Name: OECD_SIDS / SUBSTANCE : Diammonium hydrogen 2-hydroxypropane-1,2,3-

tricarboxylate / diammonium hydrogen citrate / 3012-65-5 Fri, 16 Dec 2022, 14:40:50+0900 /Legal entity owner: National Institute of Health Sciences
Printing date: 2022-12-16T14:40:50.467+09:00

iTable of Contents
0/0 ............................................................................................................................. 1
National Institute of Health Science ..................................................................... 2
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate ............................... 3
1 General information .................................................................................. 3
1.1 Identification ................................................................................. 3
Identification ............................................................................... 3
Identification ...................................................................... 3
1.10 Assessment approach (assessment entities) ................................. 4
Assessment approach (assessment entities) ................................ 4
7 Toxicological information ......................................................................... 5
7.2 Acute Toxicity ............................................................................... 5
7.2.1 Acute toxicity: oral .............................................................. 5
Acute toxicity: oral.001 ........................................................ 5
7.5 Repeated dose toxicity ................................................................... 9
7.5.1 Repeated dose toxicity: oral ................................................. 9
Repeated dose toxicity: oral.001 ........................................... 9
7.6 Genetic toxicity ............................................................................ 16
7.6.1 Genetic toxicity in vitro ...................................................... 16
Genetic toxicity in vitro.001 ................................................ 16
Genetic toxicity in vitro.002 ................................................ 21
7.8 Toxicity to reproduction ............................................................... 25
7.8.1 Toxicity to reproduction ..................................................... 25
Reproductive/developmental toxicity.001 ............................ 25
References ................................................................................................................ 33
Reference Substances ...................................................................................... 33
diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate ...................... 33
Test Materials .................................................................................................. 35
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate ...................... 35
Literatures ....................................................................................................... 36
Combined repeat dose and reproductive/developmental toxicity screening
test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate byoral administration in rats .......................................................................... 36
In Vitro Chromosomal Aberration Test of diammonium hydrogen 2hydroxypropane-1,2,3-tricarboxylate on Cultured Chinese Hamster Cells. ....... 37
Reverse Mutation Test of diammonium hydrogen 2-hydroxypropane-1,2,3tricarboxylate on Bacteria. ......................................................................... 38
Single Dose Oral Toxicity Test of diammonium hydrogen 2hydroxypropane-1,2,3-tricarboxylate in Rats ................................................. 39
Legal Entities ................................................................................................... 40
National Institute of Health Sciences .......................................................... 40ii

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

Dossier subject
Dossier subject
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate / diammonium hydrogen citrate /3012-65-5
Public nameSubmitting legal entity
National Institute of Health Science
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Fri, 16 Dec 2022, 14:40:50+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

Diammonium hydrogen 2hydroxypropane-1,2,3-tricarboxylate
General information
Identification
Identification
SUBSTANCE: Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate

UUID: IUC5-8627c376-bb47-4255-90c4-6639d07c0bb2
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Author:Date: 2022-12-16T14:40:36.984+09:00
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Substance name
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Legal entity
National Institute of Health Sciences / Kawasaki / Japan

Identification of substance
Reference substancediammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate / diammonium hydrogen citrate /3012-65-5 / 221-146-3
EC number

EC name221-146-3

EC Inventory

CAS number

CAS name

3012-65-5
IUPAC namediammonium hydrogen citrate

Role in the supply chain
Manufacturer
false
Importer
false
Only representativefalse
Downstream user
false

3

Assessment approach (assessment entities)
FIXED_RECORD: Assessment approach
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Author:Date: 2016-12-21T15:15:08.000+09:00
Remarks:4

Toxicological information
Acute Toxicity
Acute toxicity: oral
ENDPOINT_STUDY_RECORD: Acute toxicity: oral.001

UUID: IUC5-0469431d-735d-47de-8e42-89cac5c65922
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Author:Date: 2022-12-16T14:35:59.541+09:00
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Administrative data
Endpoint
acute 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
other: OECD Test Guideline study under GLP condition

Data source
Reference
Single Dose Oral Toxicity Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate in Rats /MHLW / publication
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
5Guideline
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Test material
Test material information
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-trica
rboxylate- Analytical purity: 100.0%- Lot/batch No.: 6803
- Stability under test conditions: The stability of test material was identified by analysis of theremainder.
- Storage condition of test material: At a cold place (temperature 2~7℃) in a refrigerator, with astopper.

