by oral gavage in rats, no effects on fertility were observed at any dose level. The NOAEL for reproductive effects was set at >75 mg/kg bw/day due to the absence of effects on reproductive parameters in the F0 and F1 generation, which was the highest dose level tested.

Systemic toxicity was observed in F0 and F1 animals, reflected in increased liver weights in F0 and F1 males and females at 75 mg/kg bw/day and hepatocyte hypertrophy of F0 males and F1 males and females at 75 mg/kg bw/day. In addition, the kidneys showed increased weights  in F1 animals, and mild/minimal mineralization was observed at 75 mg/kg bw/day in F0 and F1 males, as well as minimal to mild tubular basophilia in F1 males and females.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 October 2019 - 6 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Analytical method

Reason / purpose for cross-reference:

reference to other assay used for intermediate effect derivation

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Based on ECHA decision No. TPE-D-2114456461-51-01/F.

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks; 10 weeks premating exposure duration is required because there is no substance-specific information in the dossier supporting shorter premating exposure duration. Ten weeks exposure duration is supported also by the lipophilicity of the substance to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection: Within the repeated dose 90-day study (Charles River Study No. 511237) animals were dosed for 90 days at 0, 7.5, 25 and 75 mg/kg bw/day. At 75 mg/kg bw/day there were increases in the relative organ weight of the liver, kidney and adrenal weight. This was accompanied by minimal histopathological changes in the liver, spleen, thymus, thyroid, kidney and adrenals, the study concluded that these findings were considered not to be adverse. Additionally, there were small changes in albumin and creatinine in both sexes and urea in the females. It was concluded that the findings were considered not to be adverse. No toxicologically significant findings were noted in clinical appearance, functional observations, body weight, food consumption, ophthalmoscopy or macroscopic evaluations. Although none of the findings at 75 mg/kg bw/day in the 90-day toxicity study indicated a significant impairment of a vital organ function or indicated significant organ damage, based on the relative increase in several organ weights, the no observed adverse effect level (NOAEL) for 2-amylanthraquinone for the 90-day toxicity study was considered to be 25 mg/kg bw/day. Based on this study a high dose level of 75 mg/kg/day was considered to be suitable to induce moderate but not excessive effects of toxicity.
- Inclusion/exclusion of extension of Cohort 1B: not included, since the criteria lead down in column 2 of Section 8.7.3 (Annex X) are not met, i.e. there are no uses leading to significant exposure of consumers and professionals, the substance does not display any genotoxic effects in somatic cell mutagenicity tests in vivo, there are no toxicokinetic indications for a steady state of the internal dose for the active substance and/or its metabolites after extended exposure, there are no indications for an endocrine disrupting mode of action.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not included, since there are no particular concerns for developmental neurotoxicity (criteria in column 2 of 8.7.3 (Annex X) are not met).
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not included, since there are no particular concerns for developmental immunotoxicity (criteria in column 2 of Section 8.7.3 (Annex X) are not met).
- Route of administration: The oral route of administration was selected for this study as this route is considered the most appropriate route of administration for liquid substances such as the test substance.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in an incubator set to maintain 55°C.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: determined in Charles River Study No. 511240 as well as during dose formulation analysis as part of the present study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: determined in Charles River Study No. 511240.
- Reactivity of the test material with the incubation material used: glass vials were used.

Species:

rat

Strain:

Wistar

Remarks:

CRL:WI (Han)

Details on species / strain selection:

The Han Wistar rat was chosen as the animal model for this study as it is a rodent species
accepted by regulatory agencies for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, United Kingdom.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7-9 wks
- Weight at study initiation: (P) Males: 219-372 g (target 200-350 g); Females: 134-208 g (target: 125-225 g). The difference in the males' actual bodyweight and target bodyweight was considered not to impact the study as all animal were the correct age and were therefore considered to be suitable for use on this study.
- Fasting period before study: not applicable.
- Housing: Animals were initially housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with solid bottoms. Females were transferred to these cages for mating. Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from GD 20. F0 females with litters were retained in this type of cage until termination. On a suitable day after completion of mating, the males were re-housed with their original cage mates. Cohort 1A and 1B animals were housed 2 or 3 by sex per cage in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.
- Diet: SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.
- Water: water ad libitum from the public supply from water bottles which were changed as necessary throughout the course of the study.
- Acclimation period: The F0 animals were allowed to acclimate for a period of 17 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 23°C (target 19 to 23°C)
- Humidity (%): 19% to 62% (target 40 to 70%)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 October 2019 To: 7 May 2020

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations were prepared based on a method established at the Test Site (Test Site Study No. 511240) at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4°C protected from light, and dispensed daily.
The control item, Polyethylene glycol 400, was used as supplied and dispensed at least weekly, stored in a refrigerator set to maintain 4°C and protected from light, and dispensed daily for administration to Control animals.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: On Day 71 of dosing, females were housed with their allocated co-group male partner. The pairing period for each pair of animals was a maximum of 14 nights.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal lavage referred to as Day 0 of pregnancy
- If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing.
- After successful mating, females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from GD 20. F0 females with litters were retained in this type of cage until termination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis on Day 1, in Week 14 and in Week 25. For concentration analysis, samples were taken from the control and test item formulations. For homogeneity analysis, samples were taken from the test item formulations only. The homogeneity results obtained from the top, middle and bottom for the low, mid and high dose test item formulations were averaged and utilised as the concentration results.
Duplicate top, middle and bottom (duplicate middle only for control) sets of samples (500 mg) for each sampling time point were collected into cryo vials; triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. All samples were stored at <-15˚C within the established stability period. For stability analysis, a sample of test item was sent at ambient temperature, protected from light, along with the Day 1 samples. Analyses were performed by ultra performance liquid chromatography (UPLC) using a validated analytical procedure (Test Site Study No. 511240).
Concentration results were considered acceptable if sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. After acceptance of the analytical results, backup samples were discarded. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
Stability analyses performed previously in conjunction Test Site Study No. 511240 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

F0 males were treated daily for 10 weeks prior to mating until necropsy after the termination of the F0 females; total exposure 123-127 days.
F0 females were treated daily for 10 weeks prior to mating, then through mating, gestation and until at least LD 21 (termination between LD 22-24).
Cohort 1A animals were dosed daily from PND 21 until at least PND 90 (termination between PND 91 and 96).
Cohort 1B animals were dosed daily from PND 21 until at least PND 90 (termination between PND 98 and 100).

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

F0: 25 animals/sex/group
F1 weanlings - non-selected pups: 10 animals/sex/group
Cohort 1A: 20 animals/sex/group
Cohort 1B: 20 animals/sex/group, except for the high dose group, to which 21 males and 19 females were allocated. This was due mis-sexing, with a male pup inadvertently selected for the female group. This was noticed on Study Day 120. However, this animal was unable to be replaced as the dam and remaining pups from the litter had been sent for necropsy. This animal was kept on study so the same number of animals were dosed within each treatment group. There was considered to be a sufficient number of both sexes to be able to meet the objective of the study. This was therefore considered not to impact the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the repeated dose 90-day study (CRL Study No. 511237) a high dose level of 75 mg/kg bw/day was considered to be suitable to induce moderate but not excessive effects of toxicity.
- Rationale for animal assignment (if not random): F0: during the week before the commencement of dosing, individual body weights were checked to ensure that the mean body weights of each sex within each treatment group were within ± 5% of the mean weight of each sex. Cohort 1A and 1B: from each group, 40 males and 40 females were selected for post-weaning assessments, nominally by selecting up to 2 males and 2 females from each litter. Where fewer than 40 litters were weaned, the necessary additional animals were obtained by selecting an additional pup from appropriate litters; these appropriate litters were normally selected arbitrarily but attention was paid to the retention of as wide a genetic pool as possible. The selected pups were the median’th weight pups of that sex in the litter on LD 21. These pups were removed from their mother on LD 21, individually identified and housed in their new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.
- Fasting period before blood sampling for clinical biochemistry: yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study
- Cage side observations: observations for general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly starting in the week prior to the start of dosing (week -1).
- Animals were removed from the cage for examination.
- Observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also assessed.

PRE-/POST-DOSE OBSERVATIONS
Animals were observed regularly throughout the day on each day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males were weighed weekly beginning Week -1.
F0 females were weighed weekly from Week -1 until pairing for mating, and then on GD 0, 7, 14 and 20, and LD 1, 7, 14 and 21.
Litters were weighed en masse and by sex on LD 4, 7 and 14 and individually on LD 1 and 21.
F1 cohort 1A and 1B were weighed weekly from weaning, starting on a suitable day within one week of weaning for the majority of litters.
A weight was recorded on the day of scheduled necropsy for all animals.

FOOD CONSUMPTION (no feeding study):
Food consumption was measured as follows;
-for F0 animals, weekly from Week -1 for both sexes until pairing for mating.
-for F0 males weekly after mating and re-housing, and
-for mated F0 females over the periods GD 0-7, 7-14 and 14-20 and LD 1-7, 7-14 and 14-21.
-for F1 cohort 1A and 1B,weekly, starting on a suitable day within one week of weaning for the majority of litters.

WATER CONSUMPTION: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles. No inter-group differences were noted.

OBSERVATIONS OF FEMALES DURING PARTURITION AND LACTATION
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups were born) was designated LD 0. The duration of gestation in days was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it became soiled.

CLINICAL PATHOLOGY
-On the morning of necropsy, 10-13 F0 animals/sex/group were sampled for haematology, coagulation and clinical chemistry investigations. Animals were fasted prior to blood sampling. Blood was collected via the orbital sinus.
For haematology, K2EDTA was used as anticoagulant and the following parameters were analysed: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute) and Large unstained cells (absolute).
For coagulation, blood samples were collected using 3.2% (w/v) trisodium citrate as anticoagulant and processed for plasma, which was analysed for the following parameters: Activated partial thromboplastin time, Fibrinogen Prothrombin time and Sample quality.
For clinical chemistry, blood samples were collected using lithium heparin as anticoagulant and processed for plasma, which was analysed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride and Sample quality.

-In the last week of dosing, 10 animals/sex/group were housed in individual metabolism cages, with access to water only, over a period of 6 hours. Urine was collected for analysis of the following parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones and Blood.

THYROID HORMONE ANALYSIS
On the morning of necropsy, blood samples were collected from 10 F0 animals/sex/group from the jugular vein. Animals were fasted prior to sampling. Samples were analysed for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) using validated analytical procedures. T3 and T4 were measured by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a solid phase enzyme immunometric assay.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored daily for F0 females from Day 57 of dosing until day of detection of a copulatory plug in situ and/or of sperm in the lavage. Vaginal lavages were taken early each morning and the stages of estrous observed was recorded. Vaginal smears were also examined on the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries.

Sperm parameters (parental animals):

Parameters examined in F0 males:
- testis weight (both sides)
- epididymis weight (both sides)
- epidydimis (right cauda): assessment of percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL) and total sperm count expressed per cauda epididymis and per gram of cauda epididymis.
In addition, a sperm smear was prepared and stained. From the control and high dose animals (both generations), at least two hundred sperm per animal were evaluated for morphological abnormalities.
- one testis: determination of total spermatid count expressed per testis and per gram of testis.

Litter observations:

PARAMETERS EXAMINED IN F1 OFFSPRING
LD 0-LD 4
The number of live and dead pups born in each litter was recorded as soon as possible after
completion of parturition on LD 0. The live pups were counted, sexed and weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including LD 4.
Assessment of ano-genital distance was measured for both sexes on PND 1.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Where 4 males or 4 females were not available, extra pups of the opposite sex were retained to ensure a total number of 8 pups. When the total number of pups in a litter on LD 4 was ≤ 8 pups, no litter size adjustment occurred.

LD 5 - LD 21
From LD 5, the total live pups were counted daily, and were sexed and examined for abnormality again on LD 7, 14 and 21. These pups were weighed en masse, sexes separate, on LD 4, 7 and 14. On LD 1 and 21 all pups were weighed individually.
Nipple retention was assessed in males on PND 13.

SEXUAL MATURATION
Cohort 1A and 1B animals were examined for sexual maturation.
Commencing at 28 days of age, F1 females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day.
F1 females had their estrous cycles monitored daily from the day after vaginal patency (circa PND 28) until 1 estrous cycle was confirmed, then daily from at least PND 75 up to and including the day of scheduled necropsy.
Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

SPERM PARAMETERS
Parameters examined in Cohort 1A males:
- testis weight (both sides)
- epididymis weight (both sides)
- epidydimis (right cauda): assessment of percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL) and total sperm count expressed per cauda epididymis and per gram of cauda epididymis.
In addition, a sperm smear was prepared and stained. From the control and high dose animals (both generations), at least two hundred sperm per animal were evaluated for morphological abnormalities.
- one testis: determination of total spermatid count expressed per testis and per gram of testis.

