Name: OECD_SIDS / SUBSTANCE : 1,4-Bis(isopropylamino)-9,10-anthracenedione /14233-37-5 Tue, 29 Nov 2022, 14:56:31+0900 /Legal entity owner: National Institute of Health Sciences
Printing date: 2022-11-29T14:56:31.744+09:00

iTable of Contents
0/0 ............................................................................................................................. 1
National Institute of Health Science ..................................................................... 2
1,4-Bis(isopropylamino)-9,10-anthracenedione ...................................................... 3
1 General information .................................................................................. 3
1.1 Identification ................................................................................. 3
Identification ............................................................................... 3
Identification ...................................................................... 3
7 Toxicological information ......................................................................... 4
7.5 Repeated dose toxicity ................................................................... 4
7.5.1 Repeated dose toxicity: oral ................................................. 4
Repeated dose toxicity: oral. 001 .......................................... 4
7.6 Genetic toxicity ............................................................................ 14
7.6.1 Genetic toxicity in vitro ...................................................... 14
Genetic toxicity in vitro.001 ................................................ 14
Genetic toxicity in vitro.002 ................................................ 21
7.8 Toxicity to reproduction ............................................................... 25
7.8.1 Toxicity to reproduction ..................................................... 25
Toxicity to reproduction. 001 .............................................. 25
References ................................................................................................................ 36
Reference Substances ...................................................................................... 36
1,4-Bis(isopropylamino)anthraquinone ........................................................ 36
Test Materials .................................................................................................. 37
1,4-Bis(isopropylamino)anthraquinone ........................................................ 37
Literatures ....................................................................................................... 38
Combined repeat dose and reproductive/developmental toxicity screening
test of 1,4-Bis(isopropylamino)anthraquinone by oral administration inrats ........................................................................................................... 38
In Vitro Chromosomal Aberration Test of on 1,4Bis(isopropylamino)anthraquinone Cultured Chinese Hamster Cells. ............. 39
Reverse Mutation Test of 1,4-Bis(isopropylamino)anthraquinone on
Bacteria. ................................................................................................... 40
Legal Entities ................................................................................................... 41
National Institute of Health Sciences .......................................................... 41ii

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Dossier header
Dossier submission type
Name
OECD SIDS
Versioncore 7.0
Name (given by user)

Dossier subject
Dossier subject
1,4-Bis(isopropylamino)-9,10-anthracenedione / 14233-37-5
Public nameSubmitting legal entity
National Institute of Health Science
Dossier creation date/time
Tue, 29 Nov 2022, 14:56:31+0900
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LEGAL_ENTITY: National Institute of Health
Science
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General information
Legal entity nameNational Institute of Health Science21,4-Bis(isopropylamino)-9,10-anthracenedione
General information
Identification
Identification
SUBSTANCE: 1,4-Bis(isopropylamino)-9,10-anthracenedione

UUID: 2a0b5c5e-11da-4464-bb85-dee4a7be72f1
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Substance name
1,4-Bis(isopropylamino)-9,10-anthracenedione
Legal entity
National Institute of Health Sciences / Kawasaki / Japan

Identification of substance
Reference substance
1,4-Bis(isopropylamino)anthraquinone / 14233-37-5
EC number

EC name

CAS number

CAS name

14233-37-5
IUPAC nameRole in the supply chain
Manufacturer
false
Importer
false
Only representativefalse
Downstream user
false

3

Toxicological information
Repeated dose toxicity
Repeated dose toxicity: oral
ENDPOINT_STUDY_RECORD: Repeated dose toxicity: oral. 001

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Administrative data
Endpoint
short-term repeated dose toxicity: oral
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study OECD Test Guideline study under GLP condition
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study 7.8.1 Toxicity to reproduction: Toxicity to reproduction. 001
Related information
OECD / Toxicity to reproduction / Toxicity to reproduction. 001 / 1,4-Bis(isopropylamino)-9,10anthracenedione / 14233-37-5

Data source
Reference
Combined repeat dose and reproductive/developmental toxicity screening test
Bis(isopropylamin / Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5d.pdf
4

of

1,4-

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test)
Deviationsno
GLP compliance
yes
Limit test
no

Test material
Test material information
1,4-Bis(isopropylamino)anthraquinone
Specific details on test material used for the study- Name of test material (as cited in study report): 1,4-Bis(isopropylamino)anthraquinone- Analytical purity: 100%
- Storage condition of test material: Cold and dark place (3 - 6°C)
- Stability under test conditions: The stability of test material was identified by analysis of the remainder.

Test animals
Speciesratcommon rodent species
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Atsugi Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation: Male: 416 g (385-475 g), Female: 253 g (230-279 g)- Housing: Animals were individually housed in bracket-type metallic wire-mesh cages (254W × 350D
× 170H mm), from gestation day 17 to lactation day 4, Dams were bred individually or with individual
littermates in plastic cages (340W x 400D x 185H mm) and bedding.
- Diet: Solid feed (NMF: Oriental Yeast Co., ltd.) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: 19 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 23±3 (actual temperature: 22-26°C)
5

- Humidity (%): 50±20% (actual humidity: 33-50%)- Air changes (per hr): 10-15- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 7:00~19:00)

Administration / exposure
Route of administration
oral: gavage
Vehiclemethylcellulose 0.5w/v%
Details on oral exposure- Amount of vehicle (if gavage): 10 mL/kg- Dosing volume: 10 mL/kg
Analytical verification of doses or concentrations
yes
Details on analytical verification of doses or concentrations
Test suspensions at each concentration to be used for males group in week 1 and weeks 6 ad
ministration were analyzed by absorptiometry. Results showed that the concentration of test suspensi
ons in each concentration was 95.8 to 103.3% of the nominal concentration and both values were
within the acceptable range (concentration: percentage of nominal concentration, 100±10%)Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating(P) Females: 41-46 days including 14 days pre-mating, mating and gestation periods and the days
until day 4 of lactation
Female (non- mating, satellite group): 42 days
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
12

mg/kg bw/day (actual dose received)

