Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study in compliance with OECD 408 under GLP without significant deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: Japanese MHLW (1121002), METI (2), ME (031121002)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): GLDA
- Trade name: Dissolvine GL-PD
- Lot/batch No.: CFC-8112 and CFC-8112B (see CoAs in the report attached
- Appearance: white powder
- Expiration date of the lot/batch: 27 December 2008
- Stability under test conditions: stable
- Storage condition of test material:: at room temperature in the dark
- Purity: glutamic acid - N,N-diacetic acid, tetrasodium salt = 98 ± 1%; content: glutamic acid - N,N-diacetic acid, tetrasodium salt = 95 ± 1%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: approx. 190 g (males), approx. 160 g (females)
- Housing: macrolon cages with sterilised saw dust as bedding material and paper as cage enrichment; 5/sex
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 5 days before study initiation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-23.7
- Humidity (%): 30-83 (higher values due to cleaning)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 9 April 2007 To: 24 July 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Concentration in vehicle: 10 mg/mL, 30 mg/mL, 100 mg/mL (w/w)
- Amount of vehicle (if gavage): 10 mL/kg bw

Formulations were prepared daily within 4 hours prior to dosing (on a few days within approximately 6 hours prior to dosing) and were homogenised to visually acceptable levels. Formulations were stored at ambinet temperature before use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations were done in week 1, 6, and 13 (duplicate samples were analysed)
0 mg/kg day: measurement of accuracy in random sample
100 mg/kg day: measurement of accuracy and homogeneity in samples taken at top, middle and bottom
300 mg/kg day: measurement of accuracy in random sample
1000 mg/kg day: measurement of accuracy and homogeneity in samples taken at top, middle and bottom

Test substance formulations formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 93-100% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.




Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
dosis: mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on a preceding, non-glp, 14-day RF study.
- Rationale for selecting satellite groups: to observe persistance or recovery from effects 14 days after the last exposure
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): not indicated
Positive control:
Not needed.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were performed once prior to start and once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly

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: No

WATER CONSUMPTION: No data
- Time schedule for examinations: subjective appraisal of water intake

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start and in week 12 of the study
- Dose groups that were examined: prior to start (all groups), in week 12 (control and high dose recovery animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (maximally 20 hours)
- How many animals:all main groups the day after last dosing, all recovery groups on the last day of the recovery period
- Parameters examined: all parameters indicated in OECD TG 408.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all main groups the day after last dosing, all recovery groups on the last day of the recovery period
- Parameters examined: all parameters indicated in OECD TG 408.

URINALYSIS: Yes
- Time schedule for collection of urine: night before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, colour, clarity, specific gravity, pH, protein, glucose, ketone, bilirubin, blood, leukocytes, nitrite, urobilinogen, Na, K, Ca, sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in study week 12-13
- Dose groups that were examined: control and high dose recovery animals
- Battery of functions tested: sensory activity / pupillary reflex/ static righting reflex / grip strength / motor activity:

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (according to OECD TG 408)
Other examinations:
None
Statistics:
Pooled variance - Dunnett in case of normal distribution
Steel-test in case of non-normal distribution
Fisher exact test in case of frequency data

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:

HAEMATOLOGY
At the end of the 13-week study period:
- lncreased red blood cell count in males at 1000 mg/kg/day,
- Reduced mean corpuscular volume (MCV) in males and females at 1000 mg/kg/day,
- Reduced mean corpuscular haemoglobin (MCH) in males and females at 1000 mg/kg/day, and in males also at 300 mg/kg/day,
- lncreased red cell distribution width (RDW) in females at 1000 mg/kg/day,
- lncreased platelet count in females at 1000 mg/kg/day.
These changes were statistically significant; these were absent at the end of the recovery period.

CLINICAL CHEMISTRY
In animals at 1000 mg/kg/day at the end of the 13-week study period::
- lncreased albumin levels in males,
- Reduced creatinine levels in males and females,
- Reduced inorganic phosphate levels in males,
- lncreased cholesterol levels in females,
- Reduced chloride levels in females.
These changes were statistically significant; these were absent at the end of the recovery period.

URINALYSIS
At the end of the 13-week study period:
- Reduced urinary volume in females at 1000 mg/kg/day,
- lncreased specific gravity in females at 1000 mg/kg/day,
- Reduced clarity (increased score) in females at 1000 mg/kg/day,
- lncreased protein level in females at 1000 mg/kg/day,
- lncreased sodium concentration in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day (not statistically significant in males at 300 rng/kg/day),
- lncreased sodium excretion in males and females at 300 and 1000 mg/kg/day (not statistically significant for females at 300 mg/kg/day),
- lncreased potassium concentration in females at 1000 mg/kg/day.
These changes were absent at the end of the recovery period.