CLINICAL PATHOLOGY
-On the morning of necropsy, 10-14 animals/sex/group from Cohort 1A were sampled for haematology, coagulation and clinical chemistry investigations. Animals were fasted prior to blood sampling. Blood was collected via the orbital sinus. Sampling, processing and analyses were similar to those described for F0 animals.
-In the last week of dosing, 10 animals/sex/group from Cohort 1A were housed in individual metabolism cages, with access to water only, over a period of 6 hours. Urine was collected for urinanalysis. Parameters were identical to those described for F0 animals.

THYROID HORMONE ANALYSIS
The following blood samples were taken from the F1 litters:
-PND 4: culled offspring (up to 4 rats/litter), taken via cardiac puncture without anticoagulant. At least 1 mL of blood per litter (from 2 or more pups per litter, as necessary) was collected. Samples were collected from all litters at each dose level with sufficient numbers of culled pups.
-PND 22-24: unselected pups (10 rats/sex/group from 10 litters), taken from the jugular vein. At least 0.8 mL/pup was collected.
-Cohort 1A (10 rats/sex/group) on the morning of scheduled necropsy, taken from the jugular vein (1 mL). Procedures were similar as for F0 animals.
Samples were analysed for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) using validated analytical procedures, identical to those described for the F0 samples.

GROSS EXAMINATION OF DEAD PUPS:
- Animals euthanised or found dead before LD 14: where practicable, these animals were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any externally abnormal pups were fixed in 10% formalin for optional further examination. Externally normal pups were discarded.
- Animals euthanised of found dead after LD 14: These animals were subject to a gross necropsy. An external examination was followed by an inspection of the cranial, thoracic and abdominal contents. Representative samples of any abnormal tissues were preserved.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed between Study Days 123-127, after termination of the F0 females.
- Maternal animals: All surviving animals were sacrificed between LD 22-24, after weaning of the F1 litters. Females which failed to produce a litter by their expected GD 24 were sent for necropsy.
- All adult animals were euthanized by exposure to carbon dioxide, followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all F0 females were examined for signs of implantation, the number of any implantation sites were recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of all non-pregnant females were fixed, stained and examined for implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
For all groups, the tissues indicated in Table 1 were weighed and prepared for microscopic examination. Organ weights were not recorded for animals euthanised in poor condition or in extremis. Paired organs were weighed together. Terminal body weights were used for organ weight analysis. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.
For F0 animals, from each ovary, 6 step serial sections (sectioning a small amount into the ovary e.g. 25 µm before the next section was taken) were taken. One section was stained with haematoxylin and eosin and 5 sections were stained for IHC using PCNA marker for enumeration of primordial and primary follicles.
All tissue samples from all high dose and control group animals were examined for histology and histopathology. For the low and mid dose group animals, only gross lesions and the target tissues (identified as liver and kidney for males, and liver for females) were included for histology and histopathology.

Testes
A detailed qualitative examination of the testes 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 into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries
The examination of the ovaries included quantification of the primordial and growing oocytes and the confirmation of the presence or absence of corpora lutea.

Sperm evaluation
From all males from the F0 animals, the right cauda epididymis was placed in Medium 199 and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser, to assess percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL).
The cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension, a sperm smear was prepared and stained with eosin Y solution. From the high dose and control group 1 and 4 animals, at least two hundred sperm per animal were evaluated for morphological abnormalities.
One testis was decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris etc, if required. The number of homogenisation resistant spermatids in dilutions of this suspension was counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Postmortem examinations (offspring):

SACRIFICE
Timing:
- Culled pups on PND 4
- Non-selected pups at weaning between PND 22-24 (at the same time as their mother)
- Cohort 1A animals between PND 91 and 96
- Cohort 1B animals between PND 98 and 100

- Animals less than 10 days old were killed by intraperitoneal injection of sodium pentobarbitone or cervical dislocation, followed by exsanguination. Animals 10 days of age or more were euthanised by exposure to carbon dioxide, followed by exsanguination.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Non-selected pups on PND 4: these animals were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved.
-Non-selected pups on PND 22-24: from each litter, 1 male and 1 female pup (where they were available) were necropsied from 10 litters/group. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of selected tissues and any grossly abnormal tissues were preserved.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality had a gross necropsy performed and any abnormalities were preserved.
- Cohort 1A (PND 91-96 ): Necropsy was performed as described for the F0 animals.
- Cohort 1B (PND 98-100 ): Necropsy was performed as described for the F0 animals.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 (Cohort 1A), Table 2 (Non-selected pups on PND 22) and Table 3 (Cohort 1B) were weighed and prepared for microscopic examination.
From non-selected pups terminated on PND 22-24, full (limited) tissue histology and histopathology was performed for the high dose and control animals. For the low and mid dose animals, only gross lesions were included in histology and histopathology.
From Cohort 1A, all tissue samples from all high dose and control group animals were examined for histology and histopathology. For the low and mid dose group animals, only gross lesions and the target tissues (identified as liver and kidney for males, and liver for females) were included for histology and histopathology.
No histopathology was performed for samples from Cohort 1B. For control animals, all tissues were processed to paraffin blocks.

For Cohort 1 animals, from each ovary, 6 step serial sections (sectioning a small amount into the ovary e.g. 25 µm before the next section was taken) were taken. One section was stained with haematoxylin and eosin and 5 sections were stained for IHC using PCNA marker for enumeration of primordial and primary follicles.

Testes
A detailed qualitative examination of the testes 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 into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries
The examination of the ovaries included quantification of the primordial and growing oocytes and the confirmation of the presence or absence of corpora lutea.

Sperm evaluations
Sperm evaluations were performed for all Cohort 1A males, as described for the F0 males.

IMMUNOPHENOTYPING
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group of the Cohort 1A animals at necropsy and were collected into tubes containing RPMI medium and stored immediately on wet ice, and were analysed using a validated flow cytometry-based analytical method.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and have been reported at the 1%, and 5% levels. The following variables were analysed according to sex and occasion (groups with les that 3 observations were excluded: body weight (gains), food consumption, haematology variables, coagulation variables, clinical chemistry variables, urinalysis variables, TSH, T3 and T4 variables, organ weights, organ weight relative to body weight, ovarian scoring (total number of oocytes per animal). Pair-wise comparisons of the low-, mid- or high dose group relative to the control group were made.
Levene’s test was used to assess the homogeneity of group variances. Groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups were compared using a Dunnett’s test (equivalent to t-test in Nevis 2012 tables) or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012 tables).

Reproductive indices:

Female Mating Index = (Number of Females with Evidence of Mating (or no confirmed mating date and pregnant))/Number of Females Paired;
Female Fertility Index = Number of Pregnant Females / (Number of Females with Evidence of Mating (or no confirmed mating date and pregnant));
Female Pregnancy Index = Number of Pregnant Females/Number of Females Paired;
Male Mating Index = (Number of Males with Evidence of Mating (or female partner confirmed pregnant))/Number of Males Paired;
Male Fertility Index = Number of Males Impregnating a Female/(Number of Males with Evidence of Mating (or female partner confirmed pregnant));
Male Pregnancy Index = Number of Males Impregnating a Female/Number of Males Paired;
Gestation Length = Number of days from GD 0 to the day the first pup is observed;
Gestation Index (Percentage of pregnancies that resulted in birth of live litters) = Number of Animals with Live Offspring/Number of Animals Pregnant x 100;
Post-Implantation Loss/Litter = ((Number of Implants – Total Newborn Pups)/Number of Implants) x 100.

Offspring viability indices:

Live Birth Index (Percentage of pups born alive) = Number of Live Newborn Pups/Number of Newborn Pups x 100; Viability Index (Percentage of pups born that survived 4 days postpartum) = Number of Live Pups on Day 4 Postpartum/Number of Live Newborn Pups x 100;
Lactation Index (Percentage of pups that survived 21 days postpartum) = Number of Live Pups on Day 21 Postpartum/Number of Live Pups on Day 4 (postculling) Postpartum x 100;
Sex Ratio (% males) (Percentage of male pups per litter) = Number of Live Male Pups/Total Number of Live Pups x 100.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were clinical observations of salivation at 5, 20 or 75 mg/kg bw/day in the F0 males as well as at 20 or 75 mg/kg bw/day in the F0 females, whereas there were no observations in the control. There was also a higher incidence of ploughing at 5, 20 or 75 mg/kg bw/day in the F0 animals when compared to the control. Salivation and ploughing had a dose-dependent relationship as there was a higher number of incidences at increasingly higher dose levels. Both salivation and ploughing were predominantly observed immediately post-dose with only few occasions at the 0-1 hour timepoint after dosing, therefore it is considered that these findings are most likely due to the taste of the formulation so were considered not to be adverse.
All other clinical observations were considered to be incidental background findings commonly observed in this species.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no bodyweight or body weight gain effects considered to be related to the test item in the F0 animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on food consumption considered to be related to the test item in the F0 animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in haematology parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 animals. The changes are summarized in Table 3.
At 75 mg/kg bw/day there were higher numbers of reticulocytes in F0 generation animals and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. Additionally, in the F0 females there were high mean corpuscular volume and corpuscular haemoglobin which could be related to the lower levels of red blood cells. At 75 mg/kg bw/day there were higher levels of monocytes in the F0 males and lymphocytes in the F0 females. The changes in the F0 males corresponded with a higher level of white blood cells.
There were no effects in the haematology parameters considered to be related to the test item
at 5 or 20 mg/kg bw/day in the F0 animals. All other values were considered to be within normal biological variation when compared with the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in clinical chemistry parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 males, and at 20 or 75 mg/kg bw/day in the F0 females. These changes are summarised in Table 3.
There were higher levels of albumin at 75 mg/kg bw/day in the F0 males as well as lower level of globulin at 75 mg/kg bw/day in the F0 animals when compared with the control. The changes in albumin and globin corresponded with higher albumin/globulin ratio at 75 mg/kg bw/day in the F0 animals. Additionally there were lower levels of triglycerides at 20 or 75 mg/kg bw/day in the F0 females when compared with the control.
There were no effects in the clinical chemistry parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the F0 males or at 5 mg/kg bw/day in the F0 females. All other values were considered to be within normal biological variation when compared with the controls.

Coagulation
At 75 mg/kg bw/day there were lower levels of fibrinogen in the F0 males with a 0.87-times lower fold difference respectively when compared with the control. There were no test item-related effects on coagulation parameters at 5, 20 or 75 mg/kg bw/day of 2-mylanthraquinone in the F0 females or at 5 or 20 mg/kg bw/day in the F0 males.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in the thyroid hormones related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 animals. The levels of T3, T4 and TSH at 75 mg/kg bw/day in the F0 animals are summaries in Table 4.
At 75 mg/kg bw/day there were lower levels of T4 in the F0 males and T3 in the F0 males and females. There were also higher levels of TSH at 75 mg/kg bw/day in the F0 animals.
There were no test item related effects at 5 or 20 mg/kg bw/day in the in the F0 animals. All other values were considered to be within normal biological variation when compared with the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no urinalysis findings considered related to the test item at 5, 20 or 75 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related histopathological findings at 75 mg/kg bw/day and at 20 mg/kg bw/day in the F0 males. These finding have been summarised in Table 6.
At 20 or 75 mg/kg bw/day the liver showed incidences of minimal focal hepatocellular degeneration in the F0 males, whereas there were no observations within the control. At 75 mg/kg bw/day there were were incidences of minimal hepatocellular hypertrophy in the liver in the F0 males as well as mild focal hepatocellular necrosis in the F0 males.
There were no findings considered to be related to the test item at 5, 20, or 75 mg/kg bw/day in the F0 females, or at 5 mg/kg/day in the F0 males. All other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item related.

Ovarian follicle counts
There were no findings in the ovarian follicle counts that were considered to be related to the test item at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the F0 females.
There were no findings in the histopathology examination of the ovaries to indicate an increase in degenerating ovarian follicles. Therefore although there were mean ovarian follicles counts that were statistically significantly different from their respective controls, these differences were considered to be within normal biological variation for this species.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects on the estrous cycles considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no findings in the sperm straight line velocity or the percentage of motile or progressive motile sperm considered to be related to the test item at 5, 20 or 75 mg/kg bw/day
2-Amylanthraquinone in the F0 males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on the maternal performance considered to be related to the test item at 5, 20 or 75 mg/kg bw/day. There were no effects on the duration of gestation, litter performance or survival considered to be related to the dosing of test item at 5, 20 or 75 mg/kg bw/day.