Dose / conc.
60

mg/kg bw/day (actual dose received)

Dose / conc.
300

mg/kg bw/day (actual dose received)

No. of animals per sex per dose
Mating group: 12 animals/sex /dose (0, 12, 60, and 300 mg/kg bw/day). 5 males each in the 0 and
300 mg/kg bw/day were assigned to the recovery group.
6

Non-mating group (Satellite group): 10 females/dose (0 and 300 mg/kg bw/day), 5 females in eachgroup were assigned to the recovery group.
Control animals
yes, concurrent vehicle
Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the highest dose of 300
mg/kg bw/day was selected as an expected obvious toxic dose, and the lowest dose of 12 mg/kg
bw/day was selected as an expected no toxic dose. The middle dose levels of 60 mg/kg bw/day wereselected.
[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0, 100, 300 or 1000 mg/kg bw/
day). At 100 mg/kg bw/day and above, bluish discoloration of skin, dark blue feces, purplish urine,
bluish discoloration of whole body fat or organs, decreased food consumption and increased liver
weight were observed. At 1,000 mg/kg bw/day, slight suppression of body weight gain in males andfemales, low urea nitrogen in males, high total cholesterol in females and high adrenal weight in females were observed.
- Rationale for animal assignment (if not random): Body weight-balanced randomization

Examinations
Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: Yes- Time schedule: 3 times/day (before administration, immediately after administration and 1-3 hours
after administration) during the administration period. Once a day during the recovery period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule:
Males in the mating group and females in the non-mating (satellite) group: once before the start ofadministration, once every weekly during the administration and recovery periods.
Females in the mating group: once a week during the pre-mating period, on designated days duringmating, gestation, and lactation (mated females: days 1, 7, 14 and 20 of pregnancy, parturient
females: day 4 of lactation).
BODY WEIGHT: Yes- Time schedule for examinations:
Males in the mating group and females in the non-mating (satellite) group: days 1, 4, 8, 11, 15, 18, 22,
25, 29, 32, 36, 39 and 42 of administration and on the day of necropsy, and days 1, 4, 8, 11 and 14 ofrecovery and on the day of necropsy.
Females in the mating group: days 1, 4, 8, 11, and 15 of administration (uncopulated animals were
weighed on days 18 of administration as well), days 0, 7, 14 and 20 of gestation, days 0 and 4 oflactation and the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Males in the mating group and females in the non-mating (satellite) group: days 1, 4, 8, 11, 15, 32, 36,
39 and 42 of administration, and days 1, 4, 8, 11 and 14 of recovery
Females in the mating groups: days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20
of gestation and days 2 and 4 of lactation.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery p
eriod in both sexes- Anaesthetic used for blood collection: Isoflurane7- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/
day)- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mea
n corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte percentage,platelet count, white blood cell count, differential white blood cell count, absolute number of each
white blood cell, prothrombin time, activated partial thromboplastin time, fibrinogen.
CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/
day)- Parameters checked: ALP, total cholesterol, triglyceride, phospholipids, total bilirubin, glucose, bloodurea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein,
albumin, A/G ratio, AST (GOT), ALT (GPT), LDH, γ-GTP
BLOOD HORMONE: Yes- Time schedule for collection of serum: Same as clinical chemistry- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/day)
- Parameters checked: Triiodothyronine (T3), Thyroxin (T4), and thyroid stimulating hormone (THS) of
thyroid hormone
URINALYSIS OF MALES: Yes- Time schedule for collection of urine: final week of administration (days 36 to 37 of administration)and in the final week of recovery (days 8 to 9 of recovery)- Metabolism cages used for collection of urine: Yes
A urine collector to collect four-hour urine samples under fasting but ad libitum drinking conditions, fol
lowed by collection of 20-hour urine samples under ad libitum feeding and drinking conditions.
- How many animals: 5 animals/group- Parameters checked: pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, color,
sediment, urine volume (4-hour volume), osmotic pressure, urine volume (20-hour volume), water intak
e (24-hour volume).
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations:
Males in the mating groups: final week of administration (day 40 of administration)
Females in the mating groups: lactation day 4 (day 41 to day 44 of administration) after necropsy of
F1 pupsMales and females in the recovery groups: final week of administration (day 40 of administration) andin the final week of recovery (day 12 of recovery).
- Dose groups that were examined: All dose groups (5 animals/sex/group)- Battery of functions tested:
1) Manipulative Test. Auditory response, approach response, touch response, tail pinch response, pupi
llary reflex, aerial righting reflex, landing foot splay
2) Measurement of Grip Strength. Following manipulative test, grip strength of forelimb and hind limb
were measured by CPU gauge MODEL-RX-5 (AIKOH Engineering Co., Ltd.).
3) Measurement of Motor Activity. Following measurement of grip strength, motor activity wasmeasured by a motor activity sensor for experimental animals NS-AS01 (Neuro Science, Inc.). The
measurement was conducted for 1 hour, and measured values at 10-minute intervals and from 0 to
60 minutes were collected.