Test animals
Speciesratcommon species
Strainother: Crl:CD(SD)SexfemaleDetails on test animals or test system and environmental conditions
TEST ANIMALS- Source :Charles River Japan Inc.
- Age at study initiation: 8-9 weeks old- Weight at study initiation: Females, 207-224 g- Fasting period before study: Approximately 16-18 hrs- Housing:1/cage- Diet (e.g. ad libitum): Ad libitum except fasting period for 16-18 hrs before administration to 4 hrs
after administration- Water (e.g. ad libitum):Ad libitum- Acclimation period:6-16 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3 ℃(actual temperature: 19-24℃)- Humidity (%):50 ± 20% (actual humidity: 33-57%)- Air changes (per hr): Approximately 10-15 times/hr- Photoperiod (hrs dark / hrs light):12 hrs light / 12 hrs dark

Administration / exposure
Route of administration
oral: gavage
Vehicleother: 0.5%CMC-Na
Details on oral exposure- Amount of vehicle (if gavage):10 mL/kg bw
6

Doses300, 2000 mg/kg bw
No. of animals per sex per dose
3 (1st step group), 3 (2nd step group), 3 (3rd step group) and 3 (4th step group)
Control animals
noDetails on study design- Duration of observation period following administration: 14 days
- Frequency of observations: nearly successive observation (from time just to 1 hr after administration) and observation (at 2, 4 and 6 hr (only 3 & 4 steps) after administration) (day 0); twice a day (fromday 1-day13) and once a day (day14)- Frequency of weighing: just before administration (day 0), and 1,3,5,7,10 and 14 day after adminis
tration- Necropsy of survivors performed: yes

Results and discussion
Effect levels
Key result
false
Sex
female
Dose descriptor
LD50
Effect level>2000

mg/kg bw

Mortality
No deaths were observed in any group.
Clinical signs
other: At 2000 mg/kg bw, mucus feces were observed.
No changes related to the test substance were observed in any group.
Gross pathology
No changes related to the test substance were observed in any group.

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF3012 -65 -5a.pdf

Applicant's summary and conclusion
7

Executive summary
The acute oral LD50 of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate was > 2,000 mg/
kg bw in female rats based on a study conducted according to OECD TG 423. No deaths were observed
at 2,000 mg/kg bw. This substance caused mucoid stools at 2,000 mg/kg bw.

8

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

UUID: IUC5-55581c53-51f6-4e2f-a181-994678536d94
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Author:Date: 2022-12-16T14:37:06.491+09:00
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Administrative data
Endpoint
short-term repeated dose toxicity: oral combined repeated dose and reproduction / developmental
screening
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
other: GLP guideline study
Cross-reference
Reason / purpose for cross-reference
reference to same study
Remarks
7.8.1 Reproductive/developmental toxicity.001

Data source
Reference
Combined repeat dose and reproductive/developmental toxicity screening test of diammoniumhydrogen 2 / MHLW, Japan / study report
Data accessdata published9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
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-trica
rboxylate- Purity: 100.0%- Impurities (identity and concentrations):- Lot/batch No.: 6803
- Stability under test conditions: Stable- Storage condition of test material: Refrigeration- Dosing solution storage condition: Room temperature- Other: The dosing solution was used within 10 days of preparation.

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 Laboratories Japan, Inc. Atsugi- Age at study initiation: 10 weeks- Weight at study initiation: Males: 371-439 g; Females: 219-273 g- Housing: Steel wire-mesh cage (250 mm x 350 mm x 200 mm )- Diet: ad libitum- Water: ad libitum- Acclimation period: 15 days
10

ENVIRONMENTAL CONDITIONS- Temperature (°C): 21-26- Humidity (%): 42-64- Air changes: 10-15 times / hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure
Route of administration
oral: gavage
Vehiclewater
Details on oral exposure
PREPARATION OF DOSING SOLUTIONS:VEHICLE- Amount of vehicle (if gavage): 5 mL/kg bw- Lot/batch no. (if required): 6F74
Details on analytical verification of doses or concentrations
Test suspensions at each concentration to be used for males in week 1 and week 6 of administration
were analyzed by the HPLC method at Bozo Research Center Inc. Results showed that the conce
ntration of the test article in each suspension was 93.3 to 100.0% of the nominal concentration
and both values were within the acceptable range (concentration: percentage of the nominal con
centration, 100 ± 10%; C.V.: 10% or below)
Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating and mating periods, and thereafter 14 days (P) Femal
es: 41-47 days including 14 days pre-mating, mating and gestation periods, and the days until day 4 oflactation; satellite animals: 42 days.
Frequency of treatment
Once/day, 7days/week
Doses / concentrations
Remarks
Doses / Concentrations:
0 (vehicle), 100, 300, and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose
12 animals/sex/dose as a main dose group,
5 males and 5 females at 0 and 1000 mg/kg bw/day as a satellite group (without mating)Control animals
yes, concurrent vehicle
Details on study design- Dose selection rationale: Doses in this test were set based on the results of the following study: 14day repeated dose oral toxicity test (doses: 100, 300, and 1000 mg/kg bw/day). In the 14-day repeateddose oral toxicity test, abnormalities were observed in animals in the 1000 mg/kg bw/day group,
such as an increase in relative kidney weight. No effects were observed at 300 mg/kg bw/day. On th
11