The changes in the haematology, coagulation and clinical chemistry parameters are considered likely to be related to the changes in the organ weights and/or histopathology observed in the spleen, liver and kidney. These findings were considered not to be adverse as they were mild to moderate in severity and there were no associated adverse histopathological or toxicology effects.

Higher kidney weights in the F0 generation and Cohort 1A and 1B animals were considered not to be adverse as they were not associated with any adverse histopathological changes. In the liver there were test item-related mild/minimal histological findings of focal hepatocellular degeneration, hepatocellular hypertrophy and/or focal hepatocellular necrosis at ≥ 20 mg/kg bw/day in the F0 males. Hepatocyte hypertrophy in the liver often results from induction of microsomal metabolic enzymes, which is an adaptive response to xenobiotics (Maronpot et al 2010, Hall et al 2012). These histological findings were classified as either mild or minimal changes and so are considered not to be adverse. Additionally, the liver weight (as % of body weight) was 29.4 - 45.2 % higher at 75 mg/kg bw/day in the F0, Cohort 1A and 1B males and 15.1 - 34.7 % higher at 75 mg/kg bw/day in the F0, Cohort 1A and 1B females, these changes were not considered to be adverse as they were not associated with any adverse histopathological changes.
Test item-related higher spleen weights at 75 mg/kg bw/day and higher pituitary gland weights
at ≥ 20 mg/kg bw/day were not associated with any gross pathological or histological findings
therefore these organ weight changes were considered not to be adverse.
At 75 mg/kg bw/day there were lower levels of T4 and T3 as well as higher levels of TSH. These changes in the F0 males were associated with a higher thyroid weight. Given the trend of increased liver weights described above, the thyroid changes may be also related to these changes. The changes in the thyroid hormone levels were considered to be related to the test item however, under the conditions of this study no adverse effects could be linked to the changes in the thyroid hormones therefore this change was not taken into account when determining the parental NOAEL.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effect on reproduction parameters.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

PUP AND LITTER (LD 1- LD 21): There were no pup or litter observations considered to be related to the dosing of the test item at 5, 20 or 75 mg/kg bw/day.
COHORT 1A and 1B: There were clinical observations of salivation at 20 or 75 mg/kg bw/day in the Cohort 1A animals and Cohort 1B animals, whereas there were no observations in the control. There was also a higher incidence of ploughing at 5, 20 or 75 mg/kg bw/day in the Cohort 1A and 1B animals when compared to the control. Salivation and ploughing had a dose-dependent relationship as there was a higher number of incidences at increasingly higher dose levels. Both salivation and ploughing were predominantly observed immediately post-dose with only few occasions at the 0-1 hour timepoint after dosing, therefore it is considered that these findings are most likely due to the taste of the formulation so were considered not to be adverse.
All other clinical observations were considered to be incidental background findings commonly observed in this species.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

PUPS AND LITTER (LD 1 - 21): There were no effects on the litter weights or individual pup weights considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.
COHORT 1A and 1B: There were no bodyweight or body weight gain effects considered to be related to the test item in the Cohort 1A or 1B animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on food consumption considered to be related to the test item in the Cohort 1A or 1B animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in haematology parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A animals. These changes are summarised in Table 2.
At 75 mg/kg bw/day there were higher numbers of reticulocytes in Cohort 1A males and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. At 75 mg/kg bw/day there were higher levels of platelets in the Cohort 1A males.
There were no effects in the haematology parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the Cohort 1A animals. All other values were considered to be within normal biological variation when compared with the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in clinical chemistry parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A males and at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females. These changes are summarised in Table 3.
There were higher levels of albumin at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females as well as lower level of globulin at 75 mg/kg bw/day in the Cohort 1A females, when compared with the control. The changes in albumin and globin corresponded with higher albumin/globulin ratio at 75 mg/kg bw/day in the Cohort 1A females, as well as higher total protein levels at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females. Additionally there were lower levels of triglycerides at 75 mg/kg bw/day in the Cohort 1A males when compared with the control.
There were no effects in the clinical chemistry parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

Coagulation
At 75 mg/kg bw/day there were lower levels of fibrinogen in the Cohort 1A males with a 0.89 times lower fold difference respectively when compared with the control.
There were no test item-related effects on coagulation parameters at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the Cohort 1A females or at 5 or 20 mg/kg bw/day in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no urinalysis findings considered related to the test item at 5, 20 or 75 mg/kg bw/day.

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no findings related to sexual maturation (age and weight at vaginal opening or preputial separation) and no difference in time to first estrous cycle in Cohorts 1A and 1B considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no findings in anogenital distance considered to be related to the test item at 5, 20 or 75 mg bw/kg/day.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in the absolute and relative to body weights related to the administration of 2-Amylanthraquinone were observed at 20 or 75 mg/kg bw/day in the Cohort 1A and Cohort 1B animals. Test item-related organ weight differences are summarised in Table 5.
At 75 mg/kg bw/day there were higher kidney and liver weights in the Cohort 1A and 1B animals, as well as higher spleen weights in the Cohort 1B animals and Cohort 1A females, when compared with the control. At 20 mg/kg bw/day there were higher kidney weights in the Cohort 1B males and higher liver weights in the Cohort 1B males. when compared with the control.
The liver weight changes at 75 mg/kg bw/day in the Cohort 1A animals correlated with hepatocellular hypertrophy.
There no findings considered to be related to the test item at 20 mg/kg bw/day in the Cohort 1A animals or Cohort 1B females or at 5 mg/kg bw/day in the Cohort 1A or Cohort 1B animals. There were other individual organ weight values that were different from their respective controls, however, there were no patterns or correlating data (taking into account differences in sexual maturity) to suggest these values were test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross findings in the culled F1 pups on PND4, unselected F1 animals on PND22-24, Cohort 1A animals or Cohort 1B animals. Gross findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related histopathological findings at 75 mg/kg/day in the Cohort 1A animals. These findings have been summarised in Table 6.
At 75 mg/kg bw/day in the liver, there were incidences of minimal hepatocellular hypertrophy in the Cohort 1A males and females. At 75 mg/kg bw/day in the kidney there were higher incidences of minimal focal mineralization and minimal/mild accumulation of hyaline droplets in the Cohort 1A males and minimal/mild tubular basophilia in the Cohort 1A males and females.
There were no findings considered to be related to the test item at 5, 20, or 75 mg/kg bw/day in the unselected F1 pups on PND22-24, or at 5 or 20 mg/kg bw/day in the Cohort 1A males and females. All other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item related.

Ovarian follicle counts
There were no findings in the ovarian follicle counts that were considered to be related to the test item at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the Cohort 1A females. Additionally, there were no findings in the histopathology examination of the ovaries to indicate an increase in degenerating ovarian follicles. Therefore although there were mean ovarian follicles counts that were statistically significantly different from their respective controls, these differences were considered to be within normal biological variation for this species.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS F1
Changes in the thyroid hormones related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A males. The levels of T3, T4 and TSH at 75 mg/kg/day in the Cohort 1A animals are summaries in Table 4.
At 75 mg/kg bw/day there were higher levels of TSH in the Cohort 1A males. There were no test item related effects at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the F1 pups culled on PND 4 or the unselected F1 pups on PND 22-24 or the Cohort 1A females. There were also no test item related effects at 5 or 20 mg/kg bw/day in the in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

SPERM MEASURES
There were no findings in the sperm straight line velocity or the percentage of motile or progressive motile sperm considered to be related to the test item at 5, 20 or 75 mg/kg bw/day
2-Amylanthraquinone in the Cohort 1A males. There were no effects on sperm morphology, sperm counts or spermatid counts considered to be related to the test item at 5, 20 or 75 mg/kg bw/day 2-Amylanthraquinone in the Cohort 1A males.

IMMUNOPHENOTYPING
There were no test item, dose dependent or sex related effects observed for any of the immune cell populations analysed within the rat spleens at 5, 20 or 75 mg/kg bw/day (Cohort 1A).

See also Details on results (P0)
At 75 mg/kg bw/day there were test-item-related mild/minimal findings in the kidney of focal
mineralization in the Cohort 1A animals as well as accumulation of hyaline droplets and
tubular basophilia in the Cohort 1A males. In the kidney, hyaline droplet accumulation can be
induced by xenobiotics (including some hydrocarbons) that disrupt the lysosomal catabolism
of alpha-2u-globulin in the renal proximal tubules (Alden 1986, Frazier et al 2012). Because
this globulin is synthesised in especially large amounts by the male rat liver and is not present
in humans, the finding is not considered relevant to humans. These findings were therefore
considered not to be adverse as they were mild/minimal in severity and were not associated
any degenerative changes. The accumulation of hyaline droplet could be associated with the
higher kidney weights in the Cohort 1A males.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

> 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effects on developmental parameters.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

no

DOSE FORMULATION ANALYSES

A summary of the analysis of the dose formulation samples taken in study week 1, 14 and 25 is given in Table 1 below.

No test item was detected in the formulations for the control group (0 mg/mL).

 

Table 1: Results of dose formulation analysis

Dose concentration (mg/mL)	Accuracy range (%)	Mean Accuracy (%)	Homogeneity (coefficient of variation [%])
Week 1
0	n.a.	n.a.	n.a.
1	95-98	97	1.2
4	101-105	104	1.5
25	89-98	94	3.3
Week 14
0	n.a.	n.a.	n.a.
1	91-97	95	2.1
4	86-94	91	3.3
25	84-93	90	4.3
Week 25
0	n.a.	n.a	n.a.
1	101-102	102	0.59
4	91-98	96	3.1
25	95-98	97	1.0

In conclusion, all formulations were within the acceptance criteria (mean concentration within 15%, with a relative standard deviation <10%). No test item was detected in the control groups formulations. Dose levels were thus adequate.

 

HAEMATOLOGY  

Table 2 Haematology Findings in the F0 and Cohort 1A animals

	F0		Cohort 1A
	Males	Females	Males	Females
Dose Level (mg/kg/day	75	75	75	75
Haemoglobin (g/L)	x0.92	-	x0.92	-
Haematocrit (L/L)	x0.93	-	x0.94	-
Red Blood Cells (x10^12/L)	x0.93	x0.89	x0.92	-
White Blood Cells (x109/L)	x1.13	-	-	-
Monocytes (x10^9/L)	x1.30	-	-	-
Reticulocyte (x10^9/L)	x1.40	x1.57	-	-
Red Blood Cell Distribution Width (%)^a	x1.14	-	x1.16	-
Platelets (10^9/L)	-	-	x1.10	-
Reticulocyte count (10^9/L)	-	-	x1.27	x1.15
Lymphocytes (x10^9/L)	-	x1.14	-	-
Mean Corpuscular Volume (fL)	-	x1.05	-	-
Mean Corpuscular Heamoglobin (pg)	-	x1.06	-	-
A dash (-) indicates absence of a test item related change. Numerical value indicates fold difference of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05). a – describes the variation in the size of the red blood cells

 

 

 

CLINICAL CHEMISTRY

Table 3 Clinical Chemistry Findings F0 and Cohort 1A animals

	F0			Cohort 1A
	Males	Females		Males	Females
Dose Level (mg/kg/day)	75	20	75	75	5	20	75
Triglycerides (mmol/L)	-	x0.59	x0.53	x0.73	-	-	-
Albumin (g/L)	x1.10	-	-	-	x1.05	x1.10	x1.14
Globulin (g/L)	x0.82	-	x0.83	-	-	-	x0.96
Albumin/Globulin (ratio)	x1.34	-	x1.28	-	-	x1.09	x1.19
Total Protein (g/L)	-	-	-	-	x1.06	x1.08	x1.08
A dash (-) indicates absence of test item related change. Numerical value indicates fold difference of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05).

 

THYROID HORMONE ANALYSIS

Table 4: TSH, T3 and T4 Findings F0 and Cohort 1A animals

	F0		Cohort 1A
	Males	Females	Males	Females
Dose Level (mg/kg/day)	75	75	75	75
T3 (ng/mL)	x0.79	x0.80	x0.94	x1.36
TSH (ng/mL)	x2.37	x1.84	x2.53	x1.23
T4 (ng/mL)	x0.78	x0.90	x0.92	x0.97
Bolded values were statistically significant from the control group (P ≤ 0.05).

 

ORGAN WEIGHTS

Table 5: Summary Group Mean Organ Weight Data – F0, Cohort 1A and Cohort 1B animals.

