Reference type

Author

Year

Title

Bibliographic source

Testing laboratory

Report no.

Owner company

Company study no.

Report date

study report

MHLW Japan

2009

Combined repeat dose and reproductive/developmental toxicity screening test of diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate by oral administration in rats

available in the web of Japan Existing Chemical Data Base (JECDB) at http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp

BoZo Research Center

data published

7.8.1 Reproductive/developmental toxicity.001

combined repeated dose and reproduction / developmental screening

no

Qualifier

Guideline

Deviations

according to

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

no

yes

yes

Identifier

Identity

CAS number

3012-65-5

- Name of test material (as cited in study report): diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
- 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.

rat

other: Crl: CD(SD)

male/female

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

oral: gavage

water

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 6F74

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 concentration 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 concentration, 100 ± 10%; C.V.: 10% or below)

(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.

Once/day, 7days/week

0 (vehicle), 100, 300, and 1000 mg/kg bw/day

Basis

actual ingested

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)

yes, concurrent vehicle

- Dose selection rationale: Doses in this test were set based on the results of the following study: 14-day repeated dose oral toxicity test (doses: 100, 300, and 1000 mg/kg bw/day). In the 14-day repeated dose 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 the 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 approximately 3.
- Rationale for animal assignment (if not random): Body weight-balanced randomization
- Post-exposure recovery period in satellite groups: 14 days

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 measures 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 females of 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 and recovery 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. 16h-fasting) in recovery period
Females (main group): Twice a week during the precopulation period (days 1, 4, 8, 11, and 15); gestation days 0, 4, 7, 11, 14, 17, and 20; lactation days 0 and 4; and the day of necropsy (after ca. 16 h-fasting)

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 period
Males 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)

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 lactation.

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 (including bone marrows), macroscopic lesions.

Organ weight: Brian, thyroids (including parathyroids), thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymis

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 homogeneous, data was analyzed by the Student t-test, whereas heterogeneous data was analyzed by the Aspin-Welch t-test (p<0.05, two-sided).

Endpoint

Effect level

Based on

Sex

Basis for effect level / Remarks

LOAEL

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

test mat.

male/female

effects on the stomach

no effects

no effects

no effects

not examined

not examined

not examined

no effects

yes

yes

no effects

no effects

no effects

yes

not examined

CLINICAL CHEMISTRY
High 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 to temporary, dose-independent, or within the normal ranges of physiological variability.

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.

UUID

IUC5-253e1121-b70b-4f87-9374-6d5b549c946a

Dossier UUID

0

Author

dra / National Institute of Health Sciences / Tokyo / Japan

Date

2017-02-15 15:48:17 JST

Remarks

Purpose flag

key study

Study result type

experimental result

Reliability

1 (reliable without restriction)

Rationale for reliability incl. deficiencies

OECD Test Guideline study under GLP condition