e basis of these effects, a dose level of 1000 mg/kg was selected as the maximum dose expecting
to induce the toxic changes, and then dose levels of 300 and 100 mg/kg bw/day were selected as a
middle dose and a minimum dose levels, respectively, in accordance with a common ratio of approxi
mately 3.
- Rationale for animal assignment (if not random): Body weight-balanced randomization- Post-exposure recovery period in satellite groups: 14 days

Examinations
Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: Yes- Time schedule:
Males and females: once before the start of administration, 3 times/day during the administration
period, and once during the recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
The functional observational battery testing (FOB) was performed on all animals. Among the mea
sures in the FOB, detailed clinical observations were made before the initiation of dosing. Thereafter,in males of the main groups, detailed clinical observations were made once a week. Also in femalesof the main groups, detailed clinical observations were made once a week in pre-mating and mating
periods thereafter, and then those were made on days 1,7,14 and 20 of gestation, and on day 4 of lactation. For the satellite group, detailed clinical observations were made once a week in dosing andrecovery periods.
Sensory motor reflexes, forelimb and hindlimb grip strengths, and motor activity were measured
on week 6 of administration period (main/recovery group animals) and week 2 of recovery period (recovery group animals).
BODY WEIGHT: Yes- Time schedule for examinations: Males (main) & males and females (recovery group): Days 1, 4, 8,
11, 15, 22, 25, 29, 32, 36, 39, 42, and the day of necropsy (after ca. 16h-fasting) in dosing period
Males and females (recovery group): Days 1, 4, 8, 11, 14, and the day of necropsy (after ca. 16hfasting) in recovery period
Females (main group): Twice a week during the precopulation period (days 1, 4, 8, 11, and 15); gestati
on days 0, 4, 7, 11, 14, 17, and 20; lactation days 0 and 4; and the day of necropsy (after ca. 16 hfasting)
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Males (main) & males and females (recovery group): Days 1, 4, 8, 11, 15, 32, 36, and 39 in dosing
periodMales and females (recovery group): Days 1, 4, 8, 11, and 14 in recovery period
Females (main group): Days 1, 4, 8, 11, and 15; gestation days 1, 4, 7, 11, 14, 17, and 20; lactation days
2 and 4
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood: Blood was collected on the day of necropsy- Anaesthetic used for blood collection: Yes (ether)- Animals fasted: Yes, 16-20h- How many animals: 5 sex/dose/group- Parameters checked in table were examined.
CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Same as hematology- Animals fasted: Same as hematology- How many animals: Same as hematology- Parameters checked in table were examined.
12

URINALYSIS: Yes (males only)- Time schedule for collection of urine: Day 37-38 in dosing period, day 11-12 in recovery period- Metabolism cages used for collection of urine: No data
- Animals fasted: fasting and only water at libitum (4h-urine), no fasting (20h-urine)
Sacrifice and pathology
SACRIFICE:
Male animals: Rats were euthanized by exsanguination under ether anesthesia on the day after the last administration.
Maternal animals: Rats were euthanized by exsanguination under ether anesthesia on day 4 of lactati
on.
GROSS PATHOLOGY, Yes: whole organs and tissues
ORGAN WEIGHTS, Yes: Brain, thyroids(including parathyroids), thymus, heart, liver, spleen, kidneys,adrenals, testes, epididymis
HISTOPATHOLOGY, Yes: Cerebrum, cerebellum, pituitary gland, spinal cord (thoracic), sciatic nerve,
thyroid, parathyroid, adrenal glands, thymus, spleen, submandibular lymph nodes, mesenteric lymph
nodes, heart, lung (including the bronchi), stomach, duodenum, jejunum, ileum, cecum, colon, rectum,
liver, kidney, bladder, testis, epididymis, ovary, uterus, seminal vesicles, sternum and femur (includingbone marrows), macroscopic lesions.
Other examinations
Organ weight: Brian, thyroids (including parathyroids), thymus, heart, liver, spleen, kidneys, adrenals,
testes, epididymis
Statistics
The data were analyzed for homogeneity of variance by the Bartlett test. If variances were
homogeneous, data was analyzed by the Dunnett test, whereas heterogeneous data was analyzed by
the Dunnett type mean rank test (p<0.05, two-sided).
In the recovery test, these values of two groups were analyzed by F test. If variances were homogene
ous, data was analyzed by the Student t-test, whereas heterogeneous data was analyzed by the AspinWelch t-test (p<0.05, two-sided).

