2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study in Rats

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2021 to 27 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Test material form:

solid: nanoform

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxy Methyl Cellulose within the mean percent recovery range of 85 to 115
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulation
- Final preparation of a solid: Suspension

FORM AS APPLIED IN THE TEST: Liquid suspension

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on
Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA
- Age at study initiation: Minimum 9 weeks
- Weight at study initiation: Males: 260.41 to 287.31 g and Females: 212.17 to 270.12 g
- Housing: Housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: Start: 28 May 2021 to 23 June 2021.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.4
- Humidity (%): 47 to 64
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 28 May 2021 To: 19 July 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % w/v

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxy Methyl Cellulose is a routinely used and universally accepted
vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: G2: 11.1 mg/mL; G3: 33.3 mg/mL; G4: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- batch no.: SLCD3996

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The collected samples were transferred to Analytical Chemistry department of
Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at
established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were
considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤15%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 (M:F)
- Length of cohabitation: 14 days initially + 7 days extention
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19 [15 days per animal]

Frequency of treatment:

once daily

Duration of test:

15 days per animal starting from implnatation to one day prior to scheduled delivery

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item Hostaperm Red Violet ER 02. However, the NOAEL of Pigment Red 282, the structural analogue of test item is 1000 mg/kg body weight/day estimated in a 28-Day Repeated Dose Oral Toxicity in Wistar Rats performed according to OECD TG 407, (https://echa.europa.eu/registration-dossier/-/registereddossier/14755/7/6/2#).

Based on the above available information, the doses of 0, 111, 333 and 1000 mg/kg body weight/day had been selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).

Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for present prenatal developmental toxicity study in consultation with the sponsor

- Rationale for animal assignment (if not random): The body weight of mated females on each day of gestation day ‘0’ was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups and permanent identification numbers were assigned

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:Gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and
on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs along with uterus and ovaries.

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 raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analysis using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests was designated by the superscripts throughout the report as, * Statistically significant (P<0.05) change than the control group.

Parametric: One-way ANOVA with Dunnett’s post test: Maternal body weight, Percent change in maternal body weight,Corrected body weight for maternal increase,Gravid uterus weight,Maternal Feed consumption, Mean fetal weight per dam, Mean fetal crown rump length per dam, Mean fetal anogenital distance per dam, Serum T3, T4 and TSH levels and Organ weight.

Non-Parametric:Kruskal-Wallis test : No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam,Percent of early/late resorptions per dam,Sex ratio (m/f) per dam,Pre/Post implantation losses (%) per dam,Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison (Cross Tabs): Chi-square test: Frequencies comparison for No. of pregnant / non-pregnant,No. of dams with / without live fetuses,No. of dams with / without dead fetuses,No. of dams with / without resorptions.

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight

Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation sites / Total Number of Corpora lutea x 100

Post-implantation Loss (%) = Number of Implantation sites - Number of Viable Fetuses / Number of Implantation Sites X 100

Anogenital Distance (AGD) Ratio = Anogenital Distance (mm) / Cube root of fetal weight x 100

Historical control data:

Included in report where applicable

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats. (Elizabeth McInnes, 2012).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean serum T3 levels (ng/mL) were 1.567, 1.617, 1.827 and 2.092 for groups G1, G2, G3 and G4 respectively. There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in groups G3 and G4 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

The mean serum T4 levels (ng/mL) were 52.581, 51.869, 52.053 and 54.421 for groups G1, G2, G3 and G4 respectively. There were no changes noted for this parameter across the dose groups when compared to the vehicle control group.

The serum TSH levels (µIU/mL) were 1.906, 1.942, 1.870 and 1.370 for groups G1, G2, G3 and G4 respectively. There were no changes for this parameter across the dose groups when compared to the vehicle control group.

Details on results:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of general/systemic toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions noted

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-Implantation Loss: The mean percentage of pre-implantation loss per litter was 4.71, 0.00, 1.43 and 0.00 for groups G1, G2, G3 and G4 respectively.
There was no test item related changes noted for percentage of pre-implantation loss per litter in all the tested dose groups. The noted statistically significant reduction in percentage of pre-implantation loss per litter in groups G2 and G4 when compared to the vehicle control group is considered as toxicologically irrelevant.

Post-Implantation Loss: The mean percentage of post-implantation loss per litter was 5.67, 3.17, 2.64 and 5.18 for G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G4.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 - ca. 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

other: Serum T3, T4 and TSH levels

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

vitreous chamber / humor

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group (refer table no. 15). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal skeletal alterations were noted across the dose groups and vehicle control group (refer table no. 17). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal soft tissue/visceral alterations were noted across the dose groups and vehicle control group (refer table no. 16). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 - ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: Anogenital distance and Crown rump length

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test item on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate
no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

 

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

 

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤15%.

 

A total of 21 (out of 25), 22 (out of 25), 21 (out of 25), and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 88%, 84% and 88% respectively. In group G4, 1 out of 25 females was noted with total implantation loss with no evidence of live fetuses.

 

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO₂asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid

 

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

 

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no changes noted in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase, maternal feed consumption, number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam. There were no changes noted in mean gravid uterus weight in all the dose levels. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

 

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain.