Magnesium bis(2-ethylbutanoate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study. Well described protocol and testing conditions. No internationally accepted protocol (e.g. OECD) mentioned in the study report, but applied protocol highly similar to OECD 408. Neurobehaviour not examined.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no assessment of sensory reactivity to stimuli, assessment of grip strength and motor activity assessment.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: fine, white, cohesive powder
- Analytical purity: ca. 96%
- Composition of test material, percentage of components: contains 4% of moisture
- Lot/batch No.: 9601
- Storage condition of test material: stored at ambient temperatures and protected from light until required

Test animals

Species:

rat

Strain:

other: HanIbm Wistar strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac (UK) Limited, Bicester, Oxfordshire, England
- Age at study initiation: ca. 34 - 41 days
- Weight at study initiation: 79 - 95 g
- Fasting period before study: no
- Housing: five animals of one sex per gace. Cage type TR18: stainless steel body with a stainless steel mesh lid and floor. Cages were suspended above absorbent paper which was changed at appropriate intervals.
- Diet: a powdered rodent diet, RM1(E) SQC (Special Diets Services Ltd., Witham, Essex, England) was freely available, except overnight before routine blood sampling or urine collection. Diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Weighed amounts of diet were provided at intercals during each week to each cage. At the end of each treatment week the weight of uneaten food was recorded and the food discarded.
- Water (e.g. ad libitum): Water taken from the public supply was freely available, except during urine collection, via polycarbonate bottles fitted with siper tubes.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C (range: 19-25°C)
- Humidity (%): 55% (range: 40-70%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other:

Details on oral exposure:

Method of administration:
Feed
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered rodent diet RM1(€) SQC (see above)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

RSS-4 was extracted from diet mix by sonicating with water (100 mL) for 10 minutes followed by shaking for 20 minutes. The weight of diet taken was such that the water extract would contain 300 ug/mL of RSS-4, dry substance. The extract was filtered. An aliquot of filtrate (10 mL) was acidified by addition of 0.1M hydrochloric acid (2 mL) and cyclohexane added (10 mL). The parent acid was extracted into the cyclohexane layer using a tube rotator for 10 minutes. The mixture was centrifuged and clear cyclohexane extract diluted to a known volume with cyclohexane to give an expected concentration of RSS-4, dry substance (present as parent acid) within the range 20 to 40 ug/mL.


The concentration of parent acid in the filtrate was adjusted to within a nominal range before quantitative analysis by gas liquid chromatography (HP 5890 Series GC system) using a flame ionisation detector.

Duration of treatment / exposure:

Test duration: 90 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 3000, 10000 and 30000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
241.7 - 277.7 mg/kg bw day (male - female)
Basis:
other: mean achived dosage over 13 weeks, corresponding to 3000 ppm nominal dose

Doses / concentrations 3

Remarks:

Doses / Concentrations:
816.3 - 938.7 mg/kg bw day (male - female)
Basis:
other: mean achived dosage over 13 weeks, corresponding to 10000 ppm nominal dose

Doses / concentrations 4

Remarks:

Doses / Concentrations:
2643.5 - 3028.0 mg/kg bw day (male - female)
Basis:
other: mean achived dosage over 13 weeks, corresponding to 30000 ppm nominal dose

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day
Male: 10 animals at 242 mg/kg bw/day
Male: 10 animals at 816 mg/kg bw/day
Male: 10 animals at 2644 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 10 animals at 278 mg/kg bw/day
Female: 10 animals at 939 mg/kg bw/day
Female: 10 animals at 3028 mg/kg bw/day

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results obtained from a preliminary study in which there was no evidence of systemic toxicity or unpalatability of the diets at concentrations of 10000 to 30000 ppm.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice dayly
- Cage side observations checked in table 1A were included in the study report.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatisation period, on the day that treatment commenced, at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each animal, that remaining, and an estimate of any spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.