Results and discussion
Results of examinations
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
Food efficiency
not examined13

Water consumption and compound intake (if drinking water study)
not examined
Ophthalmological findings
not examined
Haematological findings
no effects observed
Clinical biochemistry findings
effects observed, treatment-related
Urinalysis findings
effects observed, treatment-related
Behaviour (functional findings)
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, treatment-related
Histopathological findings: neoplastic
not examined
Details on results
CLINICAL CHEMISTRYHigh value of glucose was observed at 1000 mg/kg bw/day at the end of dosing.
URINALYSIS (male only)
At week 6 of administration in the main group, an increased trend of acidification and a decreased
incidence of crystallization of phosphate were observed dose-dependently.
HISTOPATHOLOGY: NON-NEOPLASTIC[At the end of dosing] and [At the end of recovery period]
Stomach: Minimal or mild hyperplasia of squamous cells in the limiting ridge were observed in males
and females at 1000 mg/kg bw/day.
Any other changes with statistically significant in the tables were considered to be incidental due totemporary, dose-independent, or within the normal ranges of physiological variability.

Effect levels
Key result
false
Dose descriptor
LOAEL
Effect level300

mg/kg bw/day (actual dose received)
14Based on
test mat.
Sexmale/female
Basis for effect level
other: effects on the stomach

Target system / organ toxicity
Key result
false
Critical effects observed
not specified

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF3012 -65 -5d.pdf

Applicant's summary and conclusion
Conclusions
Based on the effects on the stomach in males and females at 1000 mg/kg bw/day, the NOAEL for
repeated oral dosing was determined to be 300 mg/kg bw/day in male and female rats.
Executive summary
A combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening
test was performed according to OECD TG 422. Male and female rats (12 animals/sex/dose) wereadministered diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate at 0, 100, 300, and 1,000
mg/kg bw/day. Males were dosed for 42 days, including a 14-day pre-mating and mating periods.
Females were dosed for 41–47 days, including a 14-day pre-mating, mating, and gestation periods andthe time until day 4 of lactation. Five animals/sex/dose administered 0 and 1,000 mg/kg bw/day weretreated as the recovery group and examined after a 14-day recovery period. After the administration
period, squamous cell hyperplasia of the boundary edge in the stomach was observed at 1,000 mg/
kg bw/day in both sexes. This change resolved after the recovery period. On the basis of the observed
stomach changes, NOAEL for repeated-dose toxicity was determined to be 300 mg/kg bw/day in maleand female rats.

15

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

UUID: IUC5-253e1121-b70b-4f87-9374-6d5b549c946a
Dossier UUID:
Author:Date: 2022-12-16T14:38:16.038+09:00
Remarks:

Administrative data
Endpoint
in vitro gene mutation study in bacteria Type of genotoxicity: gene mutation
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
other: OECD Test Guideline study under GLP condition

Data source
Reference
Reverse Mutation Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate on Bacteria. /MHLW, Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
16Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifieraccording to guideline
Guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
in vitro gene mutation study in bacteria
GLP compliance
yesType of assay
bacterial reverse mutation assay
in vitro gene mutation study in bacteria

Test material
Test material information
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-trica
rboxylate- Analytical purity: 100.0%- Lot/batch No.: 6803
- Storage condition of test material: in a hermetically sealed and light-resistant container at cool (2-8℃) place- Stability under test conditions: The stability of test material was identified by analysis of the remai
nder.

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
rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
-S9 mix and + S9 mix: 313, 625, 1250, 2500, 5000 μg/plate (all strains)
Vehicle / solvent- Vehicle(s)/solvent(s) used: water for injection
17

Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substanceother: -S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF2:TA100, TA98, WP2 uvrA), sodium azide (SA:TA1535) and 9-aminoacridine hydrochloride (9AA:TA1537). +S9 mix: 2-aminoanthracene(2AA:all strains).
Details on test system and experimental conditions
RANGE-FINDING/SCREENING STUDIES:
Concentration: 5-5000 μg/plate
Cytotoxic conc.: No.
Precipitate: No.
METHOD OF APPLICATION: Preincubation
DURATION- Preincubation period: 20 min at 37 ℃
- Exposure duration:48-49 hrs
NUMBER OF PLATES: 3
DETERMINATION OF CYTOTOXICITY- Method: other: growth inhibition
Evaluation criteria
In any strain(s) tested with or without S9 mix, when the mean number of revertant colonies per plateincreased twice more than that of the negative control and when the increase was shown to be dose-r
elated and reproducible, the chemical was judged mutagenic.
Statistics
No.