8Sacrifice and pathology
GROSS PATHOLOGY: Yes
ORGAN WEIGHT: Yes [brain, pituitary, thyroids (including parathyroids), adrenal gland, thymus, spleen,
heart, liver, kidney, testis, epididymis, prostate, seminal vesicles, ovary, uterus]
HISTOPATHOLOGY: Yes, [cerebrum, cerebellum (including pons), sciatic nerve, spinal cord (thoracic),eyeball, optic nerve, Harderian gland, pituitary, thyroid, parathyroid, adrenal glands, thymus, spleen,submandibular lymph nodes, mesenteric lymph nodes, heart, thoracic aorta, trachea, lung (including
bronchial), tongue, larynx, esophagus, stomach, duodenum, jejunum, ileum (including payer’s patch),cecum, colon, rectum, submandibular gland, sublingual gland, liver, pancreas, kidney, bladder, testis,
epididymis, ovary, uterus, vagina, prostate, seminal vesicles (including coagulating gland), mammary
gland (inguinal region), skin (inguinal region), sternum and femur (including bone marrows), femoral skeletal muscle, gross abnormalities site and Individual identification site (pinna with ear tag)]
Underlined organs and tissues are fixed and stored only.
Statistics
For quantitative data, homogeneity of variance was tested using Bartlett method first. If the variance
was homogenous, statistical difference between each treatment group and the control group was
analyzed using Dunnett method. If not homogenous, statistical difference between each treatmentgroup and the control group was tested using Steel method. For comparison of quantitative data be
tween two groups in the recovery test, homogeneity of variance was analyzed by F-test. Then, if homo
genous, student's t-test was applied. If not homogenous, Aspin-Welch's t-test was used.
Regarding clinical observation (except for frequency of urination, defecation, rearing and grooming)and sensory reactivity, Steel test was applied. Regarding auditory response, approach response, touch
response, tail pinch response, pupillary reflex, aerial righting reflex, Fisher's test was applied.

Results and discussion
Results of examinations
Clinical signs
effects observed, non-treatment-related
Description (incidence and severity)
CLINICAL SIGNS:[At the administration period]:
Blue skin, dark blue stools, and purple urine were observed in males at 60 mg/kg bw/day and above,and in females at 12 mg/kg bw/day and above, which were considered to reflect the color of the test
substance, independent of adverse reactions.
[At the recovery period]:
These findings diminished during the recovery period.
DETAILED CLINICAL OBSERVATIONS:
There were no changes related to the test substance in any groups at the administration and recovery
periods.
Mortality
no mortality observed
Body weight and weight changes
no effects observed
Food consumption and compound intake (if feeding study)
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:9

Decrease in food consumption were observed in mating females at 12 mg/kg bw/day and above.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Food efficiency
not examined
Water consumption and compound intake (if drinking water study)
not examined
Ophthalmological findings
not examined
Haematological findings
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:
Decreases in white blood cell count, neutrophil count, eosinophil count and monocyte count wereobserved in mating females at 60 mg/kg bw/day and above.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Clinical biochemistry findings
effects observed, treatment-related
Description (incidence and severity)
Including blood hormones (T3, T4, TSH)
CLINICAL BIOCHEMISTRY:[At the end of administration period]:
Increase in ALT was observed in non-mating females at 300 mg/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
BLOOD HORMONE:[At the end of administration period]:
Increases in T3 and T4 were observed in mating females at 60 mg/kg bw/day and above.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Urinalysis findings
effects observed, non-treatment-related
Description (incidence and severity)[At the administration period]:
Light purple to deep purple urine was observed in the treated groups, which were considered to reflect
the color of the test substance independent of adverse effects.
[At the recovery period]:
Light purple urine was observed in males and females at 300 mg/kg bw/day.
Behaviour (functional findings)
no effects observed
Immunological findings
not examined
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
10Description (incidence and severity)[At the end of administration period]:
Increases in absolute and relative liver weights were observed in males at 60 mg/kg bw/day andincrease in relative liver weight in males at 300 mg/kg bw/day. Increases in absolute and relative liver
and adrenal weights, decreases in absolute and relative spleen weights, and decreases in absolute
ovary weight were observed in non-mating females at 300 mg/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Gross pathological findings
effects observed, non-treatment-related
Description (incidence and severity)[At the end of administration period]:
In the treated groups, skin, adipose tissue, and organs of the whole body were blue, which were consi
dered to reflect the color of the test substance independent of adverse effects.
[At the end of recovery period]:
In the treated groups, skin and adipose tissue were blue, which were considered to reflect the color of
the test substance independent of adverse effects.
Neuropathological findings
not examined
Histopathological findings: non-neoplastic
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:
Liver: Centrilobular hypertrophy of hepatocyte was observed in males and mating females at 60 mg/
kg bw/day and above, and non-mating females at 300 mg/kg bw/day.
Adrenal gland: Hypertrophy of cortical cell was observed in non-mating females at 300 mg/kg bw/day.
Thymus: Atrophy was observed in mating females at 300 mg/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Histopathological findings: neoplastic
not examined

Effect levels
Key result
false
Dose descriptor
NOAEL
Effect level12

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
male
11

Basis for effect level
histopathology: non-neoplastic
At 60 mg/kg bw/day, centrilobular hypertrophy of hepatocyte was observed in males.
organ weights and organ / body weight ratios
At 60 mg/kg bw/day, increase in liver weight was observed in males.
Key result
false
Dose descriptor
NOAEL
Effect level12

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
femaleBasis for effect level
clinical biochemistry
At 60 mg/kg bw/day, increases in T3 and T4 were observed in mating females.
haematology
At 60 mg/kg bw/day, decreases in white blood cell count, neutrophil count, eosinophil count andmonocyte count were observed in mating females.
histopathology: non-neoplastic
At 60 mg/kg bw/day, centrilobular hypertrophy of hepatocytes was observed in females. The
centrilobular hypertrophy of hepatocyte was observed in mating females at 60 mg/kg bw/day andabove, and non-mating females at 300 mg/kg bw/day. (The non-mating females received only 2
doses of 0 and 300 mg/kg bw/day.)Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5d.pdf