	F0				Cohort 1A		Cohort 1B
	Male		Female		Male	Female	Male		Female
Dose Level (mg/kg/day)	20	75	20	75	75	75	20	75	75
Kidney
Absolute Value	-	19.7	-	10.9	12.7	9.29	-	16.1	12.7
% of body weight	9.29	25.7	-	9.83	13.2	7.62	8.92	20.8	8.69
Liver
Absolute Value	-	33.7	-	15.8	27.7	36.5	-	40.0	27.9
% of body weight	-	40.2	-	15.1	29.4	34.7	10.0	45.2	30.1
Spleen
Absolute Value	-	10.2	-	18.4	-	18.4	-	-	18.1
% of body weight	-	15.9	-	17.0	-	16.1	-	19.1	19.7
Thyroid
Absolute Value	-	30.3	-	-	-	-	-	-	-
% of body weight	20.4	36.9	-	-	-	-	-	-	-
Pituitary Gland
Absolute Value	-	-	17.3	17.1	-	-	-	-	-
% of body weight	-	-	15.6	15.2	-	-	-	-	-
A dash (-) indicates absence of test item related change. Numerical value indicates a percentage difference (%) of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05).

 

HISTOPATHOLOGY

Table 6: Summary Test Item-related Microscopic Findings – F0 and Cohort 1A animals.

	F0			Cohort 1A
	Males*			Males		Female
Dose Level (mg/kg/day)	0	20	75	0	75	0	75
Liver (No. Examined)	(25)	(25)	(25)	(20)	(20)	(20)	(20)
Minimal focal hepatocellular degeneration	0	1	2	-	-	-	-
Minimal hepatocellular hypertrophy	0	-	9	0	5	0	8
Mild focal hepatocellular necrosis	0	-	1	-	-	-	-
Kidney (No. Examined)	(25)	(25)	(25)	(20)	(20)	(20)	(20)
minimal focal mineralization	-	-	-	2	10	-	-
minimal/mild tubular basophilia	-	-	-	4	16	4	7
minimal/mild accumulation of hyaline droplets	-	-	-	11	20	-	-
A dash (-) indicated the absence of test item related change. Where a test item related change was observed the number of incidence in the control was also included in the table to display the difference in the number of incidences in the control compared to the test item group. *In F0 females, like for F0 males no notable changes in histopathology findings  relative to the control animals were done.

 

Conclusions:

Based on the results of an extended one generation reproduction toxicity study with 2-Amylanthraquinone in rats performed according to OECD guideline 443 and following GLP principles, the NOAEL for systemic toxicity for the F0 and the F1 generation was 20 mg/kg bw/day. The NOAEL for reproduction toxicity was 75 mg/kg bw/day. The NOAEL for F1 developmental toxicity was at least 75 mg/kg bw/day.

Executive summary:

An extended one generation study was performed in rats with 2-Amylanthraquinone according to OECD guideline 443 and under GLP principle. Male and female rats from the F0 and F1 generation were exposed to the test substance by oral gavage at dose levels of 5, 20 or 75 mg/kg bw/day. The dose levels were established based on a 90-day repeated dose toxicity study. Dose formulation analyses were performed and demonstrated accurate dosing.

F0 males were treated daily for 10 weeks prior to mating until necropsy after the termination of the F0 females. F0 females were treated daily for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. Cohort 1A and 1B animals were then dosed daily from Postnatal Day (PND) 21 until at least PND 90.

The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, estrous cycling, mating performance, observations of females and litters during lactation, pre-weaning physical development of F1 animals, clinical pathology parameters (haematology, coagulation, clinical chemistry, urinalysis, thyroid stimulating hormone, T3 and T4), gross necropsy findings, organ weights, sperm evaluations and histopathological examinations.

There was a dose dependent increase in the incidence of ploughing and/or salivation at ≥ 5 mg/kg bw/day in the F0, Cohort 1A and 1B animals. These findings were predominantly observed immediately post-dose.

At 75 mg/kg bw/day there were higher numbers of reticulocytes in F0 generation animals and Cohort 1A males and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. Additionally, in the F0 generation females there were high mean corpuscular volume and corpuscular haemoglobin which could be related to the lower levels of red blood cells. At 75 mg/kg bw/day there were higher levels of monocytes in the F0 males, platelets in the Cohort 1A males and lymphocytes in the F0 females the changes, in the F0 males corresponded with a higher level of white blood cells.
At 75 mg/kg bw/day there were lower levels of fibrinogen in the F0 and Cohort 1A males. There were higher levels of albumin at 75 mg/kg bw/day in the F0 males and at ≥ 5 mg/kg bw/day in the Cohort 1A females as well as lower level of globulin at 75 mg/kg bw/day in the F0 animals and Cohort 1A females. The changes in albumin and globulin corresponded with a higher albumin/globulin ratio at 75 mg/kg bw/day in the F0 animals and Cohort 1A females as well as a higher total protein levels at ≥ 5 mg/kg bw/day in the Cohort 1A females. Additionally, there were lower levels of triglycerides at ≥ 20 mg/kg/day in the F0 females and 75 mg/kg bw/day in the Cohort 1A males.
The changes in the haematology, coagulation and clinical chemistry parameters were considered not to be adverse as they were mild to moderate in severity and there were no associated adverse histopathological or toxicology effects.

At 75 mg/kg bw/day there were test item-related histological findings in the kidney of focal mineralization and accumulation of hyaline droplets the Cohort 1A males and tubular basophilia in the Cohort 1A males and females. There were also higher kidney weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and at 20 mg/kg bw/day in F0 and Cohort 1B males. These changes were considered not to be adverse as they were not associated with any adverse histopathological changes.
In the liver at ≥ 20 mg/kg bw/day test item-related histological findings of focal hepatocellular degeneration in the F0 males. Additionally, at 75 mg/kg bw/day there were test item-related histological findings in the liver of hepatocellular hypertrophy in the F0 males and Cohort 1A animals as well as focal hepatocellular necrosis in the F0 males. The histological findings in the liver were considered to be mild or minimal changes therefore considered not to be adverse. Additionally, there were also higher liver weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and in the Cohort 1B males at 20 mg/kg bw/day. These changes in the weights of the liver were not considered to be adverse as they were not associated with any adverse histopathological changes.
There were higher spleen weights at 75 mg/kg bw/day in the F0 animals, Cohort 1B animals and Cohort 1A females and higher pituitary gland weights at ≥ 20 mg/kg bw/day in the F0 females. There were no associated gross pathological or histological findings with these organ weight changes therefore they were considered not to be adverse.

At 75 mg/kg bw/day there were lower levels of T4 in the F0 males and T3 in the F0 animals. There were also higher levels of TSH at 75 mg/kg bw/day in the F0 animals and Cohort 1A males. The changes in the levels of the thyroid hormones was associated with a higher thyroid weight at 75 mg/kg bw/day in the F0 males.

Given the trend of increased liver weights described above, the thyroid changes may be also related to these changes. The changes in the thyroid hormone levels were considered to be related to the test item however, under the conditions of this study no adverse effects could be linked to the changes in the thyroid hormones therefore this change was not taken into account when determining the parental NOAEL.
There were no treatment related findings related to bodyweights or food consumption in the F0 generation or Cohort 1A and 1B animals. There were no treatment related effects seen estrous cycling, mating performance, observations of females and litters during lactation, preweaning physical development of F1 animals or sexual maturation of the F1 animals. There were also no effects on the number of ovarian follicles in the ovaries of the F0 or Cohort 1A females.

In conclusion, the administration of 2-Amylanthraquinone by once daily oral gavage was considered to have a systemic No Observed Adverse Effect Level (NOAEL) of 20 mg/kg bw/day for the F0 and F1 generation, due to the higher organ weights of the kidney and liver and associated minimal to mild histopathology  findings in both liver and kidney, as well as mild to moderate changes in the haematology and clinical pathology parameters at 75 mg/kg bw/day. Findings in the kidney were minimal mineralisation and minimal to mild tubular basophilia; findings in the liver were minimal hepatocellular hypertrophy and degeneration, especially in males. Although individually, these findings were considered not to be adverse and did not indicate significant organ damage or impaired function, the combined changes within these parameters were considered to be adverse at 75 mg/kg bw/day.
The NOAEL for reproduction toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on any of the reproductive endpoints examined within this study. The NOAEL for F1 developmental toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on the developmental endpoints examined within the study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Reliable without restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In a GLP-compliant OECD Guideline 414 study with rats no signs of developmental toxicity were observed at any dose level. The NOAEL for developmental effects was set at the highest dose level of 75 mg/kg bw/day. Maternal toxicity was observed at this dose level, manifested in slightly lower body weights and statistically significantly lower body weight gain during the treatment period, as well as reduced food consumption. Relative liver weights were statistically significantly increased by 15% at 75 mg/kg bw/day compared to the concurrent vehicle controls. Based on these observations the NOAEL for maternal toxicity was set at the mid-dose of 25 mg/kg bw/day.

A GLP-compliant OECD Guideline 414 study with 2-Amylanthraquionone administered at 3.75, 7.5 or 15 mg/kg bw/day by oral gavage in rabbits was performed. All doses up to and including the highest dose of 15 mg/kg bw/day were well-tolerated and showed that there were no adverse fetal findings. The top dose resulted in only a minor reduction in the total body weight gain. Based on these observations the maternal and embryofetal NOAELs were considered to be at least 15 mg/kg bw/day.

Overall, no developmental toxicity was seen in pre-natal developmental toxicity studies performed in rat (rodent species) and a second non-rodent species, rabbit.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 August 2019 to 18 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Version 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was found to solidify at ambient temperature. The test item storage conditions were changed during this study to maintain the clear homogeneous liquid state. Subsamples of the test item were analysed by the Sponsor following changes in storage conditions and the test item was found to be stable. Summary of storage conditions: ambient temperature (from receipt on 31May2019 until 20Nov2019); in an incubator set to maintain 37°C (from 21Nov2019 until 05Dec2019); in an incubator set to maintain 55°C (from 06Dec2019 until study completion).

- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable during storage under above-mentioned conditions and for indicated periods of time.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Whilst it is not known if the test item has affinity to adhere to plastic, this was considered possible based on the formulation analysis results. The results obtained when using glass sample vials were considered a more accurate reflection as the formulations used for dosing were maintained in glass containers during the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- No correction for purity was performed.

Species:

rabbit

Strain:

New Zealand White

Remarks:

Time-mated The rabbit was chosen as the animal model for this study as it is a species accepted by regulatory agencies for developmental toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Shaw’s Farm, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 4-5 months
- Weight at study initiation: 3.1-3.9 kg (dose-range finding study) and 3.0-4.1 kg (main study)
- Fasting period before study: n.a.
- Housing: Females were housed individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated floor. The housing provided an area for hiding. A tray containing absorbent paper was suspended beneath each cage.
- Diet: Envigo Diet available ad libitum. Each animal was also offered a supplement of hay at least 3 times per week and a selection of fruits and/or vegetables up to twice per study week.
- Water: water from the public supply ad libitum.
- Acclimation period: 3 days (from arrival until start dosing on GD6)
- Animal enrichment: For psychological/environmental enrichment, animals were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study
procedures/activities. The animals were given a period of exercise in a separate floor pen (a minimum of 30 minutes, and no longer than 45 minutes) up to 3 times per week.

It was considered that there were no contaminants in the supplied diet, water, bedding and enrichment items that influenced the outcome of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17°C to 20°C (target 19°C to 23°C)
- Humidity (%): 28% to 70% (target 40% to 70%)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From 19 August 2019 (initiation of dosing dose-range finding study) to 5 March 2020 (end of in-life main study)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Site for formulation analysis at appropriate concentrations to meet dose level requirements. The required amount of test item was weighed and approximately 80% of the control item was added. The formulation was mixed mechanically until visibly homogeneous, and then the appropriate amount of control item was added to make the final volume. This formulation was magnetically stirred until visibly homogeneous. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4oC, protected from light and dispensed daily.
The control item, Polyethylene glycol 400, was prepared at least weekly by measuring suitable daily aliquots that were stored in a refrigerator set to maintain 4°C, and dispensed as ̀required for administration to control animals.
The dosing formulations and prepared control item were removed from the refrigerator and stirred for at least 30 minutes before and continuously during dosing.