Results and discussion
Test results
Key result
false
Species / strain
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
bacteria
Metabolic activation
with and without
Genotoxicity
negative18

Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated 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
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
Positive controls validity
valid
Additional information on results
Contamination with any other bacterias was not found.
Remarks on result
other: all strains/cell types tested Migrated from field 'Test system'.

Any other information on results incl. tables
Field content is not in a valid XML format and thus ignored!

Applicant's summary and conclusion
Conclusions
Interpretation of results (migrated information):negative
Executive summary
In a bacterial reverse mutation assay using S. typhimurium TA100, TA1535, TA98, and TA1537 andE. coli WP2uvrA (OECD TG 471), diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate wasnegative with or without metabolic activation.
19

20

ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.002

UUID: IUC5-043e5217-bdd4-4262-941f-19e239d9bdc0
Dossier UUID:
Author:Date: 2022-12-16T14:39:15.804+09:00
Remarks:

Administrative data
Endpoint
in vitro cytogenicity / chromosome aberration study in mammalian cells Type of genotoxicity:
chromosome aberration
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
other: OECD Test Guideline study under GLP condition

Data source
Reference
In Vitro Chromosomal Aberration Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
on / MHLW, Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)in vitro cytogenicity / chromosome aberration study in mammalian cells
21

Deviations
no
Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviationsno
GLP compliance
yesType of assay
in vitro mammalian chromosome aberration test
chromosome aberration

Test material
Test material information
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-trica
rboxylate- Analytical purity: 100.0%- Lot/batch No.: 6803
- Storage condition of test material: in a hermetically sealed and light-resistant container at cool (2-8℃) place- Stability under test conditions: The stability of test material was identified by analysis of the remai
nder.

MethodTarget gene
Chromosome
Species / strain
Species / strain / cell typeother: Chinese hamster lung(CHL/IU) cells
Metabolic activation
with and without
Metabolic activation system
rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
-S9 mix (short-term treatment): 0, 565, 1130, 2260 ug/mL+S9 mix (short-term treatment): 0, 565, 1130, 2260 ug/mL-S9 mix (continuous treatment, 24 h): 0, 283, 656, 1130, 1695, 2260 ug/mL
Vehicle / solvent- Vehicle(s)/solvent(s) used: water for injection
22

Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substancebenzo(a)pyrene
mitomycin C
Remarks
mitomycin C (without S9 mix), benzo[a]pyrene (with S9 mix)
Details on test system and experimental conditions
METHOD OF APPLICATION: Exposure duration: [continuous treatment]: 24 hrs [short-term treatment]:6 hrs + 18 hr
SPINDLE INHIBITOR: Colcemid
NUMBER OF CELLS EVALUATED: 200 cells / dose
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 cells with chromosomal aberrations: Negative (-): < 5%; equivocal (±): 5-10%;
positive (+): > 10%.
Finally, the substance is positive when the incidence is considered to be dose-related and repro
ducible.
Statistics
not used.

Results and discussion
Test results
Key result
false
Species / strainother: Chinese hamster lung (CHL/IU) cells
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
23

Vehicle controls validity
valid
Untreated negative controls validity
not examined
Positive controls validity
valid
Key result
false
Species / strainother: Chinese hamster lung (CHL/IU) cells
Metabolic activation
without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity 50% cell growth inhibition: 2260 ug/mL (24h continuous)
Vehicle controls validity
valid
Untreated 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.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF3012 -65 -5f.pdf

Applicant's summary and conclusion
Executive summary
An in vitro chromosomal aberration test using CHL/IU cells (OECD TG 473) showed negative result withor without metabolic activation.

24

Toxicity to reproduction
Toxicity to reproduction
ENDPOINT_STUDY_RECORD: Reproductive/developmental toxicity.001

UUID: IUC5-c1ff5a74-f962-4d95-a7cd-a59a30a70cb5
Dossier UUID:
Author:Date: 2022-12-16T14:40:21.935+09:00
Remarks:

Administrative data
Endpoint
screening for reproductive / developmental toxicity based on test type (migrated information)
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
other: OECD Test Guideline study under GLP condition
Cross-reference
Reason / purpose for cross-reference
reference to same study
Remarks
7.5.1 Repeated dose toxicity: oral.001

Data source
Reference
Combined repeat dose and reproductive/developmental toxicity screening test of diammoniumhydrogen 2 / MHLW, Japan / study report
Data accessdata published25

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
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-trica
rboxylate- Purity: 100.0%- Impurities (identity and concentrations):- Lot/batch No.: 6803
- Stability under test conditions: Stable- Storage condition of test material: Refrigeration- Dosing solution storage condition: Room temperature- Other: The dosing solution was used within 10 days of preparation.