Applicant's summary and conclusion
Conclusions
Based on the effects of 1,4-bis(isopropylamino)anthraquinone on the liver, the no observed adverse effect levels (NOAELs) for repeated oral dosing were determined to be 12 mg/kg bw/day in male andfemale rats.
Executive summary
In the combined repeated dose and reproductive/developmental screening test (OECD TG422), SD rats
were treated orally with the test substance at the doses of 0, 12, 60 and 300 mg/kg bw/day, Males were
dosed for 42 days including 14-days pre-mating and mating periods. Females were dosed during theperiods of pre-mating, mating, gestation and days until day 4 of lactation (41 to 46 days).
No animals died in any of the treatment groups, and there were no treatment-related effects on clinicalsigns, detailed clinical observations, body weight, functional tests, grip strength, motor activity, or12urinalysis. The following findings were observed in examination at the end of administration period. In
the haematological examination, decreases in white blood cell count, neutrophil count, eosinophil countand monocyte count were observed in mating females at 60 mg/kg bw/day and above. In the clinicalchemistry, increases in T3 and T4 were observed in mating females at 60 mg/kg bw/day and above.
An increase in ALT was observed in non-mating females at 300 mg/kg bw /day. In the organ weights,
increases in liver weight were observed in males at 60 mg/kg bw/day and above. Increases in liver
and adrenal weights, decreases in spleen and ovary weights were observed in non-mated females at
300 mg/kg bw/day. In the histopathological examination, centrilobular hypertrophy of hepatocyte wasobserved in males and mating females at 60 mg/kg bw/day and above, and non-mating females at 300
mg/kg bw/day. Hypertrophy of cortical cell of adrenal gland was observed in non-mating females at
300 mg/kg bw/day. Atrophy of thymus was observed in mating females at 300 mg/kg bw/day. At theend of recovery period, all changes observed in haematological examination, clinical chemistry, organ
weight and histopathological examination were disappeared or reduced.
Based on the above results, NOAELs for repeated dose toxicity of 1,4-bis(isopropylamino)anthraquinone
were determined to be 12 mg/kg bw/ day in male and female rats.

13

Genetic toxicity
Genetic toxicity in vitro
ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.001

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Author:Date: 2022-11-14T15:04:14.000+09:00
Remarks:

Administrative data
Endpoint
in vitro gene mutation study in bacteria
Type of information
experimental study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study
Reliability 1

Data source
Reference
Reverse Mutation Test of 1,4-Bis(isopropylamino)anthraquinone on Bacteria. / Ministry of Health,Labour and Welfare (MHLW), Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
14

in vitro gene mutation study in bacteria
Deviations
no
Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
genetic toxicity in vitro, otherDeviationsno
GLP compliance
yesType of assay
bacterial reverse mutation assay
in vitro gene mutation study in bacteria

Test material
Test material information
1,4-Bis(isopropylamino)anthraquinone
Specific details on test material used for the study
Purity 99.73%

MethodSpecies / strain
Species / strain / cell typeS. typhimurium TA 1535, TA 1537, TA 98 and TA 100
bacteria
Species / strain / cell type
E. coli WP2 uvr A
bacteria
Metabolic activation
with and without
Metabolic activation system
S9 mix: SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
-S9 mix:
2.44, 4.88, 9.77, 19.5, 39.1, 78.1 μg/plate (TA100 strain)
9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA1535, TA98, TA1537 strains)
313, 625, 1250, 2500, 5000 μg/plate (WP2uvrA strain)+S9 mix:
313, 625, 1250, 2500, 5000 μg/plate (TA100, TA1535, TA1537 strains)15

156, 313, 625, 1250, 2500, 5000 μg/plate (TA98 strain)
313, 625, 1250, 2500, 5000 μg/plate (WP2uvrA strain)
Maximum concentration was established based on the result of the preliminary test at concentration
up to 5000 ug/plate. In this test, the growth inhibition was observed at 78.1 μg/plate and above for S.
typhimurium TA100 without S9 mix, at 313 μg/plate and above for S. typhimurium TA1535, TA98, TA
1537 without S9 mix.
Vehicle / solvent- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substancesodium azide
SAZ
benzo(a)pyrene
furylfuramide
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide
other: ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine 2HCl, 2AA: 2-a
minoanthracene
Remarks-S9 mix: AF-2: (TA 100, TA98 and WP2 uvrA), SAZ: (TA1535), ICR-191: (TA1537).
+S9 mix: 2AA: (TA1535 and WP2 uvrA), benzo(a)pyrene (TA100, TA98 and TA1537)
Details on test system and experimental conditions
METHOD OF APPLICATION: Preincubation
DURATION- Preincubation period: 20 min at 37°C
- Exposure duration: 48 or 48.5 hrs
NUMBER OF PLATES: 3
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY- Method: other: growth inhibition
Evaluation criteria
A chemical was judged to be mutagenic when the mean number of revertant colonies per plateincreased more than twice that of the negative control and when the dose-related and reproducible increase was observed.
StatisticsnoResults and discussion
16

Test results
Key result
false
Species / strain
S. typhimurium TA 1535
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity -S9 mix: 313 μg/plate
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 1537
bacteria
Metabolic activation
with
Genotoxicity
positive
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
false
17

Species / strain
S. typhimurium TA 1537
bacteria
Metabolic activation
without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity -S9 mix: 156 μg/plate and above
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 98
bacteria
Metabolic activation
with
Genotoxicity
positive
Cytotoxicity / choice of top concentrations
cytotoxicity +S9 mix: 5000 μg/plate
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
false
Species / strain
S. typhimurium TA 98
bacteria
18

Metabolic activation
without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity -S9 mix: 313 μg/plate
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
false
Species / strain
S. typhimurium TA 100
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity -S9 mix: 78.1 μg/plate and above
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
false
Species / strain
E. coli WP2 uvr A
bacteria
Metabolic activation
with and without
Genotoxicity
negative
19

Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Additional information on results
The maximum specific activity of mutation was 750 revertants/mg, which was observed in platesof Salmonella typhimurium TA98 treated with the test article at 156 μg/plate without metabolic a
ctivation.

Any other information on results incl. tables
Figures and Tables (in Japanese) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5e.pdf
Please also see the attached files (Tables in English)

Applicant's summary and conclusion
Conclusions
Interpretation of results (migrated information): positive with metabolic activation.
Executive summary
In a bacterial reverse mutation assay using Salmonella typhimurium TA100, TA1535, TA98, and TA
1537, and Escherichia coli WP2uvrA (OECD TG 471), 1,4-bis(isopropylamino)anthraquinone was positive
for TA98 and TA 1537 with metabolic activation. The maximum specific activity of mutation was 750
revertants/mg/plate, which was observed in plates of Salmonella typhimurium TA98 treated with thetest article at 156 μg/plate without metabolic activation.