VEHICLE
- Concentration in vehicle: 3.75, 7.5 and 15 mg/mL
- Amount of vehicle (if gavage): 1 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected during the dose-range finding study from the dosing solutions meant for dosing on GD 6 of Batch 1 animals (Day 1 of dosing). Dose formulation samples were collected during the main study from the dosing solutions meant for Batch 3 animals on GD6 (=on Day 1 of dosing) and on GD25.
>Concentration: all treatment groups
>Homogeneity: all test item formulations

Duplicate sets of top, middle and bottom (duplicate middle only for control) samples (500 mg) for each sampling time point were sent to the analytical laboratory. Triplicate sets of top, middle and bottom (triplicate middle only for control) samples (500 mg) for each sampling time point were collected as backup samples. The samples were collected into cryogenic or glass scintillation vials and stored in a freezer set to maintain -20°C, protected from light and shipped on dry ice to the Test Site for formulation analysis within the established stability period.
Analyses were performed by ultra-performance liquid chromatography (UPLC) with spectrophotometric detection using a validated analytical procedure (Test Site Study No. 511240).

Acceptance: Concentration results were considered acceptable if sample concentration results were within ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ±10% for each group. Batch 1 GD 6 backup samples were analysed for confirmation of original results. The homogeneity results obtained from top/middle/bottom test item formulation preparations for the main study were averaged per phase and utilised as concentration results.

Details on mating procedure:

Animals were purchased in mated condition.

Duration of treatment / exposure:

From GD 6 to 28 (where GD 0 was the day of detection of mating).

Frequency of treatment:

once daily

Duration of test:

Approximately 3 weeks, females were sacrificed on GD 29.

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

Dose-range finding study: 6 females
Total number of animals per group was divided into subsets of 3 females (attributed to batches 1 and 2) that were dosed consecutively at least 1 day apart. All batches contained a similar number of animals from each dosing group.

Main study: 22 females
Total number of animals per group was divided into subsets of 3 females (attributed to batches 3-9) and 1 female (batch 10) that were dosed consecutively at least 1 day apart. All batches contained a similar number of animals from each dosing group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for this study were selected based on findings from the preliminary tolerability study in rabbits (Test Site Study No. 499948) in which 2-Amylanthraquinone was administered to groups of 3 non-pregnant rabbits at dose levels of 5, 10, 30, 100 or 300 mg/kg bw/day. The following parameters and endpoints were observed: clinical observations, body weights, food consumption and necropsy findings. Dose levels of 100 and 300 mg/kg bw/day were not tolerated due to signs of liquid faeces and low food consumption. At 300 mg/kg bw/day clinical observations of tremors, decreased respiratory rate, coldness to touch and sunken eyes. At 30 mg/kg bw/day, on Day 2, prior to dosing all animals had low food consumption and reduced faecal output, additionally, one animal was euthanised on Day 3 due to clinical observations of liquid faeces. At 5 and 10 mg/kg bw/day there were no effects on food consumption or body weight and no clinical signs were observed.
Based on the tolerability study, dose levels of 5, 10 and 20 mg/kg bw/day were considered to be
suitable for Phase 1 (dose range finding) in this study. Groups of 6 pregnant females were dosed with the test item from GD 6 until GD 29 (dosing volume 1 mL/kg; dosing solution concentrations of 5, 10 and 20 mg/mL). Observations, examinations, procedures and statistics were similar to dose described for the main study, except for fetal examinations: visceral examinations, skeletal examinations, fixed head examination and sex determination were not performed for the dose-range finding study. Based on the findings of one animal at 20 mg/kg bw/day (sent to unscheduled euthanasia; for details on design and results, see section 'Any other information on results', dose levels of 3.75, 7.5 and 15 mg/kg bw/day were selected for the main part of this study (Phase 2).
- Rationale for animal assignment (if not random): Mated females were allocated to dose groups based on parentage and siring information. Allocations were made based on mating information to avoid the allocation of females inseminated by the same male to the same treatment group. Body weights were assessed to ensure that the group mean body weights of the test item groups were within ± 5% of the control group mean body weight.
- Other: The study was performed in consecutive batches (batch 3 - batch 10). Dosing of the batches was at least one day apart.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study
- Animals were checked for general health, mortality and moribundity.
- On each day of dosing, animals were observed regularly throughout the day for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during pre-treatment (GD 5), weekly from GD 6 and on GD 29.

BODY WEIGHT: Yes
- Time schedule for examinations: once during pretreatment (on GD 5) and on GD 6, 9, 12,
15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured daily from GD 6 (the first weighed quantity was offered on GD 5). Where food consumption for an animal was below 20 g per day, hay consumption and consumption of vegetables and/or fruit were monitored to assess the welfare of the animals. In addition, where deemed necessary, if an animal had food consumption below 20 g per day it was given additional time (a minimum of 30 minutes, and no longer than 45 minutes) in the exercise area.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: 29
- Organs examined: reproductive tract, organs with abnormalities
Adult animals surviving until scheduled euthanasia were euthanised by an intravenous overdose of sodium pentobarbitone, weighed and exsanguinated. Animals were not fasted before their scheduled necropsy. Fetuses were killed by an overdose from an intrathoracic or intraperitoneal injection of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tract was dissected out and weighed intact.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (implantation sites)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae were weighed from live implants (en masse) and abnormalities in size, colour or shape were recorded. The number and distribution of live and dead fetuses were recorded.
- Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no sign of maceration), or a late resorption (macerated tissue identifiable as an embryo fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable palcentae), or an early resorption (discrete, formless, discoloured tissue mass attached to the internal uterine wall; possibly of varying size).

Blood sampling:

Not performed

Fetal examinations:

- External examinations: Yes: [all per litter], including examination of the oral cavity.
- Soft tissue examinations: Yes: [all per litter]. Prior to fixation, the fetuses were sexed and examined by open dissection for abnormalities of the thoracic and abdominal viscera. The internal structures of the heart and kidneys of all fetuses were examined. The thoracic and abdominal viscera were then discarded and the fetuses were fixed in industrial denatured alcohol (IDA) 99%.
- Skeletal examinations: Yes: [all per litter] All of the eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S and then the fetuses cleared with aqueous glycerol solutions. All preparations were then examined for the presence of skeletal abnormalities and for the extent of ossification.
- Head examinations: Yes: [half per litter] For half of the fetuses, macroscopic examination of the eyes and cranial bones was undertaken, following removal of the skin from these areas, and the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. For the remaining fetuses, the head was removed from the spine (as close to the head as possible) and placed in Bouin’s fluid for subsequent serial sectioning and evaluation. They were examined for soft tissue abnormalities using a free hand sectioning technique.

Fetal abnormalities were classified as follows:
- Malformation: A structural defect in the body due to abnormal embryonic or fetal development.
- Variation: A delay in the embryo or fetal development that would be expected to be completed with time and/or differences in development between fetuses that would be seen within a normal
population of individuals.
- Incidental: Being likely to have ensued as a change or minor consequence of another finding or a result of the handling or processing of a specimen.

Statistics:

Descriptive statistical analysis: Means, standard deviations, percentages, numbers, and/or incidences have been reported, as appropriate by dataset.
Inferential statistical methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1% and 5% levels, unless otherwise noted. The pairwise comparisons of interest are listed below:
Low dose group vs. Control group
Mid dose group vs. Control group
High dose group vs. Control group
Analyses were performed as indicated below when possible but excluded any group with less than 3 observations. Parameters determined at caesarian section/late gestation excluded animals euthanised preterminally).
-Parametric/non-parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test. This analysis was done for body weight (gains), food consumption (excluded animals not pregnant), gravid uterine weight and corrected maternal body weights, litter means (presented for males, females and sexes combined; live fetuses only) and placental weights.
Non-parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test. This analysis was done for ovarian and uterine content, Liter% of fetuses with gross/external/visceral/skeletal abnormalities and mean fetal ossification sites.
Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest. This analysis was done for parental indices and mortality.

Indices:

-Pre-implantation loss = ((no. of corpora lutea - no. of implants)/no. of corpora lutea) x 100
-Post-implantation loss = ((no. of implants)/no. of live fetuses)/ no. of implants) x 100
-Sex ratio (% males) = (no. of male fetuses/total no. of fetuses) x 100
-Litter% of fetuses with abnormalities = (no. of fetuses with a given finding/no of fetuses in litter examined) x 100

Historical control data:

Historical control data for skeletal variations in litters obtained at the Test Site in 11 studies started between March 2017 and December 2019 from for New Zealand Rabbit GD 29 were used for comparison with concurrent controls.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At dose levels up to and including 15 mg/kg bw/day, there were no clinical observations considered related to administration of 2-Amylanthraquinone.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths at any dose level up to and including 15 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight (changes):
At 15 mg/kg bw/day, the mean body weight gains over GD 6 to GD 29 were slightly lower than the control (-42.5 g, 12% lower). However, this difference was minor as a proportion of the total body weight in rabbits and considered not adverse. There were no other noteworthy effects on body weight or body weight gains.

Gravid uterine weights and corrected body weights:
There were no 2-Amylanthraquinone-related changes in gravid uterus weights or corrected maternal body weights in this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes in food consumption considered related to administration of 2-Amylanthraquinone. Any slight intergroup differences were considered incidental and too small to be attributed to the test item.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Placental weight:
There were no effects on placental weights considered related to 2-Amylanthraquinone. Slight intergroup differences were noted, however as the differences were minor, within normal biological variation and not dose-related, they could not be positively attributed to the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no visible lesions in any animal.

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions in any of the study groups.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on pre- and post-implantation loss. Slight intergroup differences were noted (e.g. slightly higher post-implantation in all test item groups), however as the differences were minor, within normal biological variation and not dose-related, they could not be positively
attributed to the test item.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The proportion of females with resorptions was higher in some dose level groups (statistically significant at 7.5 mg/kg bw/day). However as there was no pattern of dose-response and the post-implantation loss was within normal biological variation, this was considered unrelated to 2-Amylanthraquinone.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no 2-Amylanthraquinone-related effects on maternal performance in this study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal weights were slightly lower than control at 7.5 and 15 mg/kg bw/day (42.9 g and
43.7 g, respectively, compared with 45.6 g in the control) but this was not dose-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of fetuses was 6.8, 7.6, 6.7 and 7.3 for the control group and the low, mid and high dose groups, respectively.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on fetal sex ratio. Slight intergroup differences were noted , however as the differences were minor, within normal biological variation and not dose-related, they could not be positively attributed to the test item.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no 2-Amylanthraquinone-related fetal malformations at any dose level. All of the malformations that occurred were considered unrelated to the test item because they were seen in only 1 or 2 fetuses per group and there was no dose-relationship.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The incidence of some skeletal variations indicative of slightly delayed ossification (particularly of the forepaw phalanges, sternebra and cervical vertebra) was higher at 15 mg/kg bw/day and to a lesser extent at 7.5 mg/kg bw/day (incomplete ossification of sternebra), compared with the concurrent control. The incidence of unossified forepaw phalanges and minimal incomplete ossification of cervical centrum at 15 mg/kg bw/day was slightly higher than Test Facility’s historical control data but the other variations were comparable to the background data. These fetal variations are indicative of slightly retarded development but they are expected to be transitory and compensated for during postnatal development. Therefore, they were considered to be not adverse in the context of this study.
The incidence of these fetal variations is summarised in Table 'Summary of Salient Fetal Variations (Skeletal and Skeletal Body Examinations combined)'.

The mean number of paired ribs was similar in all groups, including controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were observed in the groups exposed to the test item.

Details on embryotoxic / teratogenic effects:

The fetal skeletal variations seen in the study are indicative of slightly retarded development but they are expected to be transitory and compensated for during postnatal development. Therefore, they were considered to be not adverse in the context of this study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The fetal skeletal variations are indicative of slightly retarded development but they are expected to be transitory and compensated for during postnatal development. Therefore, they were considered to be not adverse.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

RESULTS DOSE FORMULATION ANALYSIS

Dose-range finding study

The GD 6 formulation concentrations (sampled from formulations for Batch 1 animals) were within the acceptance criteria for concentration for the low dose group (5 mg/mL). For the mid dose group (10 mg/mL) and the high dose group (20 mg/mL), mean concentrations were below the target concentration (i.e. 87% and 88% of target) and not all the individual sample concentration results were within 85-115%. The backup samples were analysed but the concentrations remained below the target concentration (i.e. 80% and 88% of target) and not all the individual sample concentration results were within 85-115%.
All formulations were within the acceptance criteria for homogeneity.

Main study

The GD 6 formulation concentrations (sampled from formulations for batch 3 animals) were below the target concentration (i.e. 82%, 88% and 86% of target for formulations at 3.75, 7.5 and 15 mg/mL, respectively) and not all the individual sample concentration results were within 85-115%.
Except for the low dose group formulation (3.75 mg/mL; Coefficient of Variation: 16%), the formulations were homogeneous.
All GD 25 formulation concentrations (sampled for batch 3) were within the acceptance criteria for both concentration and homogeneity.

All formulations were prepared, handled and analysed in the same manner, with the only differences being in the total volumes prepared for each study week (adjusted based on the number of animals being dosed) and that GD 25 formulation samples (sampled for batch 3), which were within specification, were collected into glass vials instead of plastic cryogenic vials. Whilst it is not known if the test item has affinity to adhere to plastic, this was considered possible based on these formulation analysis results. The results obtained when using glass sample vials were considered a more accurate reflection as the formulations used for dosing were maintained in glass containers during the study. Overall it is concluded that dosing was accurate.

RESULTS DOSE-RANGE FINDING STUDY

Clinical observations, moribundity and mortality

At 20 mg/kg bw/day, one animal was euthanised on GD 23 due to sustained low food consumption, low fecal output and associated body weight loss.

At 5 or 10 mg/kg bw/day, there were no clinical observations considered related to administration of 2-Amylanthraquinone.

Body weight

At 20 mg/kg bw/day, one animal lost weight from the start of dosing until unscheduled euthanasia on GD 23 (-491 g from GD 6 (13%)). There were no body weight effects in any other animal.

Food consumption

At 20 mg/kg bw/day, food consumption was low (less than 20 g) in one animal from GD 13 and this continued until unscheduled euthanasia on GD 23 (10 days). In all other animals, the food consumption was similar to the control.

Gross maternal pathology

The 20 mg/kg bw/day animal euthanised early on GD 23 had abnormal content in
the colon (brown fluid filled substance) and red mucosa in the stomach.

Maternal performance (See attached Table 3)

There were no 2-Amylanthraquinone-related effects on maternal performance in this study. It was noted that the proportion of females with resorptions was higher in some dose level groups (particularly at 20 mg/kg bw/day), not achieving statistical significance. However as there was no pattern of dose-response and the post-implantation loss was within normal biological variation in the main study,  
this was considered unrelated to 2-Amylanthraquinone.

Ovarian and uterine examinations and litter observations (See attached Table 1 below)

At 10 or 20 mg/kg bw/day, the post-implantation losses were higher than the control (19% or 20%, respectively, compared with 4% in the control). Pre-implantation loss was also slightly higher than control at both of these dose levels, but this was considered to be incidental as the values were within the ranges of normal biological variation.

Fetal abnormalities

There were no fetal external abnormalities noted.

At 20 mg/kg bw/day, one animal was sent for unscheduled euthanasia on GD 23 due to sustained inappetence accompanied by body weight loss and low faecal output. At necropsy, this animal had abnormal content in the colon (brown liquid filled substance) and red mucosa in the stomach. There were no noteworthy effects in any of the other animals including animals dosed at 20 mg/kg bw/day.

Based on the death of one animal at 20 mg/kg bw/day, it was agreed not to use this dose level for further testing. Following the review of dose-range finding results and with consultation with the Sponsor, dose levels of 3.75, 7.5 and 15 mg/kg bw/day were selected for the main study.

RESULTS TABLES

Table 1: Caesarean section data – dose-range finding study#

		Controls	5 mg/kg bw/day	10 mg/kg bw/day	20 mg/kg bw/day
No. of animals examined	Total	6	6	6	6
No. of dams pregnant	Total	5	6	5	6
No. of dams with live fetuses	Total	5	6	5	5^b
No. of corpora lutea	(mean ± SD) N	(9.8±2.2) 5	(7.5±1.0) 6	(8.4±1.5) 5	(11.4±2.9) 5
No. of implantations	(mean ± SD) N	(9.4±1.9) 5	(7.3±1.4) 6	(7.8±2.3) 5	(10.6±3.6) 5
Pre-implantation loss	(mean ± SD) N	(3.6±5.0) 5	(2.8±6.8) 6	(8.6±12.8) 5	(9.1±11.7) 5
Post-implantation loss	(mean ± SD) N	(3.8±5.2) 5	(2.1±5.1) 6	(18.5±34.8) 5	(19.7±23.4) 5
No. of dead fetuses	(mean ± SD) N	(0.0±0.0) 5	(0.0±0.0) 6	(0.0±0.0) 5	(0.0±0.0) 5
No. of early resorptions	(mean ± SD) N	(0.0±0.0) 5	(0.2±0.4) 6	(1.0±1.7) 5	(1.0±1.2) 5
No. of late resorptions	(mean ± SD) N	(0.4±0.5) 5	(0.0±0.0) 6	(0.0±0.0) 5	(0.6±0.9) 5
No. of dams with resorptions	Total (%)	2 (40.0)	1 (16.7)	2 (40.0)	5 (83.5)
No. of live fetuses	(mean ± SD) N	(9.0±1.7) 5	(7.2±1.3) 6	(6.8±3.7) 5	(9.0±4.1) 5
Body weight (g)	Male and female (mean ± SD)	38.9±2.0	44.2±3.3	43.9±8.0	39.2±4.0
Placental weight (g)	Male and female (mean ± SD)	51.7±15.8	44.1±10.8	35.4±17.54	58.3±29.0

^a None of the findings were statistically significantly different from the control ^b One female from the 20 mg/kg bw/day group underwent unscheduled euthanasia due to moribundity, and was not included in maternal and fetal examinations.

Table 2: Caesarean section data – main study

		Controls	3.75 mg/kg bw/day	7.5 mg/kg bw/day	15 mg/kg bw/day
No. of animals examined	Total	22	22	22	22
No. of dams pregnant	Total	18	21	21	21
No. of dams with live fetuses	Total	18	21	21	21
No. of corpora lutea	(mean ± SD) N	(8.7±1.8) 18	(9.5±1.5) 21	(9.0±1.9) 21	(9.3±1.9) 21
No. of implantations	(mean ± SD) N	(7.2±2.1) 18	(8.1±2.5) 21	(7.8±2.9) 21	(7.8±2.8) 21
No. of dead fetuses	(mean ± SD) N	(0.0±0.0) 18	(0.0±0.0) 21	(0.0±0.0) 21	(0.0±0.0) 21
No. of early resorptions	(mean ± SD) N	(0.3±0.8) 18	(0.4±0.7) 21	(0.6±1.0) 21	(0.4±0.8) 21
No. of late resorptions	(mean ± SD) N	(0.1±0.2) 18	(0.2±0.4) 21	(0.5±0.9) 21	(0.1±0.4) 21
No. of dams with resorptions	Total (%)	4 (22.2)	9 (42.9)	12 (57.1)^a	6 (28.6)
No. of live fetuses	(mean ± SD) N	(6.8±2.0) 18	(7.6±2.4) 21	(6.7±2.7) 21	(7.3±2.8) 21
Sex ratio	% males (mean ± SD) N	(55.5±21.4) 18	50.8±14.8) 21	(54.4±20.8) 21	(53.4±26.0) 21
Body weight (g)	Male and female (mean ± SD)	45.64±4.8	44.1±5.0	42.9±6.1	43.7±6.8
Placental weight (g)	Male and female (mean ± SD)	40.9±14.1	50.4±20.3	42.6±15.4	45.4±16.9
No. of live fetuses with malformations	Fetuses/ litters (number of fetuses/ litters examined)	2/2 (123/18)	0/0 (159/21)	0/0 (141/21)	0/0 (153/21)

^a Significantly different from the control (Fischer’s Exact; p ≤ 0.05)

Table 3: Summary of Salient Fetal Variations (Skeletal and Skeletal Body Examinations combined) - main study

		0 mg/kg bw/day	3.75 mg/kg bw/day	7.5 mg/kg bw/day	15 mg/kg bw/day
Number of fetuses/litters examined		123/18	159/21	141/21	153/21
Forelimb, Forepaw phalanges, Unossified ^a	Fetuses N (%) Litters N (%)	4 (3.25%) 3 (16.7%)	5 (3.14%) 4 (19.1%)	8 (5.67%) 6 (28.6%)	20 (13.1%) 10 (47.6%)
Sternebra, Unossified ^b	Fetuses N (%) Litters N (%)	19 (15.4%) 7 (38.9%)	25 (15.7%) 13 (61.9%)	23 (16.3%) 13 (61.9%)	41 (26.8 %) 13 (61.9%)
Sternebra, Incomplete ossification ^b	Fetuses N (%) Litters N (%)	24 (19.5%) 14 (77.8%)	33 (20.8%) 14 (66.7%)	42 (29.8%) 15 (71.4%)	46 (30.1%) 18 (85.7%)
Vertebra, Cervical centrum, Incomplete ossification, Minimal ^c	Fetuses N (%) Litters N (%)	1 (0.81%) 1 (5.56%)	2 (1.26%) 2 (9.52%)	2 (1.42%) 2 (9.52%)	9 (5.88%) 6 (28.6%)

^a. Right, left and both sides combined. Sternebra 1-6 combined. ^c. Cervical centrum 2-7 combined.

Conclusions:

Based on the results of a prenatal developmental toxicity study in rabbits, performed according to OECD guideline 414 and under GLP principles, the maternal and embryofetal NOAEL was concluded to be at least 15 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was performed in rabbits with 2-Amylanthraquinone according to OECD guideline 414 and under GLP principles. To assess potential toxicity, pregnant rabbits were exposed by oral gavage from GD 6 to GD 28 to 0, 3.75, 7.5 or 15 mg/kg bw/day. The dose levels were established based on the results of a dose-range finding study, in which pregnant rabbits were exposed to 5, 10 or 20 mg/kg bw/day. Based on mortality and body weight loss at 20 mg/kg bw/day, this dose level was concluded to exceed the maximum tolerated dose and the highest dose level was thus chosen to be 15 mg/kg bw/day in the main study. Furthermore, post-implantation losses were higher at 10 and 20 mg/kg bw/day than the control (19% or 20%, respectively, compared with 4% in the control). The high dose in the main study was chosen to allow further assessment of this potential effect. It is noted that there were no fetal external abnormalities observed in the dose-range finding study (no visceral or skeletal examinations were done).
Dose formulation analyses were performed in the dose-range finding study and the main study, which showed accurate dosing.
In the main study, the following parameters and end points were evaluated: clinical observations, body weights, food consumption, gravid uterine and corrected body weights, maternal performance, ovarian and uterine examinations, gross maternal pathology, fetal weights and external examinations, fetal sex, visceral and skeletal examinations, and mean fetal ossification sites.
The mean total body weight gain over GD 6 to GD 29 was slightly lower than the control at 15 mg/kg bw/day (12% lower). The mean fetal weights were slightly lower at 7.5 and 15 mg/kg bw/day (up to 2.7 g). The incidence of skeletal variations, indicative of slightly delayed development, was increased at 15 mg/kg bw/day (forepaw phalanges, sternebra and cervical vertebra) and to a lesser extent at 7.5 mg/kg bw/day (sternebra only), which although attributed to the test item, was considered not adverse, since they were expected to be transitory and compensated for during postnatal development.
For all other parameters, there were no changes considered related to administration of 2- Amylanthraquinone. Post-implantation losses in the main study were slightly higher for the test item exposed groups with 3.95%, 7.18%, 11.71% and 6.90% for the control group and the groups dosed at 0, 3.75, 7.5 and 15 mg/kg bw/day, respectively. In light of the minimal magnitude, the absence of an apparent dose-relationship and since the percentages are within normal biological variation, this is concluded not to be test-item related.
In conclusion, administration of 2-Amylanthraquinone to pregnant rabbits by once daily oral gavage at 15 mg/kg bw/day was well-tolerated and resulted in only a minor reduction in the total body weight gain. There were no adverse fetal findings. Based on these results, the maternal and embryofetal no observed-adverse-effect levels (NOAELs) were considered to be at least 15 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2016 - 28 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer (≤-15°C) is confirmed over the concentration range 1 to 200 mg/mL.

Species:

rat

Strain:

other: Crl:WI (Han), outbred, SPF-quality

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation, g: mean (SD), day 2 post-coitum: controls: 209 (14.3), low-dose: 209 (14.2), mid-dose 210 (10.6), high dose 208 (17.1)
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS (set to maintain):
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 July 2016 To: 28 July 2016 (main study)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for specific gravity of the test item (1.15) and vehicle (1.125). No correction was made for the purity/composition of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (12 July 2016), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Analysis was based on the analytical method validated for the test item and was conducted by a UPLC method.