Test animals
Speciesrat
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Laboratories Japan, Inc. Atsugi- Age at study initiation: 10 weeks- Weight at study initiation: Males: 371-439 g; Females: 219-273 g- Housing: Steel wire-mesh cage (250 mm x 350 mm x 200 mm )- Diet: ad libitum- Water: ad libitum- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
26

- Temperature (°C): 21-26- Humidity (%): 42-64- Air changes: 10-15 times / hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure
Route of administration
oral: gavage
Vehiclewater
Details on exposure
PREPARATION OF DOSING SOLUTIONS:VEHICLE- Amount of vehicle (if gavage): 5 mL/kg bw- Lot/batch no. (if required): 6F74
Details on mating procedure- M/F ratio per cage:1:1- Length of cohabitation:up to 14 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 in week 1 and week 6 of administration
were analyzed by the HPLC method at Bozo Research Center Inc. Results showed that the conce
ntration of the test article in each suspension was 93.3 to 100.0% of the nominal concentration
and both values were within the acceptable range (concentration: percentage of the nominal con
centration, 100 ± 10%; C.V.: 10% or below)
Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating and mating periods, and thereafter 14 days(P) Females: 41-47 days including 14 days pre-mating, mating and gestation periods, and the days until day 4 of lactation; satellite animals: 42 days.
Frequency of treatment
Once/day, 7days/week
Doses / concentrations
Remarks
Doses / Concentrations:
0 (vehicle), 100, 300, and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose
12 animals/sex/dose (main dose group), 5 males and 5 females at 0 and 1000 mg/kg bw/day as asatellite group (without mating).
27

Control animals
yes, concurrent vehicle

Examinations
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes- Time schedule:
Males and females: once before the start of administration, 3 times/day during the administration
period, and once during the recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
The functional observational battery testing (FOB) was performed on all animals. Among the mea
sures in the FOB, detailed clinical observations were made before the initiation of dosing. Thereafter,in males of the main groups, detailed clinical observations were made once a week. Also in femalesof the main groups, detailed clinical observations were made once a week in pre-mating and mating
periods thereafter, and then those were made on days 1,7,14 and 20 of gestation, and on day 4 of lactation. For the satellite group, detailed clinical observations were made once a week in dosing andrecovery periods.
Sensory motor reflexes, forelimb and hindlimb grip strengths, and motor activity were measured
on week 6 of administration period (main/recovery group animals) and week 2 of recovery period (recovery group animals).
BODY WEIGHT: Yes- Time schedule for examinations: Males (main) & males and females (recovery group): Days 1, 4, 8,
11, 15, 22, 25, 29, 32, 36, 39, 42, and the day of necropsy (after ca. 16h-fasting) in dosing period
Males and females (recovery group): Days 1, 4, 8, 11, 14, and the day of necropsy (after ca. 16hfasting) in recovery period
Females (main group): Twice a week during the precopulation period (days 1, 4, 8, 11, and 15); gestati
on days 0, 4, 7, 11, 14, 17, and 20; lactation days 0 and 4; and the day of necropsy (after ca. 16 hfasting)
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Males (main) & males and females (recovery group): Days 1, 4, 8, 11, 15, 32, 36, and 39 in dosing
periodMales and females (recovery group): Days 1, 4, 8, 11, and 14 in recovery period
Females (main group): Days 1, 4, 8, 11, and 15; gestation days 1, 4, 7, 11, 14, 17, and 20; lactation days
2 and 4
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood: Blood was collected on the day of necropsy- Anaesthetic used for blood collection: Yes (ether)- Animals fasted: Yes, 16-20h- How many animals: 5 sex/dose/group- Parameters checked in table were examined.
CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Same as hematology- Animals fasted: Same as hematology- How many animals: Same as hematology- Parameters checked in table were examined.
URINALYSIS: Yes (males only)- Time schedule for collection of urine: Day 37-38 in dosing period, day 11-12 in recovery period- Metabolism cages used for collection of urine: No data
- Animals fasted: fasting and only water at libitum (4h-urine), no fasting (20h-urine)
28