20

ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.002

UUID: 27e78527-2620-4760-bb8d-1ce5b7fa4523
Dossier UUID:
Author:Date: 2021-03-08T16:43:54.000+09:00
Remarks:

Administrative data
Endpoint
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study under GLP condition
Reliability 1

Data source
Reference
In Vitro Chromosomal Aberration Test of on 1,4-Bis(isopropylamino)anthraquinone Cultured Chinese
Ham / Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
21

in vitro cytogenicity / chromosomal aberration study in mammalian cells (from 26 September 2014)
Deviations
no
Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
genetic toxicity in vitro, otherDeviationsno
GLP compliance
yesType of assay
in vitro mammalian chromosome aberration testin vitro cytogenicity / chromosome aberration study in mammalian cells

Test material
Test material information
1,4-Bis(isopropylamino)anthraquinone
Specific details on test material used for the study
Purity: 99.73%

MethodSpecies / strain
Species / strain / cell type
Chinese hamster lung (CHL/IU)
mammalian cell line
Metabolic activation
with and without
Metabolic activation system
S9 mix: SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
Cell growth inhibition study-S9 mix (short-term treatment): 25.8, 51.6, 103, 206, 413, 825, 1650, 3300 ug/mL+S9 mix (short-term treatment): 25.8, 51.6, 103, 206, 413, 825, 1650, 3300 ug/mL-S9 mix (continuous treatment, 24hr): 25.8, 51.6, 103, 206, 413, 825, 1650, 3300 ug/mL-S9 mix (continuous treatment, 48hr): 25.8, 51.6, 103, 206, 413, 825, 1650, 3300 ug/mL
Main study-S9 (short-term treatment): 825, 1650, 3300 ug/mL+S9 (short-term treatment): 825, 1650, 3300 ug/mL
Vehicle / solvent- Vehicle(s)/solvent(s) used: 0.5%CNC Na
22

Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substancecyclophosphamide+S9
mitomycin C-S9Details on test system and experimental conditions
METHOD OF APPLICATION: Exposure duration: [short-term treatment]:6 hrs + 18 hr
SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa stain (2 v/v%) for 15 min.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 + 100 cells /concentration
DETERMINATION OF CYTOTOXICITY- Method: relative total growth
Evaluation criteria
For the evaluation of the frequencies of structural aberrations and of polyploidy induced, the following
criteria were employed. Appearance incidence of cell with chromosomal aberrations: Negative (-):
less than 5%, Equivocal(±): 5% or more and less than 10%, Positive(+): 10% or more
StatisticsnoResults and discussion
Test results
Key result
trueSpecies / strain
Chinese hamster lung (CHL/IU)
mammalian cell line
Metabolic activation
with and without
Genotoxicity
positive
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
23

Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validityvalid

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5f.pdf

Applicant's summary and conclusion
Conclusions
Positive with or without metabolic activation
Executive summary
In an in vitro chromosomal aberration test using CHL/IU cells (OECD TG 473), 1,4Bis(isopropylamino)anthraquinone induced chromosome numerical aberrations but did not induce
structural chromosomal aberrations under the conditions of this study.

24

Toxicity to reproduction
Toxicity to reproduction
ENDPOINT_STUDY_RECORD: Toxicity to reproduction. 001

UUID: 32dedd3b-9b7d-4733-83b9-32b7011bf119
Dossier UUID:
Author:Date: 2022-11-29T14:48:32.584+09:00
Remarks:

Administrative data
Endpoint
reproductive toxicity, other A combined repeated dose/reproductive developmental toxicity study
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study OECD Test Guideline study under GLP condition
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study 7.5.1 Repeated dose toxicity: oral: Repeated dose toxicity: oral. 001
Related information
OECD / Repeated dose toxicity: oral / Repeated dose toxicity: oral. 001 / 1,4-Bis(isopropylamino)-9,10anthracenedione / 14233-37-5

Data source
Reference
Combined repeat dose and reproductive/developmental toxicity screening test
Bis(isopropylamin / Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5d.pdf
25

of

1,4-

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test)
Deviationsno
GLP compliance
yes
Limit test
no

Test material
Test material information
1,4-Bis(isopropylamino)anthraquinone
Specific details on test material used for the study- Name of test material (as cited in study report): 1,4-Bis(isopropylamino)anthraquinone- Analytical purity: 100%

Test animals
Speciesrat
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Atsugi Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation: Male: 416 g (385-475 g), Female: 253 g (230-279 g)- Housing: Animals were individually housed in bracket-type metallic wire-mesh cages (254W × 350D
× 170H mm), from gestation day 17 to lactation day 4, Dams were bred individually or with individual
littermates in plastic cages (340W x 400D x 185H mm) and bedding.
- Diet: Solid feed (NMF: Oriental Yeast Co., ltd.) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: 19 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 23±3 (actual temperature: 22-26°C)- Humidity (%): 50±20% (actual humidity: 33-50%)- Air changes (per hr): 10-15- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 7:00~19:00)

26

Administration / exposure
Route of administration
oral: gavage
Vehicleother: 0.5w/v% methylcellulose
Details on exposure- Amount of vehicle (if gavage): 10 mL/kg- Dosing volume: 10 mL/kg
Details on mating procedure- M/F ratio per cage:1/1- Length of cohabitation: up to 5 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations
yes
Details on analytical verification of doses or concentrations
Test suspensions at each concentration to be used for males group in week 1 and weeks 6 ad
ministration were analyzed by absorptiometry. Results showed that the concentration of test suspensi
ons in each concentration was 95.8 to 103.3% of the nominal concentration and both values were
within the acceptable range (concentration: percentage of nominal concentration, 100±10%)Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating(P) Females: 41-46 days including 14 days pre-mating, mating and gestation periods and the days
until day 4 of lactation
Female (non-mating, satellite group): 42 days
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
12

mg/kg bw/day (actual dose received)