Details on mating procedure:

- Impregnation procedure: not specified, performed at the supplier. The animals were at Day 0 or 1 post-coitum on arrival at the Test Facility.
- Proof of pregnancy: [vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From days 6 to day 20 post-coitum, inclusive

Frequency of treatment:

Once dayly for 7 days/week

Duration of test:

14 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle controls

Dose / conc.:

7.5 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected based on results of the dose range finding study.
- Rationale for animal assignment: by computer-generated random algorithm according to body weight, with all animals within ± 20% of the mean per subgroup. Females which were mated on the same day are classified in the same subgroup.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION : Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic and abdominal examination, with special attention being paid to the reproductive organs. Furhtermore the livers of all animals were dissected and examined.
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus
- The sex of each fetus from the ano-genital distance (during necropsy) and also from
gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total
resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Preimplantation loss (%) = ((number of corpora lutea - number of implantation)/number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100
Viable fetuses affected/litter (%) = (number of viable fetuses affted/litter / number of viable fetuses/litter) x 100

Historical control data:

Historical control data for rats of this strain from the testing laboratory are available from 2014-2015.

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental findings that were noted included alopecia, salivation and piloerection. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and in absence of a dose related-trend, these findings were not considered to be treatment related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg bw/day, mean body weights were slightly lower than controls from Day 9 post-coitum onwards. This change was not statistically significant. Mean body weight gain was statistically significantly lower at 25 mg/kg bw/day on Day 12 post-coitum, and at 75 mg/kg bw/day on Days 9-18 post-coitum when compared to controls.
Body weight gain corrected for gravid uterus was statistically significantly lower at 75 mg/kg bw/day when compared to controls.
No toxicologically relevant changes in body weights, body weight gain or weight gain corrected for gravid uterus were noted by treatment at 7.5 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative food consumption were significantly reduced at 75 mg/kg bw/day from Day 6 post-coitum onwards (not statistically significant for relative food consumption on Days 15-18 post-coitum). At 25 mg/kg bw/day, absolute and relative food consumption were statistically significantly reduced on Days 6-12 post-coitum and on Days 6-9 post-coitum, respectively.
Food consumption before or after correction for body weight at 7.5 mg/kg bw/day were comparable to control levels

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights (both absolute and relative to terminal body weights) were statistically significantly increased at 75 mg/kg bw/day. An increase of 15% was noted for relative liver weights. No toxicologically relevant changes in liver weights were noted at 7.5 and 25 mg/kg bw/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related non-adverse microscopic findings were noted in the liver of the 75 mg/kg bw/day group. An increased glycogen content was present in the liver at 75 mg/kg bw/day up to slight degree. This was considered to be within the range of background pathology encountered in pregnant non-fasted female rats of this age and strain and for this study type and therefore non-adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In the 75 mg/kg bw/day group, a left lateral liver lobe grown together with the stomach and a diaphragmatic hernia of the right median liver lobe were noted for single females. Moreover, one control female showed a discolored thymus and one control female showed alopecia, confirming the clinical sign observed during the in-life phase. At the incidence observed, these findings were considered to be incidental and not toxicologically relevant.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on pre- or post-implantation loss by treatment up to 75 mg/kg bw/day. Although (not statistically significant) higher number of pre-implanation losses was observed in high-dose group (10.9% vs 4.5% in controls), it still fell within the historical control values.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on the number of litters lost by resorption by treatment up to and including 75 mg/kg bw/day.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on the number of early or late resorptions by treatment up to and including 75 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on the number of dead fetuses by treatment up to and including 75 mg/kg bw/day.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on the pregnancy duration by treatment up to and including 75 mg/kg bw/day/
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There were no effects on pregnancy duration.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were pregnant and had litters with viable fetuses.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to and including 75 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 75 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no reductions in numbers of live offspring at any dose level.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to and including 75 mg/kg bw/day.
Mean sex ratios (males:females) were 54:46, 57: 43, 58:42 and 50:50 for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.5, 11.2, 10.9 and 11.0 fetuses/litter for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to and including 75 mg/kg bw/day.
The only external malformation that occurred among fetuses in this study was observed in Group 3 fetus. This fetus had an inwards rotated hind limb whereby no structural abnormality could be detected skeletally and as it occurred singly, it was considered to be a chance finding.
In addition, a late resorption with generalized edema (anasarca) was noted in the control group and as such considered toxicologically irrelevant.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to and including 75 mg/kg bw/day.
Malformations that were revealed at skeletal examination included in Group 4 severely malaligned sternebrae and vertebral anomaly without associated rib anomaly, and in Group 3 bent scapula and humerus. No malformations were observed in Group 2 and in the control Group, one fetus had a vertebral anomaly with associated rib anomaly. The single occurrence and group distribution of these malformations do not suggest any treatment relationship. Moreover, all were previously seen in historical controls.
Skeletal variations occurred at an incidence of 79.5%, 82.4%, 84.1% and 82.6% per litter in Groups 1, 2, 3 and 4, respectively. All variations noted were not considered treatment related as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to and including 75 mg/kg bwday.
Visceral malformations observed in groups treated with test item were a combination of interrupted aortic arch and atrial septum defect in one Group 4 fetus and a small eye in one Group 3 and one Group 4 fetuses. As these malformations occurred singly and there even was a control fetus that missed an eye, these were considered chance findings.
Remaining visceral malformations occurred in a control fetus that had situs inversus and abnormal lung lobation. The variations in this study occurred at low incidences, in the absence of a dose-related incidence trend or were observed in a control fetus only. Therefore, these visceral malformations and variations were not considered to be treatment related.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects observed at the highest tested dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Accuracy and homogeneity

The concentrations analysed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (i.e. mean accuracies 91% for group 2 (n = 6) and 92% for group 3 (n = 2)).

The mean accuracy of the formulation of Group 4 was slightly below the target concentration (i.e. 89% of target). The accuracy of preparation of the formulation is considered acceptable

since the deviation is small and in line with the results of the QC samples at the similar concentration level of 15 mg/g.

The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation 0.86% for group 2 and 2.6% for group 4, n= 6).

Table 1. Summary of developmental effects in the study.

Dose level (mg/kg bw/day)	0	8	25	75
Pregnant/total dams	22/22	22/22	22/22	22/22
-early resorptions -late resorptions (% per litter)	8.1 0.4	2.7 0.0	4.1 0.0	5.0 0.0
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only	0	0	0	0
Pre-implantation loss (number and percent)	13 (4.5%)	26 (8.5%)	27 (8.9%)	34 (10.9%)
Post-implantation loss (number and percent)	17 (8.5%)	7 (2.7%)	11 (4.1%)	13 (5.0%)
Body weight on day 21 (g)	332	335	327	320
Body weight gain day 6-21 (%)	46	48	44	42
Gravid uterine weight (g)	74.6	77.3	75.4	74.0
Mean live offspring (number)	10.5	11.2	10.9	11.0
Live offspring (percent)	91.5	97.3	95.9	95.0
Mean fetal/pup body weight males (g)	5.4	5.3	5.4	5.2
Mean fetal body weight females	5.1	5.1	5.2	5.0
Mean fetal body weight (sexes combined)	5.3	5.3	5.3	5.1
Malformations (including runts) number and percent of fetuses per litter	3 (2.5%)	0 (0.0%)	3 (2.3%)	4 (3.2%)
Variations (% per litter) -external -soft tissue -skeletal	0 8.3 79.5	0 3.8 82.4	0 13.5 97.6	0 5.0 87.6

Table 2. Historical control data of fetal examinations

2-AMYLANTHRAQUINONE							Project 511244
Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21		Mean of Study Means
Study Date Range: 2014 - 2015
Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	13
Total No. of Animals in the Control Group	304
No. of Animals that Died	0
No. of Animals that were Euthanized	0
No. of Animals that Aborted or Delivered	3
Percent Pregnant		98.8	2.73	100.0	90.9	100.0	97.1	100.0
No. of Animals Examined at Laparohysterectomy	301
No. Nongravid	4
No. Gravid	297
No. with Only Resorptions	2
No. of Dams with Live Fetuses	295
Mean No. Viable Fetuses/Dam		10.7	0.71	10.6	9.1	11.6	10.3	11.2
Total No. Viable Fetuses	3194
Viable Fetuses (%/Litter)		95.2	2.63	95.9	88.9	98.4	93.6	96.8
Mean No. Postimplantation Loss/Dam		0.5	0.15	0.4	0.2	0.7	0.4	0.6
Total No. Postimplantation Losses	134
Postimplantation Loss (%/Litter)		4.8	2.63	4.1	1.6	11.1	3.2	6.4
Dead Fetuses (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Early Resorptions (%/Litter)		4.7	2.62	4.1	1.6	11.1	3.2	6.3
Late Resorptions (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Mean No. Implantations/Dam		11.2	0.69	11.1	9.6	12.0	10.8	11.6
Mean No. Corpora Lutea/Dam		11.9	0.71	11.7	10.9	13.2	11.5	12.3
Mean No. Preimplantation Loss/Dam		0.7	0.32	0.6	0.2	1.3	0.5	0.9
Total No. Preimplantation Losses	207
Preimplantation Loss (%/Litter)		6.2	3.43	5.8	2.0	14.5	4.2	8.3
Total No. Male Fetuses	1617
Total No. Female Fetuses	1577
% Males/Litter		50.8	2.12	50.7	46.6	53.7	49.5	52.0
% Female/Litter		49.2	2.12	49.3	46.3	53.4	48.0	50.5
Mean Fetal Body Weight (g)		5.2	0.08	5.2	5.1	5.3	5.1	5.2
Mean Male Body Weight (g)		5.4	0.10	5.4	5.2	5.5	5.3	5.4
Mean Female Body Weight (g)		5.1	0.07	5.1	5.0	5.2	5.0	5.1
Mean Male Placenta Weight (g)^1		0.46	0.01	0.47	0.44	0.47	0.4	0.5
Mean Female Placenta Weight (g)^1		0.44	0.01	0.44	0.42	0.45	0.4	0.5

¹Based on 4 datasets

2-AMYLANTHRAQUINONE								Project 511244
APPENDIX 5
Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21
Study Date Range: 2014 - 2015
No. of Studies	13
Total No. Fetuses/Litters Examined Externally	3194	295
Total No. Fetuses/Litters Examined Viscerally	2061	295
Total No. Fetuses/Litters Examined Skeletally	2059	295
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
Total External Malformations								1	1
Total Visceral Malformations								7	7
Total Skeletal Malformations								15	15
Total Malformations								22	22
EXTERNAL
Exencephaly	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Eye(s)- Open	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
VISCERAL
Diaphragmatic Hernia	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Eye(s)- Absent and/or Small	0.1	0.26	0.0	0.0	0.9	0.0	0.2	3	3
Hydrocephaly- External	0.0	0.12	0.0	0.0	0.5	0.0	0.1	1	1
Situs Inversus	0.2	0.34	0.0	0.0	1.0	0.0	0.4	3	3
SKELETAL
Jaw- Upper Jaw Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Jaw- Lower Jaw Absent or Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Limb Bone(s)- Bent	0.3	0.44	0.0	0.0	1.1	0.0	0.5	4	4
Rib Anomaly	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Skull Anomaly	0.1	0.34	0.0	0.0	1.2	0.0	0.3	2	2
Sternebra(e)- Fused	0.1	0.29	0.0	0.0	1.0	0.0	0.3	2	2
Sternebra(e) Malaligned (Severe)	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Sternoschisis	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Vertebral Anomaly With or Without Associated Rib Anomaly	0.2	0.53	0.0	0.0	1.9	0.0	0.5	3	3
Vertebral Centra Anomaly	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1

2-AMYLANTHRAQUINONE								Project 511244
Historical Control Data Rat: Crl:WI(Han) (outbred. SPF-Quality)
Gestation Day 21
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Kidney(s)- Renal Papilla(e) Absent and/or Small	0.1	0.25	0.0	0.0	0.9	0.0	0.2	2	2
Liver- Appendix	1.2	0.56	1.3	0.3	2.3	0.9	1.6	23	21
Liver- Discolored	0.1	0.30	0.0	0.0	1.0	0.0	0.3	3	3
Liver- Small Supernumerary Lobe(s)	4.0	1.96	4.0	1.3	7.7	2.8	5.2	69	58
Spleen- Supernumerary	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Thymus- Partially Undescended Horn(s)	1.3	1.55	0.8	0.0	4.3	0.3	2.2	34	23
Thyroid- Discolored	0.1	0.36	0.0	0.0	1.3	0.0	0.3	1	1
Ureter(s)- Convoluted	1.0	2.39	0.0	0.0	8.7	0.0	2.5	43	28
Ureter(s)- Dilated	0.9	2.33	0.0	0.0	8.5	0.0	2.3	44	19
SKELETAL
7th Cervical Rudimentary Rib(s)	1.7	1.34	1.2	0.0	4.4	0.9	2.5	30	26
7th Cervical Full Rib(s)	0.1	0.36	0.0	0.0	1.1	0.0	0.4	2	2
14th Full Rib(s)	5.7	4.65	5.2	0.0	13.1	2.9	8.5	88	64
14th Rudimentary Rib(s)	44.1	19.84	54.4	19.0	72.0	32.1	56.1	798	250
Metacarpal(s) and/or Metatarsal(s) Unossified	2.2	1.97	1.0	0.0	6.3	1.0	3.4	41	24
Pelvic Girdle- Caudal Shift	6.6	3.77	7.1	1.7	12.8	4.3	8.9	127	71
Rib(s)- Bent	10.6	7.78	10.2	0.8	22.3	5.9	15.3	162	85
Rib(s)- Short	0.0	0.06	0.0	0.0	0.2	0.0	0.0	1	1
Skull- Reduced Ossification	2.7	2.55	1.8	0.0	7.0	1.2	4.3	81	46
Skull- Supernumerary Site	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Sternebra(e) #1, #2, #3 and/or #4 Unossified	0.2	0.31	0.0	0.0	0.8	0.0	0.3	3	3
Sternebra(e) #5 and/or #6 Unossified	0.9	1.33	0.0	0.0	4.1	0.1	1.7	37	23
Sternebrae- Malaligned (Slight or Moderate)	11.1	5.72	8.9	4.4	21.3	7.6	14.5	188	131
Sternum- Supernumerary Ossification Site	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Vertebral Centra- Reduced Ossification	0.6	0.88	0.4	0.0	3.0	0.1	1.2	12	12

Conclusions:

In a developmental toxicity study with rats, oral exposure to the test substance 2-amylanthraquinone during days 6-20 of gestation did not induce any developmental effects up to and including the top dose of 75 mg/kg bw/day. The NOAEL for maternal toxicity was 25 mg/kg bw/day, based on the changes in body weights and food consumption, as well as liver weight increase at the highest dose level.