Oestrous cyclicity (parental animals)
Vaginal smears were collected from all females in the main 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 P male parental generations: testes weight, epididymides weight
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, body weight, andbody weight gain.
GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities.
Postmortem examinations (parental animals)
SACRIFICE:
Male animals: Rats were euthanized by exsanguination under ether anesthesia on the day after the last administration.
Maternal animals: Rats were euthanized by exsanguination under ether anesthesia on day 4 of lactati
on.
GROSS PATHOLOGY, Yes: whole organs and tissues
ORGAN WEIGHTS, Yes: Brain, thyroids(including parathyroids), thymus, heart, liver, spleen, kidneys,adrenals, testes, epididymis
HISTOPATHOLOGY, Yes: Cerebrum, cerebellum, pituitary gland, spinal cord (thoracic), sciatic nerve,
thyroid, parathyroid, adrenal glands, thymus, spleen, submandibular lymph nodes, mesenteric lymph
nodes, heart, lung (including the bronchi), stomach, duodenum, jejunum, ileum, cecum, colon, rectum,
liver, kidney, bladder, testis, epididymis, ovary, uterus, seminal vesicles, sternum and femur (includingbone marrows), macroscopic lesions.
Postmortem examinations (offspring)
GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, andabdominal viscera.
Statistics
The data were analyzed for homogeneity of variance by the Bartlett test. If variances were
homogeneous, data was analyzed by the Dunnett test, whereas heterogeneous data was analyzed by
the Dunnett type mean rank test (p<0.05, two-sided).
In the recovery test, these values of two groups were analyzed by F test. If variances were homogene
ous, data was analyzed by the Student t-test, whereas heterogeneous data was analyzed by the AspinWelch t-test (p<0.05, two-sided).
Reproductive indices
Each parameter was determined by the following equations:
Copulation index (%) = (No. of copulated animals/No. of co-housed animals) × 100
Fertility index (%) = (No. of pregnant females/No. of copulated females) × 100
Insemination index (%) = (No. of pregnant females/No. of copulated males) × 100
Duration of gestation (days) = day 0 of lactation – day 0 of gestation
Delivery index (%) = (No. of females delivered liveborn pups/No. of pregnant females) × 100
Implantation index (%) = (No. of implantation sites/No. of corpora lutea) × 100
Stillborn index (%) = (No. of stillborn pups/Total No. of pups born) × 100
Liveborn index (%) = (No. of liveborn pups/Total No. of pups born) × 100
External abnormalities (%) = (No. of pups with external abnormalities/No. of liveborn pups) × 100
29

Sex ratio = No. of liveborn male pups/(No. of liveborn male pups + No. of liveborn female pups)

Offspring viability indices
Viability index (%) = (No. of surviving pus on day 4 after birth/No. of liveborn pups on day 0 after birth)
× 100

Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
Clinical signs
no effects 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
Description (incidence and severity)
on reproductive organs
Gross pathological findings
no effects observed
Description (incidence and severity)
on reproductive organs
Histopathological findings: non-neoplastic
no effects observed
Description (incidence and severity)
on reproductive organs

Reproductive function / performance (P0)
Reproductive function: oestrous cycle
no effects observed
Reproductive function: sperm measures
not examined
Reproductive performanceno effects observed
Description (incidence and severity)
on reproductive organs

Effect levels (P0)30Key result
false
Dose descriptor
NOAEL
Effect level300

mg/kg bw/day (actual dose received)

Sexmale/female
Basis for effect level
other: Effects of stomach (see repeated dose toxicity)
Key result
false
Dose descriptor
NOAEL
Effect level1000

mg/kg bw/day (actual dose received)

Sexmale/female
Basis for effect level
other: no effects on reproduction

Results: F1 generation
General toxicity (F1)Clinical signs
no effects observed
Mortality / viability
no mortality observed
Body weight and weight changes
no effects observed
Sexual maturation
not examined
Organ weight findings including organ / body weight ratios
not examined
Gross pathological findings
no effects observed
Histopathological findings
not examined

Effect levels (F1)31Key result
false
Dose descriptor
NOAEL
Generation
F1
Effect level1000

mg/kg bw/day (actual dose received)

Sexmale/female

Overall reproductive toxicity
Key result
false
Reproductive effects observed
not specified

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF3012 -65 -5d.pdf

Applicant's summary and conclusion
Conclusions
NOAEL for rat reproductive/developmental toxicity was determined to be 1000 mg/kg bw/day.

Executive summary
In the combined repeated oral dose toxicity study with the reproduction/developmental toxicity
screening test (0, 100, 300, and 1,000 mg/kg bw/day) (OECD TG 422), no effects of this substance on
reproductive and developmental parameters were observed at 1,000 mg/kg bw/day. NOAEL for the rat
reproductive/developmental toxicity of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
was regarded as 1,000 mg/kg bw/day, the highest dose tested.

