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Toxicological information

Developmental toxicity / teratogenicity

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
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 item
for 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.