L-Ascorbic acid, 2-(dihydrogen phosphate), sodium salt (1:3)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-08-25 to 1998-03-06

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

96/54/EEC

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted July 27th, 1995

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan/MHW: Guidelines for Toxicity Testing of Chemicals; twenty-eight-day repeated dose toxicity in mammalian species; MITI/MHW, 1987 (Translation), p. 183-188

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-180-1

EC Name:

-

Cas Number:

66170-10-3

Molecular formula:

C6 H9 O9 P . 3 Na (CAS) C6 H6 Na3 P O9 (Hill)

IUPAC Name:

trisodium mono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl)phosphate

Details on test material:

- Name of test material (as cited in study report): Sodium ascorbyl phosphate
- Physical state: powder / light brown
- Analytical purity: 85.4% (method: HPLC-method with UV-Detection)
- Purity test date: 1997-06-25
- Lot/batch No.: 28600/37-9
- Date of production: 1997-05-02
- Stability under test conditions: proven by reanalysis after the in life phase of the study (purity: 86.8%)
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
-Age at arrival: males 40 days, femals 39 days
- Age at study initiation: 49 days
- Weight at study initiation: males: 217.3 g; females 158.6 g
- Fasting period before study: no
- Housing:The rats were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2). Underneath the cages, waste trays were fixecl containing absorbent material (type 3/4 dust free embedding, supplied by SSNIFF, Soest, FRG). The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm') and small amounts of absorbent material. The animals were housed in a fully air-conditioned room.
- Diet: The food used was ground Kliba maintenance diet rat/mouse/hamster, meal, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland (ad libitum)
- Water: from water bottles, ad libitum
- Acclimation period: 9-day (males) or 10-day (females)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.


IN-LIFE DATES: From: 1997-08-25 To: 1997-10-17

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution in drinking water. The test substance and drinking water were weighed in depending on the dose group. The solutions were mixed with a magnetic stirrer for about 5 minutes. The drinking water solutions were prepared at least twice a week. Drinkin water consumption of the animals was measured.

DIET PREPARATION (Drinking water)
- Rate of preparation of diet (frequency): at least twice a week.

VEHICLE
- Concentration in vehicle: 0; 1000; 5000; 15000 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water was tested over a period of 4 days at room temperature. Concentration analyses were performed in samples of all concentrations at the start of the administration period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 82.8, 424.1, 1426.0 mg/kg bw/day (male)
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 90.3, 512.0, 1661.7 mg/kg bw/day (female)
Basis:
nominal in water

No. of animals per sex per dose:

5 male, 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the expected low toxicity of the test substance
- Rationale for animal assignment: Five days prior to the start of the administration period of males the animals, separated by sex, were distributed according to weight among the individual test groups. The list of randomization instructions was compiled with a computer (laboratory data processing, Department of Toxicology, BASF Aktiengesellschaft).
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.


DETAILED CLINICAL OBSERVATIONS: Yes
Further clinical examinations were carried out once a day.
Open field observations, functional observational battery (FOB), motor activity measurements

BODY WEIGHT: Yes
The body weight was determined before the start of the administration period in order to randomize the animals. During the administration period and the recovery period the body weight was determined on day0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.


FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Water consumption was determined weekly over a period of 3 and 4 days. The values were calculated as grams per animal per day.


OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the end of the administration period
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked:
leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the administration period
- Animals fasted: No
- How many animals: all animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test RL, Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 mociel Boehringer, Mannheim, FRG).
The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically.
The following examinations were carried out:
- volume
- color
- turbidity
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- specific gravity
- sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The cages were cleaned prior to each use. The animals were put into the cages in a randomized order. The measurements started at about 2.00 p.m. in main groups respectively at about 3.00 p.m. in recovery groups. The number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights The weight of the anesthetized animals as well as the weights of liver, kidneys, adrenal glands, testes, epididymiaes, ovaries, brain, thymus, heart, and spleen from all animals sacrificed at scheduled dates were determined.

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% formaldehyde solution:
- all gross lesions
- brain
- pituitary gland
- thyroid glands with parathyroid glands
- thymus
- trachea
- lungs
- heart
- liver
- spleen
- kidneys
- adrenal glancis
- testes/ovaries
- uterus/vagina
- accessory genital organs (epiciidyniides, prostate gland, seininal vesicle)
- stomach (glandular and non-glandular)
- duodenum, jejunum, ileum
- cecun, colon, rectum
- urinary bladder
- mandibular and mesenteric lymph nodes
- sciatic nerve
- bone marrow (femur)
- eyes
- spinal cord (cervical, thoracic and lumbar cord)

Other examinations:

no

Statistics:

Parameter:
food consumption, water consumption, food efficiency, body weight, body weight change
Statistical test:
F-test, DUNNET's test, Student's t-test

Parameter:
feces rearing, grip strenght fore limbs, grip strength hind limbs, landing foot-splay test, motor activity
Statistical test:
KRUSKAL-WALLIS test, MANN-WHITNEY U-test, Student's t-test

Parameter:
clinical pathology parameters, exept differential blood count
Statistical test:
MANN-WHITNEY U-test

Parameter:
urinalysis except volume, color and turbidity
Statistical test:
FISHER's exact test

Parameter:
weight parameters
Statistical test:
KRUSKAL-WALLIS test, WILCOXON-test

Results and discussion

Results of examinations

Details on results:

The following substance-related findings were observed:

15,000 ppm (1426.0 mg/kg body weight in males, 1661.7 mg/kg body weight in females):
- increased water consumption in males and females
- focal or diffuse hyperplasia of the transitional epithelium in the urinary bladder of all male rats
- cystitis in four male rats
- increased number of starry sky cells in the cortex of the thymus of four female rats

5,000 ppm (424.1 mg/kg body weight in males, 512.0 mg/kg body weight in females):
- increased number of starry sky cells in the cortex of the thymus of three female rats

1,000 ppm (82.8 mg/kg body weight in males, 90.3 mg/kg body weight in females):
- no substance-related effects

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

424.1 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: water consumption, histopathology changes in the urinary bladder (high dose).

2

Dose descriptor:

NOAEL

Effect level:

90.3 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: water consumption (high dose group), histopathology changes in the thymus (mid and high dose group).

Target system / organ toxicity

Critical effects observed:

not specified