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

Developmental toxicity / teratogenicity

Some information in this page has been claimed confidential.

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

other: Analytical method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Test material form:

solid

Remarks:

The test item was found to solidify at ambient temperature.

Details on test material:

- Appearance: Solid at room temperature. At temperatures of 37°C - 55°C the test item was a yellow/light brown liquid.

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.

Test animals

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)

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.