Dose / conc.
60

mg/kg bw/day (actual dose received)

Dose / conc.
300

mg/kg bw/day (actual dose received)

No. of animals per sex per dose
Mating group: 12 animals/sex /dose (0, 12, 60, and 300 mg/kg bw/day). 5 males each in the 0 and
300 mg/kg bw/day were assigned to the recovery group.
27

Non-mating group (Satellite group): 10 females/dose (0 and 300 mg/kg bw/day), 5 females in eachgroup were assigned to the recovery group.
Control animals
yes, concurrent vehicle
Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the highest dose of 300
mg/kg bw/day was selected as an expected obvious toxic dose, and the lowest dose of 12 mg/kg
bw/day was selected as an expected no toxic dose. The middle dose levels of 60 mg/kg bw/day wereselected.
[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0, 100, 300 or 1000 mg/kg bw/day).
At 100 mg/kg bw/day and above, bluish discoloration of skin, dark blue feces, purplish urine, bluishdiscoloration of whole body fat or organs, decreased food consumption and increased liver weight
were observed. At 1,000 mg/kg bw/day slight suppression of body weight gain was also observed inmales and females, low urea nitrogen in males, high total cholesterol in females and high adrenal wei
ght in females.
- Rationale for animal assignment (if not random): Body weight-balanced randomization

Examinations
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes- Time schedule: 3 times/day (before administration, immediately after administration and 1-3 hours after administration) during the administration period. Once a day during the recovery period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule:
Males in the mating group and females in the non-mating (satellite) group: once before the start of ad
ministration, once every weekly during the administration and recovery periods.
Females in the mating group: once a week during the pre-mating period, on designated days duringmating, gestation, and lactation (mated females: days 1, 7, 14 and 20 of pregnancy, parturient
females: day 4 of lactation).
BODY WEIGHT: Yes- Time schedule for examinations:
Males in the mating group and females in the non-mating (satellite) group: days 1, 4, 8, 11, 15, 18, 22,
25, 29, 32, 36, 39 and 42 of administration and on the day of necropsy, and days 1, 4, 8, 11 and 14 ofrecovery and on the day of necropsy.
Females in the mating group: days 1, 4, 8, 11, and 15 of administration (uncopulated animals were weighed on days 18 of administration as well), days 0, 7, 14 and 20 of gestation, days 0 and 4 of lactationand the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Males in the mating group and females in the non-mating (satellite) group: days 1, 4, 8, 11, 15, 32, 36,
39 and 42 of administration, and days 1, 4, 8, 11 and 14 of recovery
Females in the mating groups: days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20
of gestation and days 2 and 4 of lactation.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Anaesthetic used for blood collection: Isoflurane28

- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/day)
- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte percentage,platelet count, white blood cell count, differential white blood cell count, absolute number of each
white blood cell, prothrombin time, activated partial thromboplastin time, fibrinogen.
CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/day)
- Parameters checked: ALP, total cholesterol, triglyceride, phospholipids, total bilirubin, glucose, bloodurea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein,
albumin, A/G ratio, AST (GOT), ALT (GPT), LDH, γ-GTP
BLOOD HORMONE: Yes- Time schedule for collection of serum: Same as clinical chemistry- Animals fasted: Yes- How many animals:
All animals/sex/group (Control and 300 mg/kg bw/day), 5 animals/sex/group (12 and 60 mg/kg bw/
day)- Parameters checked: Triiodothyronine (T3), Thyroxin (T4), and thyroid stimulating hormone (THS) of
thyroid hormone
URINALYSIS OF MALES: Yes- Time schedule for collection of urine: final week of administration (days 36 to 37 of administration)and in the final week of recovery (days 8 to 9 of recovery)- Metabolism cages used for collection of urine: Yes
A urine collector to collect four-hour urine samples under fasting but ad libitum drinking conditions, fo
llowed by collection of 20-hour urine samples under ad libitum feeding and drinking conditions.
- How many animals: 5 animals/group- Parameters checked: pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, color,
sediment, urine volume (4-hour volume), osmotic pressure, urine volume (20-hour volume), water intake (24-hour volume).
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations:
Males in the mating groups: final week of administration (day 40 of administration)
Females in the mating groups: lactation day 4 (day 41 to day 44 of administration) after necropsy of
F1 pupsMales and females in the recovery groups: final week of administration (day 40 of administration) andin the final week of recovery (day 12 of recovery).
- Dose groups that were examined: All dose groups (5 animals/sex/group)- Battery of functions tested:
1) Manipulative Test. Auditory response, approach response, touch response, tail pinch response, pupillary reflex, aerial righting reflex, landing foot splay
2) Measurement of Grip Strength. Following manipulative test, grip strength of forelimb and hind limb
were measured by CPU gauge MODEL-RX-5 (AIKOH Engineering Co., Ltd.).
3) Measurement of Motor Activity. Following measurement of grip strength, motor activity wasmeasured by a motor activity sensor for experimental animals NS-AS01 (Neuro Science, Inc.). The me
asurement was conducted for 1 hour, and measured values at 10-minute intervals and from 0 to 60
minutes were collected.