FOOD EFFICIENCY:
Weekly group mean food conversion efficiencies were calculated for each week of treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
Before commencement of treatment both eyes of all rats were examined by means of an indicrect ophthalmoscope, after the instillation of 0.5% tropicamide. The structures examined inlcuded the following: palpebrae and adjacent structures; conjunctiva; cornea and sclera; anterior chamber and iris; lens and vitreous: ocular fundus. Animals with moderatley severe ophthalmic lesions were rejected and replaced with spare animals drawn from the same batch. After 12 weeks of treatment all surviving animals from groups 1 (control group) and 4 (dose group 30000 ppm) were similarly examined.
- Dose groups that were examined: before treatment: all; after 12 weeks: groups 1 and 4.

HAEMATOLOGY: Yes
During week 13 of treatment, blood samples were obtained from all animals, after overnight starvation. Blood samples were withdrawn from the retro-orbital sinus, with the animals held under halothane/nitrous oxide anaesthesia, and collected into EDTA as anticoagulant. All samples were examined for the following characteristics, using a Technicon H1 analyser: packed cell volume (PCV), haemoglobin concentration (HB), erythrocyte count (RBC), total and differential leucocyte count (WBC), platelet count (PLAT), mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MCH) and mean cell volume (MCV). Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types including normoblasts. Additional blood samples were taken into citrate anticoagulant and examined in respect of Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
At the same time as for peripheral haematology further blood samples were taken and collected into lithium heparin as anticoagulant. After separation the plasma was examined in the respect of: alkaline phosphatase activity (ALP); Alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), gamma-glutamyl transpeptidase activity (GGT), glucose concentration (GLUC), total bilirubin concentration (BILT), total cholesterol concentration (CHOL), creatinine concentration (CREA), urea concentration (UREA), total protein concentration (TP), albumin concentration (CHEM ALB), albumin/globulin ratio (A/G), sodium (Na), potassium (K), chloride (Cl), calcium concentration (Ca) and inorganic phosphorus (PHOS).

URINALYSIS: Yes
Overnight urine samples were collected from all animals during week 12 of treatment. Each rat was placed in an individual metabolism cage for urine collection under conditions of food and water deprivation. The individual samples were examined in respect of: appearance (APP), volume (VOL), pH, specific gravity (SG), protein (PROT), glucose (GLUC), ketones (KET), bilirubin (BIL) and blood. Microscopy - the sediment from centrifugation was examined for epithelial cells (EP), polymorphonuclear leucocytes (P), erythrocytes (RBC), crystals (CRY), spermatozoa and precursors (S) or other abnormalities (A). Grading of populations was on a 4-point scale ranging from 0 = none seen, to 3 = many in all fields examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Animals were killed by carbon dioxide inhalation.
The sequence in which the animals were killed after completion of treatment was selected to allow satisfactory inter-group comparison.
All animals were subjected to a detailed necropsy. Organ weights were recorded and expressed as a percentage of the body weight recorded immediately before necropsy. Tissue samples were preserved for and subjected to histopathologic examination.

Statistics:

Standard deviations were calculated, as considered appropriate, using the sample statistic.
For bodyweight changes and organ weights, homogeneitay of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perfom pairwise comparisons, otherwise a Dunnet(s test was used.
The significance of inter-group differences for haematology examinations (excluding the incidence of morphological abnormalities evident on the blood smears), blood chemistry and urinalysis (volume, pH and specifiy gravity only) was assessed by Student's t-test using a pooled error variance. Statistical significances for reticulocyte, eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed.
Fisher's Exact test was applied as a two-tailed test, where appropriate, to the distribution of macroscopic or microscopic pathological entities.
Unless stated, group mean values or incidences for the treated goups were not significantly different from those of the controls (p<0.05)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Staining (brown, yellow and urine)

Mortality:

mortality observed, treatment-related

Description (incidence):

Staining (brown, yellow and urine)

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight decrease

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

not specified

Details on results:

Signs and mortality:
There were no deaths.
Nine of the 10 males and all the females receiving 30000 ppm had brown staining around the head and sorsal body srufaces, although the highest incidence was seen on the head of the affected animals. Brown staining was first apparent in week 3 for males and week 5 for females. The highest incidences were generally aparent from week 8. Brown staining was seen at a low incidence in other groups, including controls.
In six males and all females receiving 10000 ppm and nine males and all females receiving 30000 ppm, yellow staining of the tail was seen. In addition, perigenital urine staining was seen in seven males and one female receiving 30000 ppm. The yellow or urine staining was seen from week 5 of the study.