ORGAN WEIGHTS
- A statistically significant increase in absolute and relative (organ to body weight ratio) kidney weight in males at 1000 mg/kg/day at the end of the treatment period (but not at the end of the recovery period)
- A statistically significant increase in absolute and relative kidney weight in females at 1000 mg/kg/day at the end of the recovery period (but not at the end of the treatment period)
- A statistically significant lower absolute brain weight (but not relative brain weight) in females at 1000 mg/kg/day at the end of the recovery period (but not at the end of the treatment period)
- A statistically significant higher relative liver (but not absolute liver weight) in females at 1000 mg/kg/day at the end of the recovery period (but not at the end of the treatment period)

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: haematology; clinical chemistry; urinalysis; organ weights

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
At 300 mg/kg/day, changes were limited to a slightly reduced mean corpuscular haemoglobin level, and increased urinary sodium concentration/
excretion. At 1000 mglkglday, a range of more pronounced changes in blood and urine, and including increased absolute and relative kidney weight, were noted. Although no concurrent morphologic evidence of organ dysfunction was obtained for these changes, given the number of alterations at 1000 mglkglday a No Observed Adverse Effect Level (NOAEL) for GLDA-Na4 of 300 mg/kg/day was established.
Executive summary:

Repeated dose 90 -day oral toxicity study with GLDA-Na4 by daily gavage in the rat, followed by a 14-day recovery period.

The study was based on the following guidelines.

- EC Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001.

- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

- OPPTS 870.31 00, EPA 71 2-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

- Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No. 2) and ME (No. 031 121002).

The rationale for the dose levels was based an the results of a preceding, non-glp,14 -day range finding study (NOTOX Project 483233), the dose levels for this 90 -day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg/day.

The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. An extra 10 animals per sex in the control and high dose group were

allowed 14 days of recovery.

Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 12-13; body weight and food consumption weekly; ophthalmoscopy at pretest and in week 12; clinical pathology, urinalysis and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test substance in water were prepared accurately and homogenously.

Treatment up to 1000 mg/kg/day was well tolerated; no treatment-related changes were noted in clinical appearance, functional observations, body weight and food consumption. Blood analyses revealed a number of changes, primarily at 1000 mg/kg/day. These consisted of changes in red blood cell parameters (increased red blood cell count in males at 1000 mg/kg/day, reduced mean corpuscular volume in males and females at 1000 mg/kg/day, reduced mean corpuscular haemoglobin in males and females at 300 and 1000 mg/kg/day, increased red cell distribution width in females at 1000 mg/kg/day), and increased platelet count in females at 1000 mg/kg/day. At 300 mg/kg/day, haematological changes were confined to reduced mean corpuscular haemoglobin in both sexes. Changes in clinical biochemistry parameters at 1000 mg/kg/day at the end of treatment included increased albumin and cholesterol levels, and reduced creatinine, inorganic phosphate and chloride levels in males and/or females. These blood changes were within or just outside the range considered normal for rats of this age and strain), and were absent at the end of the recovery period. Also, no supportive histopathological lesions were observed. Therefore, these changes were considered to be of limited toxicological significance. A number of changes were recorded in the urine of animals at 1000 mg/kg/day, which primarily included an increased sodium concentration/excretion in males and females at 300 and 1000 mg/kg/day, but also a reduced urinary volume and clarity in females, and increased specific gravity, protein level and potassium concentration in females. These changes were absent at the end of the recovery period, indicating that these were reversible in nature. Histopathologically, there was no evidence of an effect on kidney function, and no

morphological correlate was obtained for the slightly increased kidney weight and kidney to body weight ratio in males at 1000 mg/kg/day at the end of the treatment phase. At the end of the recovery phase kidney weights were similar to control levels. In females at 1000 mg/kg/day, kidney weight and kidney to body weight ratio was increased at the end of the recovery period, but not at the end of the treatment period. These results indicate that the test substance has an effect on kidney function. No toxicologically significant changes were noted during macroscopic and microscopic

examination.

At 300 mg/kg/day, changes were limited to a slightly reduced mean corpuscular haemoglobin level, and slightly increased urinary sodium concentration/excretion. At 1000 mg/kg/day, a range of more pronounced changes in blood and urine were noted. Although no concurrent

morphologic evidence of organ dysfunction was obtained for these changes, given the number of alterations at 1000 mg/kg/day a No Observed Adverse Effect Level (NOAEL) for GLDA-Na4 of 300 mg/kg/day was established.