29

Oestrous cyclicity (parental animals)
Vaginal smears were collected from all females in the mating groups and microscopically examined
every day from the day after the start of administration until the day copulation was confirmed.
During the pre-mating administration period, vaginal smear pictures were classified as proestrus,
estrus, metestrus or diestrus and examined for the frequency of estrus and interval between estruses(estrous cycle). During the mating period, vaginal smears were examined for the presence of sperm.
Sperm parameters (parental animals)
Parameters examined in all P male parental generations: testis weight, epididymis weight, histopatho
logical examinations for testes, epididymides, seminal vesicle and ventral prostate.
Litter observations
PARAMETERS EXAMINED: The following parameters were examined in F1 offspring: Number and sexof pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, and weight gain.
GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities.
Postmortem examinations (parental animals)
METHOD OF SACRIFICED: All animals were sacrificed by exsanguination under isoflurane anesthesia.
SACRIFICE: Males in the mating groups and female in the non-mating (satellite) groups: On next day
after the last administration (Day 43), Maternal animals: on Day 4 of lactation, and Males and females
in the recovery groups: on Day 14 of recovery.
GROSS PATHOLOGY: Yes
ORGAN WEIGHT: Yes [brain, pituitary, thyroids (including parathyroids), adrenal gland, thymus, spleen,
heart, liver, kidney, testis, epididymis, prostate, seminal vesicles, ovary, uterus]
HISTOPATHOLOGY: Yes, [cerebrum, cerebellum (including pons), sciatic nerve, spinal cord (thoracic),eyeball, optic nerve, Harderian gland, pituitary, thyroid, parathyroid, adrenal glands, thymus, spleen,submandibular lymph nodes, mesenteric lymph nodes, heart, thoracic aorta, trachea, lung (including
bronchial), tongue, larynx, esophagus, stomach, duodenum, jejunum, ileum (including payer’s patch),cecum, colon, rectum, submandibular gland, sublingual gland, liver, pancreas, kidney, bladder, testis,
epididymis, ovary, uterus, vagina, prostate, seminal vesicles (including coagulating gland), mammary
gland (inguinal region), skin (inguinal region), sternum and femur (including bone marrows), femoral
skeletal muscle, gross abnormalities site and Individual identification site (pinna with ear tag)]
Underlined organs and tissues are fixed and stored only.
Postmortem examinations (offspring)
SACRIFICE- The F1 offsprings were euthanized on PND4 by exsanguination under isoflurane anesthesia.
GROSS NECROPSY：Yes- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, andabdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS- Not examined.
Statistics
For quantitative data, homogeneity of variance was tested using Bartlett method first. If the variance
was homogenous, statistical difference between each treatment group and the control group was
analyzed using Dunnett method. If not homogenous, statistical difference between each treatmentgroup and the control group was tested using Steel method. For comparison of quantitative data be
tween two groups in the recovery test, homogeneity of variance was analyzed by F-test. Then, if homo
genous, student's t-test was applied. If not homogenous, Aspin-Welch's t-test was used.
Regarding clinical observation (except for frequency of urination, defecation, rearing and grooming)and sensory reactivity, Steel test was applied. Regarding implantation index, stillborn index, live birthindex, viability index and external abnormalities, Steel test was applied. Regarding copulation index,
30

insemination index, fertility index, and delivery index, auditory response, approach response, touch
response, tail pinch response, pupillary reflex, aerial righting reflex, Fisher's test was applied.
Reproductive indices
Each parameter was determined by the following equations:
Copulation index (%) = (No. of copulated animals / No. of mated animals) × 100
Fertility index (%) = (No. of pregnant females / No. of copulated females) × 100
Insemination index (%) = (No. of males which impregnated females / No. of copulated males) × 100
Gestation length (days) = No. of days from pregnancy day 0 to parturition day
Delivery index (%) = (No. of females which delivered liveborns / No. of pregnant females) × 100
Implantation index (%) = (No. of implantation sites / No. of corpora lutea) × 100
Stillborn index (%) = (No. of stillborn / No of liveborns and stillborns) × 100
Live birth index (%) = (No. of liveborn / No. of implantation sites) × 100
External abnormalities (%) = (No. of pups with external abnormalities / No. of liveborns) × 100
Sex ratio = No. of liveborns males / No. of liveborns
Sex ratio of live pups on day 4= No. of live males on day 4/ No. of live pups on day 4
Offspring viability indices
Viability index on postnatal day 4 (%) = (No. of live pups on day 4 / No. of liveborns on day 0) × 100

Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
Clinical signs
effects observed, non-treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Mortality
no mortality observed
Body weight and weight changes
no effects observed
Food consumption and compound intake (if feeding study)
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Food efficiency
not examined
Water consumption and compound intake (if drinking water study)
not examined
Ophthalmological findings
not examined
Haematological findings
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
31

Clinical biochemistry findings
effects observed, treatment-related
Description (incidence and severity)
See 7.5 Repeated dose toxicity.001
Urinalysis findings
effects observed, non-treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Behaviour (functional findings)
no effects observed
Immunological findings
not examined
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Gross pathological findings
effects observed, non-treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Neuropathological findings
not examined
Histopathological findings: non-neoplastic
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity.001
Histopathological findings: neoplastic
not examined

Reproductive function / performance (P0)
Reproductive function: oestrous cycle
no effects observed
Reproductive function: sperm measures
no effects observed
Reproductive performance
effects observed, treatment-related

Details on results (P0)General toxicity: See 7.5.1 Repeated dose toxicity.001
Reproductive function / performance: Decreased nursing behavior was observed in dams at the 12
mg/kg bw/day and above. Death of all nursing infants in almost all dams were observed at 300 mg/
kg bw/day.
32

Effect levels (P0)
Key result
false
Dose descriptor
NOAEL
Effect level>=300

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
male
Basis for effect level
other: No effects on reproduction
Key result
true
Dose descriptor
NOAEL
Effect level<12

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
femaleBasis for effect level
food consumption and compound intake
Decreased food consumption was observed in dams during lactation at the 12 mg/kg bw/day.
reproductive performance
Decreased nursing behavior was observed in dams at the 12 mg/kg bw/day.