Bodyweight:
From week 1, weight gains of males receiving 30000 ppm were lower than those of the controls. This effect was most marked during the first 5 weeks of the study, although weight gains thereafter were still slightly lower than in the controls. A similar effect on weight gain was seen in females, although the effect was not as consistent as that seen in the males. Overall, the weihgt gains in males and females receiving 30000 ppm were approximately 26 and 30% lower than those seen in the controls. Amongst rats receiving 10000 ppm, a slight effect on weight gain was noted in both males and females. Overall gains in these animals were 8 and 12% lower than those of the controls for males and females respectively.
The bodyweight gains of animals receiving 3000 ppm are considered to be unaffected by treatment.

Food consumption:
The total food consumption of animals receiving 10000 to 30000 ppm was slightly lower than that of controls. Overall, the food intake was between 7 and 8% lower in treated animals when compared to control values. There was no clear dosage-relationship. A less marked effect on food intake was apparent in males and females receiving 3000 ppm.

Food conversion efficiency:
The overall food conversion efficiency of animals receiving 30000 ppm was lower than their controls. No clear effect was seen in other treated groups.

Ophthalmoscopy:
there were no ophthalmoscopic abnormalities which could be attributed to treatment.

Haematology:
During week 13, significant low platelet counts (p<0.05 or less) were seen in males and females receiving 30000 ppm. A similar, less marked, effect was also seen on platelet counts in males receiving 10000 ppm, although this did not achieve statistical significance. There were no other changes in the haematological composition of the blood which were related to treatment and, in absence of any associated pathological change, the significance of these changes is unclear.
A number of inter-group differences also achieved statistical significance (p<0.05). They were, however, minor, confined to one sex or did not show any dosage-relationship. These changes were therefore considered to represent normal biological varation and are unrelated to treatment.

Blood chemistry:
A number of changes in the biochemical composition of the plasma was seen in treated animals. These included changes in alkaline phosphatase activity and urea, cholesterol, bilirubin, calcium and phosphorous concentrations.
Significantly high alkaline phosphatase activities (p<0.001) were seen in males and females receiving 30000 ppm.Other treated animals were unaffected.
High plasma urea concentrations (p<0.01) were seen in both males and females receiving 30000 ppm. There was no clear effect on urea concentrations in animals receiving 3000 or 10000 ppm.
Slightly low plasma cholesterol concentrations were seen in males receiving 30000 ppm (pw0.05). Marginally low plasma cholesterol concentrations were also seen in females receiving this dietary concentration.
In comparison with controls, there was a general trend for low plasma bilirubin concentrations in females receiving 10000 or 30000 ppm. Overall, the group mean values were only slightly different, however the incidence of each concentration varied between the groups.
Slightly, but significantly, low plasma calcium concentrations were seen in males and females given 30000 ppm (p<0.001). Similar, but less marked effects were seen in males receiving 3000 ppm and in males and females receiving 10000 ppm. High plasma phosphorus concentrations were seen in males and females receiving 30000 oppm (p<0.001) and, to a lesser extent, in females receiving 10000 ppm, although this did not achieve statistical significance.
A number of inter-group differences also achieved statistical significance (p<0.05). They were, however, minor, confined to one sex or did not show any dosage-relationship. These changes were therefore considered to represent normal biological varation and are unrelated to treatment.

Urinalysis:
There were no changes in the composition of the urine which could be attributed to treatment.