32

References
Reference Substances
REFERENCE_SUBSTANCE: diammonium hydrogen 2hydroxypropane-1,2,3-tricarboxylate
UUID: ECB5-399f7091-4bb7-447c-b011-267f474d9c96
Dossier UUID:
Author:Date: 2016-12-21T15:14:55.000+09:00
Remarks:
Reference substance namediammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
IUPAC namediammonium hydrogen citrate

Inventory
Inventory number
Inventory namediammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
Inventory
EC Inventory
Inventory number
221-146-3
CAS number
3012-65-5
Molecular formula
C6H8O7.2H3N
Description
CAS number
3012-65-5

Synonyms
Synonyms
Identity
1,2,3-Propanetricarboxylic acid, 2-hydroxy-, diammonium salt
Identity
1,2,3-Propanetricarboxylic acid, 2-hydroxy-, diammonium salt

33

Identity
1,2,3-Propanetricarboxylic acid, 2-hydroxy-, diammonium salt

Molecular and structural information
Molecular formula
C6H8O7.2H3N
Molecular weight
225.1772
SMILES notation[NH4+].[NH4+].OC(CC(=O)[O-])(CC(=O)[O-])C(=O)[O-]
InChl
InChI=1/C6H8O7.2H3N/c7-3(8)1-6(13,5(11)12)2-4(9)10;;/h13H,1-2H2,(H,7,8)(H,9,10)(H,11,12);2*1H3/
p-1
Structural formula

Related substancesGroup / category information
DSL Category: Organics

34

Test Materials
TEST_MATERIAL_INFORMATION: Diammonium hydrogen
2-hydroxypropane-1,2,3-tricarboxylate
UUID: 80817e13-2fe0-3773-a70c-2e2ef08eef5c
Dossier UUID:
Author:Date: 2022-12-12T11:00:40.265+09:00
Remarks:
Name
Diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate

Composition
Composition
Type
Constituent
Reference substancediammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate / diammonium hydrogen citrate /3012-65-5 / 221-146-3
EC number

EC name221-146-3

EC Inventory

CAS number

CAS name

3012-65-5
IUPAC namediammonium hydrogen citrate

35

Literatures
LITERATURE: Combined repeat dose and reproductive/
developmental toxicity screening test of diammoniumhydrogen 2-hydroxypropane-1,2,3-tricarboxylate by oral
administration in rats
UUID: 5871efdc-ac0e-3345-bf1f-4eaf58d59637
Dossier UUID:
Author:Date: 2022-12-12T11:12:26.146+09:00
Remarks:

General information
Reference Typestudy report
Title
Combined repeat dose and reproductive/developmental toxicity screening test of diammoniumhydrogen 2-hydroxypropane-1,2,3-tricarboxylate by oral administration in rats
Author
MHLW, Japan
Year
2009
Bibliographic source
available in the web of Japan Existing Chemical Data Base (JECDB) at http://dra4.nihs.go.jp/
mhlw_data/jsp/SearchPageENG.jsp
Testing facility
BoZo Research Center

36

LITERATURE: In Vitro Chromosomal Aberration Testof diammonium hydrogen 2-hydroxypropane-1,2,3tricarboxylate on Cultured Chinese Hamster Cells.
UUID: ed9f5c39-923d-3218-a027-aa04dad90a22
Dossier UUID:
Author:Date: 2017-02-15T15:49:29.000+09:00
Remarks:

General information
Reference Typestudy report
Title
In Vitro Chromosomal Aberration Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarb
oxylate on Cultured Chinese Hamster Cells.
Author
MHLW, Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
Testing facility
Safety Research Institute for Chemical Compounds Co., Ltd.

37

LITERATURE: Reverse Mutation Test of diammoniumhydrogen 2-hydroxypropane-1,2,3-tricarboxylate on
Bacteria.
UUID: 435d0925-d6a4-3611-87a0-697e7ce9d1bb
Dossier UUID:
Author:Date: 2017-02-15T15:48:17.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Reverse Mutation Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate on Bacteria.
Author
MHLW, Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
Testing facility
Safety Research Institute for Chemical Compounds Co., Ltd.

38

LITERATURE: Single Dose Oral Toxicity Test of
diammonium hydrogen 2-hydroxypropane-1,2,3tricarboxylate in RatsUUID: 711f2de5-2173-37ae-b376-f1c8b65aca3e
Dossier UUID:
Author:Date: 2017-02-15T15:51:02.000+09:00
Remarks:

General information
Reference Typepublication
Title
Single Dose Oral Toxicity Test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate in Rats
Author
MHLW
Year
2011
Bibliographic source
available in the web of Japan Existing Chemical Data Base (JECDB) at http://dra4.nihs.go.jp/
mhlw_data/jsp/SearchPageENG.jsp
Testing facility
Safety Research Institute for Chemical Compounds Co., Ltd.

39

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 systemIUCLID440

ID16558402024DIV750

41