Results: F1 generation
General toxicity (F1)Clinical signs
no effects observed
Mortality / viability
mortality observed, treatment-related
Description (incidence and severity)
Decrease in viability index on PND 4 was observed at 60 mg/kg bw/day and above.
viability index on PND 4:0, 12, 60, 300 mg/kg bw/day: 85.8% (+/-30.6), 87.4% (+/-27.8), 29.8% (+/-25.4), 0.6% (+/-2.0)33

All pups died during the lactation period in 1/10, 1/12, 2/12, 11/12 litters at the 0, 12, 60 and 300
mg/kg bw/day.
Body weight and weight changes
effects observed, treatment-related
Description (incidence and severity)
The body weight of pups on PND 0 was lower at 60 mg/kg bw/day and above, and the body weight ofpups on PND 4 was lower at 12 mg/kg bw/day and above.
Gross pathological findings
effects observed, non-treatment-related
Description (incidence and severity)
Bluish discoloration of organs/tissues or gastrointestinal contents was observed. These were conside
red to reflect the color of the test substance.

Details on results (F1)
In the offspring, death of all nursing infants in almost all dams were observed at 300 mg/kg bw/day,decreased viability index on PND 4 was observed at 60 mg/kg bw/day and above, decreased bodyweights on PND 4 were observed at 12 mg/kg/day and above.

Effect levels (F1)
Key result
true
Dose descriptor
NOAEL
Generation
F1
Effect level<12

mg/kg bw/day (actual dose received)

Based ontest mat.
Sexmale/female
Basis for effect level
body weight and weight gain
In the offspring, decreased body weights on PND 4 were observed at 12 mg/kg/day and above.

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5d.pdf

Applicant's summary and conclusion
34

Conclusions
In the combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) described above, decreases in nursing behavior were observed in dams at 12
mg/kg bw/day and above, and deaths of all nursing infants were observed in almost all dams at 300
mg/kg bw/day. Also, food consumption at doses of 12 mg/kg bw/day and above during lactation w
ere significantly lower. In offspring, a decrease in body weight was observed at 60 mg/kg bw/day on
postnatal day (PND) 0 and at 12 mg/kg bw/day or more on PND 4, and a decrease in viability index on
PND 4 was observed at 60 mg/kg bw/day or more.
The NOAEL for the reproductive/developmental toxicity was determined to be less than 12 mg/kg bw/
day based on decreased nursing behavior in dams during lactation, decreased body weight in offsp
ring at the 12 mg/kg bw/day dose, which was the lowest dose tested.

35

References
Reference Substances
REFERENCE_SUBSTANCE: 1,4Bis(isopropylamino)anthraquinone
UUID: 6a24b2e9-eb9e-4f35-9ee6-32197dfe3c12
Dossier UUID:
Author:Date: 2020-12-29T14:25:33.000+09:00
Remarks:
Reference substance name
1,4-Bis(isopropylamino)anthraquinone

Inventory
CAS number
14233-37-5

Molecular and structural information
Molecular formula
C20H22N2O2
Molecular weight
322.4

36

Test Materials
TEST_MATERIAL_INFORMATION: 1,4Bis(isopropylamino)anthraquinone
UUID: a128e41f-ac9d-4fe1-b4f8-ca2042de1b42
Dossier UUID:
Author:Date: 2020-12-29T14:26:29.000+09:00
Remarks:
Name
1,4-Bis(isopropylamino)anthraquinone

Composition
Composition
Reference substance
1,4-Bis(isopropylamino)anthraquinone / 14233-37-5
EC number

EC name

CAS number

CAS name

14233-37-5
IUPAC nameConcentration
100

37

Literatures
LITERATURE: Combined repeat dose and reproductive/
developmental toxicity screening test of 1,4Bis(isopropylamino)anthraquinone by oral administrationin rats
UUID: 37839122-27b9-4487-944a-14d4e960cee6
Dossier UUID:
Author:Date: 2020-12-29T14:17:59.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Combined repeat dose and reproductive/developmental toxicity screening test of 1,4-Bis(isopropylamino)anthraquinone by oral administration in rats
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2012
Bibliographic source
available in the web of Japan Existing Chemical Data Base (JECDB) at https://dra4.nihs.go.jp/
mhlw_data/home/pdf/PDF14233-37-5d.pdf
Testing facility
BoZo Research Center
Report number
R-106838

LITERATURE: In Vitro Chromosomal Aberration Test of on
1,4-Bis(isopropylamino)anthraquinone Cultured Chinese
Hamster Cells.
UUID: 8c138619-d7fd-4829-99bc-88d75ed25201
Dossier UUID:
Author:Date: 2021-03-08T15:44:08.000+09:00
Remarks:

General information
Reference Typestudy report
Title
In Vitro Chromosomal Aberration Test of on 1,4-Bis(isopropylamino)anthraquinone Cultured Chinese
Hamster Cells.
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5f.pdf
Testing facility
Bozo Research Center Inc.
Report number
M-143339

LITERATURE: Reverse Mutation Test of 1,4Bis(isopropylamino)anthraquinone on Bacteria.
UUID: 7f2bc441-c2d5-407d-87a9-c04460e7efb8
Dossier UUID:
Author:Date: 2021-03-08T10:17:41.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Reverse Mutation Test of 1,4-Bis(isopropylamino)anthraquinone on Bacteria.
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF14233-37-5e.pdf
Testing facility
Bozo Research Center Inc.
Report number
T-057740

Legal Entities
LEGAL_ENTITY: National Institute of Health Sciences
UUID: IUC4-b036ff75-0f3c-323b-b200-ed5f46cf5101
Dossier UUID:
Author:Date: 2022-11-07T15:49:29.000+09:00
Remarks:

General information
Legal entity nameNational Institute of Health Sciences
Remarks
Disclaimer: The contents in this document were created based on the MHLW (Ministry of Health,Labour and Welfare) peer reviewed study reports (in Japanese) in JECDB (Japan Existing Chemical
Database) at http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp. Authorship is in the Divisionof Risk Assessment, the National Institute of Health Sciences, and the contents do not reflect any official MHLW opinions or any other regulatory policies.

Address
Address 1
Tonomachi 3-25-26
Address 2
Kawasaki-ku
Postal code
210-9501
Town
KawasakiRegion / State
Kanagawa
CountryJapan
JP

Identifiers
Other IT system identifiers
IT system
LEO
ID
10767
IT systemIUCLID441

ID
16558402024DIV750

42