Organ weights:
High kidney weights were seen in males given 10000 or 30000 ppm, although these only attained statistical significance in respect of bodyweight-relative weights. High bodyweight-relative kidney weights were also seen in females given 30000 ppm.
Significantly high liver weights (p<0.01) were seen in males receiving 30000 ppm, compared to their controls , although the effect on absolute weights was slight. Bodyweight-relative liver weights in females given this dietary concentration were also high (p<0.01).

Macropathology:
There were no macroscopic abnormalities after 13 weeks of treatment which were attributed to the treatment.

Histopathology:
Findings considered to be related to treatment were seen in kidneys and livers of male rats.
In the kidneys there were hyaline protein droplets in the corical tubular epithelium in rats given 10000 or 30000 ppm of the test substance. The incidence and severity of the change was highest in those given 30000 ppm. No protein droplets were seen in control males or males given 3000 ppm, or in any of the female rats.
There was a reduced incidence of cortico-meullary mineralistion in the kidneys of female rats given 10000 or 30000 ppm. This only achieved significance (p<0.01) at the highest concentration.
Three male rates given 30000 ppm had periacinair hypertrophy of hepatocytes. This finding was not observed in any of the other rats on the study.
There was one case of malignant lymphoma in a female rat given 30000 ppm of the test substance. This is a spontaneous lesion seen occasionally in young rats and is considered to be of no toxicological significance in this study.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

3

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: nominal concentration

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of RSS-4 dry substance to HanIbm Wistar rats for 13 weeks was associated with changes in the kidney and liver at concentrations of 10000 or 30000 ppm and reduced weight gain, food intake and food conversion efficiencies.

The NOAEL in this study was 3000 ppm (nominal), corresponding to actual intake doses of 241.7 mg/kg bw day for males and 277.7 mg/kg bw day for females.

Executive summary:

Three groups of 10 male and 10 female HanIbm Wistar rats received RSS-4 via thet diet at concentrations of 3000, 10000 or 30000 ppm for a scheduled treatment period of 13 weeks. A similarly constituted control group received untreated diet.

Results:

There were no deaths.

Brown staining on the head and dorsal surface was seen in animals receiving 30000 ppm. Yellow staining on the tail was seen in males and females receiving 10000 or 30000 ppm. Perigenital urine staining was seen in most males and one female receiving 30000 ppm.

Weight gains of females and males receiving 30000 ppm were lower when compared to control values, especially during the first 5 weeks of treatment. A less marked effect in weight gain was seen in animals receiving 10000 ppm.

Food intake in males and females receiving 10000 or 30000 ppm was slightly lower than that of the controls, although there was no clear dosage-relationship.

The food conversion efficiency of males and females receiving 30000 ppm was low.

There were no treatment-related ophthalmic abnormalities.

Low platelet counts were seen in males receiving 10000 ppm and males and females receiving 30000 ppm.

In the plasma of animals receiving 30000 ppm, high alkaline phosphatase activities, high urea concentrations and low cholesterol, bilirubin (females only), calcium and phosphorous concentrations were seen. Low bilirubin concentrations were also seen in females receiving 10000 ppm. Low plasma calcium concentrations were seen in males receiving 3000 ppm and males and females receiving 10000 ppm. High phosphorous concentrations were seen in females receiving 10000 ppm.

There were no treatment-related changes in the composition of the urine.

High kidney weights were seen in males given 10000 or 30000 ppm. High bodyweight-relative kidney weights were seen in females given 30000 pppm. High liver weights were seen in females and males receiving 30000 ppm.

There were no treatment-related macroscopic abnormalities.

Histopathological findings related to treatment included hyaline protein droplets in the kidneys of males given 10000 or 30000 ppm and hepatocytic periacnair hypertrophy in the livers of 3 males given 300000 ppm.

In conclusion, administration of RSS-4 to HanIbm Wistar rats for 13 weeks was associated with changes in the kidney and liver at concentrations of 10000 or 30000 ppm and reduced weight gain, food intake and food conversion efficiencies.

The NOAEL in this study was 3000 ppm.