Executive summary:

In a GLP-compliant OECD Guideline 414 study, the test substance 2 -amylanthraquinone was administered to pregnant rats by oral gavage during days 6 -20 of gestation. Maternal toxicity was observed at the highest dose level of 75 mg/kg bw/day, manifested in slightly lower body weights and statistically significantly lower body weight gain during the treatment period, as well as reduced food consumption. Relative liver weights were statistically significantly increased by 15% at 75 mg/kg bw/day compared to the concurrent vehicle controls. Based on these observations the NOAEL for maternal toxicity was set at the mid-dose of 25 mg/kg bw/day.

No developmental toxicity was observed at any dose level. There were also no toxicologically relevant effects on the numbers of pregnant females, corpora lutea and implantation sites, or in pre- or post-implantation loss by treatment up to and including the dose of 75 mg/kg bw/day. Based on this, the NOAEL for developmental toxicity was set at 75 mg/kg bw/day, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restrictions

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction

An extended one generation study was performed in rats with 2-Amylanthraquinone according to OECD guideline 443 and under GLP principle. Male and female rats from the F0 and F1 generation were exposed to the test substance by oral gavage at dose levels of 5, 20 or 75 mg/kg bw/day. The dose levels were established based on a 90-day repeated dose toxicity study. Dose formulation analyses were performed and demonstrated accurate dosing.

F0 males were treated daily for 10 weeks prior to mating until necropsy after the termination of the F0 females. F0 females were treated daily for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. Cohort 1A and 1B animals were then dosed daily from Postnatal Day (PND) 21 until at least PND 90.

The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, estrous cycling, mating performance, observations of females and litters during lactation, pre-weaning physical development of F1 animals, clinical pathology parameters (haematology, coagulation, clinical chemistry, urinalysis, thyroid stimulating hormone, T3 and T4), gross necropsy findings, organ weights, sperm evaluations and histopathological examinations.

There was a dose dependent increase in the incidence of ploughing and/or salivation at ≥ 5 mg/kg bw/day in the F0, Cohort 1A and 1B animals. These findings were predominantly observed immediately post-dose.

At 75 mg/kg bw/day there were higher numbers of reticulocytes in F0 generation animals and Cohort 1A males and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. Additionally, in the F0 generation females there were high mean corpuscular volume and corpuscular haemoglobin which could be related to the lower levels of red blood cells. At 75 mg/kg bw/day there were higher levels of monocytes in the F0 males, platelets in the Cohort 1A males and lymphocytes in the F0 females the changes, in the F0 males corresponded with a higher level of white blood cells.
At 75 mg/kg bw/day there were lower levels of fibrinogen in the F0 and Cohort 1A males. There were higher levels of albumin at 75 mg/kg bw/day in the F0 males and at ≥ 5 mg/kg bw/day in the Cohort 1A females as well as lower level of globulin at 75 mg/kg bw/day in the F0 animals and Cohort 1A females. The changes in albumin and globulin corresponded with a higher albumin/globulin ratio at 75 mg/kg bw/day in the F0 animals and Cohort 1A females as well as a higher total protein levels at ≥ 5 mg/kg bw/day in the Cohort 1A females. Additionally, there were lower levels of triglycerides at ≥ 20 mg/kg/day in the F0 females and 75 mg/kg bw/day in the Cohort 1A males.
The changes in the haematology, coagulation and clinical chemistry parameters were considered not to be adverse as they were mild to moderate in severity and there were no associated adverse histopathological or toxicology effects.

At 75 mg/kg bw/day there were test item-related histological findings in the kidney of focal mineralization and accumulation of hyaline droplets the Cohort 1A males and tubular basophilia in the Cohort 1A males and females. There were also higher kidney weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and at 20 mg/kg bw/day in F0 and Cohort 1B males. These changes were considered not to be adverse as they were not associated with any adverse histopathological changes.
In the liver at ≥ 20 mg/kg bw/day test item-related histological findings of focal hepatocellular degeneration in the F0 males. Additionally, at 75 mg/kg bw/day there were test item-related histological findings in the liver of hepatocellular hypertrophy in the F0 males and Cohort 1A animals as well as focal hepatocellular necrosis in the F0 males. The histological findings in the liver were considered to be mild or minimal changes therefore considered not to be adverse. Additionally, there were also higher liver weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and in the Cohort 1B males at 20 mg/kg bw/day. These changes in the weights of the liver were not considered to be adverse as they were not associated with any adverse histopathological changes.
There were higher spleen weights at 75 mg/kg bw/day in the F0 animals, Cohort 1B animals and Cohort 1A females and higher pituitary gland weights at ≥ 20 mg/kg bw/day in the F0 females. There were no associated gross pathological or histological findings with these organ weight changes therefore they were considered not to be adverse.

At 75 mg/kg bw/day there were lower levels of T4 in the F0 males and T3 in the F0 animals. There were also higher levels of TSH at 75 mg/kg bw/day in the F0 animals and Cohort 1A males. The changes in the levels of the thyroid hormones was associated with a higher thyroid weight at 75 mg/kg bw/day in the F0 males.

Given the trend of increased liver weights described above, the thyroid changes may be also related to these changes. The changes in the thyroid hormone levels were considered to be related to the test item however, under the conditions of this study no adverse effects could be linked to the changes in the thyroid hormones therefore this change was not taken into account when determining the parental NOAEL.
There were no treatment related findings related to bodyweights or food consumption in the F0 generation or Cohort 1A and 1B animals. There were no treatment related effects seen estrous cycling, mating performance, observations of females and litters during lactation, preweaning physical development of F1 animals or sexual maturation of the F1 animals. There were also no effects on the number of ovarian follicles in the ovaries of the F0 or Cohort 1A females.

In conclusion, the administration of 2-Amylanthraquinone by once daily oral gavage was considered to have a systemic No Observed Adverse Effect Level (NOAEL) of 20 mg/kg bw/day for the F0 and F1 generation, due to the higher organ weights of the kidney and liver and associated minimal to mild histopathology  findings in both liver and kidney, as well as mild to moderate changes in the haematology and clinical pathology parameters at 75 mg/kg bw/day. Findings in the kidney were minimal mineralisation and minimal to mild tubular basophilia; findings in the liver were minimal hepatocellular hypertrophy and degeneration, especially in males. Although individually, these findings were considered not to be adverse and did not indicate significant organ damage or impaired function, the combined changes within these parameters were considered to be adverse at 75 mg/kg bw/day.
The NOAEL for reproduction toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on any of the reproductive endpoints examined within this study. The NOAEL for F1 developmental toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on the developmental endpoints examined within the study.

Developmental Toxicity 

In a GLP-compliant OECD Guideline 414 study, the test substance 2 -amylanthraquinone was administered to pregnant rats by oral gavage during days 6 -20 of gestation. Maternal toxicity was observed at the highest dose level of 75 mg/kg bw/day, manifested in slightly lower body weights and statistically significantly lower body weight gain during the treatment period, as well as reduced food consumption. Relative liver weights were statistically significantly increased by 15% at 75 mg/kg bw/day compared to the concurrent vehicle controls. Based on this the NOAEL for maternal toxicity was set at the mid-dose of 25 mg/kg bw/day.

No developmental toxicity was observed at any dose level. There were also no toxicologically relevant effects on the numbers of pregnant females, corpora lutea and implantation sites, or in pre- or post-implantation loss by treatment up to and including 75 mg/kg bw/day. Based on this, the NOAEL for developmental toxicity was set at 75 mg/kg bw/day, the highest dose tested.

A prenatal developmental toxicity study was performed in rabbits with 2-Amylanthraquinone according to OECD guideline 414 and under GLP principles. To assess potential toxicity, pregnant rabbits were exposed by oral gavage from GD 6 to GD 28 to 0, 3.75, 7.5 or 15 mg/kg bw/day. The dose levels were established based on the results of a dose-range finding study, in which pregnant rabbits were exposed to 5, 10 or 20 mg/kg bw/day. Based on mortality and body weight loss at 20 mg/kg bw/day, this dose level was concluded to exceed the maximum tolerated dose and the highest dose level was thus chosen to be 15 mg/kg bw/day in the main study. Furthermore, post-implantation losses were higher at 10 and 20 mg/kg bw/day than the control (19% or 20%, respectively, compared with 4% in the control). The high dose in the main study was chosen to allow further assessment of this potential effect.

It is noted that there were no fetal external abnormalities observed in the dose-range finding study (no visceral or skeletal examinations were done). Dose formulation analyses were performed in the dose-range finding study and the main study, which showed accurate dosing.
In the main study, the following parameters and end points were evaluated: clinical observations, body weights, food consumption, gravid uterine and corrected body weights, maternal performance, ovarian and uterine examinations, gross maternal pathology, fetal weights and external examinations, fetal sex, visceral and skeletal examinations, and mean fetal ossification sites.
The mean total body weight gain over GD 6 to GD 29 was slightly lower than the control at 15 mg/kg bw/day (12% lower). The mean fetal weights were slightly lower at 7.5 and 15 mg/kg bw/day (up to 2.7 g). The incidence of skeletal variations, indicative of slightly delayed development, was increased at 15 mg/kg bw/day (forepaw phalanges, sternebra and cervical vertebra) and to a lesser extent at 7.5 mg/kg bw/day (sternebra only), which although attributed to the test item, was considered not adverse, since they were expected to be transitory and compensated for during postnatal development.
For all other parameters, there were no changes considered related to administration of 2- Amylanthraquinone. Post-implantation losses in the main study were slightly higher for the test item exposed groups with 3.95%, 7.18%, 11.71% and 6.90% for the control group and the groups dosed at 0, 3.75, 7.5 and 15 mg/kg bw/day, respectively. In light of the minimal magnitude, the absence of an apparent dose-relationship and since the percentages are within normal biological variation, this is concluded not to be test-item related.
In conclusion, administration of 2-Amylanthraquinone to pregnant rabbits by once daily oral gavage at 15 mg/kg bw/day was well-tolerated and resulted in only a minor reduction in the total body weight gain. There were no adverse fetal findings. Based on these results, the maternal and embryofetal no observed-adverse-effect levels (NOAELs) were considered to be at least 15 mg/kg bw/day.

The absence of adverse effects in the pre-natal developmental toxicity study in rabbit supports the absence findings in the prenatal developmental toxicity study for rats.

 

 

 

Toxicity to reproduction: other studies

Description of key information

No information available.

Mode of Action Analysis / Human Relevance Framework

Not applicable, as no reproductive/developmental effects were observed at the highest tested dose level.

Justification for classification or non-classification

Based on a reliable extended one-generation reproductive toxicity study in rats and two reliable pre-natal developmental toxicity studies with rats and rabbits, in which no effects were observed at the highest tested dose levels of 75 mg/kg bw/day for rats and 15 mg/kg bw/day for rabbit, the substance does not need to be classified for reproductive/developmental toxicity in accordance with Regulation 1272/2008/EC.