Zinc, [1,2,3-propanetriolato(2-)-.kappa.O1,.kappa.O2]-, homopolymer, stereoisomer

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Ltd., Blackthorn, Bicester
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 166.3 – 210.3 g (males), 130.7 – 167.6 g (females)
- Fasting period before study: no
- Housing: During accommodation, five animals per cage; during experimental phase, individually in stainless steel cages with stainless steel mesh floors and fronts.
- Diet ad libitum: ESL Modified AIN-76A diet; the animals were given free access to food at all times (except for a selection of rats during urine collection).
- Water ad libitum: potable tap water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The experimental diets were based on the ESL Modified AIN-76A diet (MODAIN purified diet ) and were prepared weekly. The experimental diets, in sealed bags, were UV sterilised for a minimum of 10 hours and were then stored at 4°C prior to use. The test substance was added to the diet mix to produce test diets with the following concentrations: 0, 0.05, 0.2 and 1 %.
Diets prepared for feeding at week 1, week 7 and week 13 were analysed to confirm the achieved concentrations. These samples of diets were also analysed for zinc levels. The samples of week 7 were analysed for moisture, protein, fat, fibre and ash content to confirm dietary composition.
The composition of the diets, as well as the nominal dietary concentrations of the test substance was confirmed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in purified diet was investigated in the study AH930558. The study report was not available to us, but major results were given:
Homogeneity of the test substance in diet at concentrations from 0.05% to 5.00% (w/w) was demonstrated by zinc analysis. The test substance was shown to undergo a small degree of hydrolysis immediately on addition to diet at concentrations of 1.00% and 5.00% (w/w). However, no further hydrolysis occurred on storage at ambient or refrigerated temperatures for 14 days and the degree of hydrolysis was less than 10% of the total test substance present. Thus, the test substance was regarded as stable under these conditions. Stability could not be confirmed at dietary levels below 1.00% (w/w) due to limitations of the analytical methodology, although even at a concentration of 0.05% (w/w) it was demonstrated that the test substance did not completely hydrolyse in diet.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

The animals were given free access to the test diets at all times.

Dose / conc.:

500 ppm

Remarks:

corresponding to 33.7 mg/kg bw/d

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 136.7 mg/kg bw/d

Dose / conc.:

10 000 ppm

Remarks:

corresponding to 695.1 mg/kg bw/d
reduced to 5000 ppm from day 58 onwards. All rats were sacrificed on day 64.

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale
The dose selection was based on the results of a 14 day palatability study (ESL FP930559, IUCLID Chapter 7.5.1)
- Rationale for animal assignment
The animals were assigned to the treatment groups based on pre-test body weight using a computer program.
- Further details
Animals of the high dose group showed signs of severe toxicity. Therefore, the dietary level was reduced to 0.5% test substance on day 58. However, the remaining rats were sacrificed due to humanity reasons on day 64 of the study.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND MORTALITY
All animals were checked at least twice each day (once on Saturdays, Sundays and public holiday) for signs of toxicity and mortality.

BODY WEIGHT
Each animal was weighed on the day of treatment starting and then at weekly intervals for the duration of the study.

FOOD CONSUMPTION AND WATER CONSUMPTION
Food and water consumptions were measured and recorded twice-weekly.

OPHTHALMOSCOPIC EXAMINATION
All animals were given an ophthalmoscopic examination prior to the start of the study. In the week prior to study termination all animals from the control group and the animals fed with 0.2 % test substance in diet were re-examined.

HAEMATOLOGY
All rats fed the high dietary level of the test substance, which were sacrificed on humane reasons on day 64 of the study and all surviving rats at the end of the 13 week period were bled by cardiac puncture under Halothane anaesthesia. Approximately 1 ml of blood, for clotting factors and 1ml of blood for general haematological analysis was used with exception for animals of the high dose level. Due to the poor general physical state 0.5ml of blood was available for general haematological analysis, only.
The following haematological indices were analysed:
Red blood cell count, Haemoglobin, Platelets, White cell count (WBC) and differential WBC (including neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells) Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocytes; MCV - mean cell volume (mean of red blood cell volume histogram), HCT - haematocrit (red blood cell count multiplied by the MCV), MCH - mean cell haemoglobin (haemoglobin divided by the red blood cell count), MCHC - mean cell haemoglobin concentration (haemoglobin divided by the HCT), RDW - red cell size distribution width, HDW - red cell haemoglobin distribution width, MPV - mean platelet volume, PDW - platelet size distribution width, PCT - plateletcrit

CLINICAL CHEMISTRY
Upon cardiac puncture blood was collected into heparinized glass tubes for plasma chemistry analysis and for mineral analysis (Zn, Cu and Fe) via Flame Atomic Absorption Spectrometry. Blood for serum parameter was collected in plain glass tubes. The following parameters were considered: Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Triglyceride, Total cholesterol, Urea, Glucose, Creatinine, Total bilirubi, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Lactate dehydrogenase (LDH), Hydroxy butyrate dehydrogenase (HBDH), Creatine Kinase (CK), Alkaline Phosphatase (ALP), Pseudocholinesterase, 5´-Nucleotidase, Total protein, Albumin/globulin ratio, Albumin, alpha1-globulin, alpha2-globulin, beta-globulin gamma-globulin.

URINALYSIS
Urine and feces samples were collected from 5 males and 5 females from each group during weeks 4, 7 and 13 of the study (except sacrificed animals at the high dietary level). The animals were housed individually in metabolism cages for approximately 24 hours. A 4 and 20 hour urine sample as well as a 24 hour feces sample was taken, stored at -20°C. The following urine parameters were measured on the 4 hour specimen: Urine volume, Refractive index, Leukocytes, Nitrite, pH, Glucose, Protein, Blood and Hemoglobin. The following urine parameters were measured on the 20 hour specimen: Urine volume, Refractive index, Creatinine, Urea, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Alanine aminopeptidase (AAP), y-glutamyltransferase (GGT), N-acetyl-ß-D-glucosaminidase (NAG), Zinc

OTHER
Feces for mineral analysis were collected daily from underneath each cage for 10 male and 10 female animals from each dose group during week 6 of the treatment period. The collected feces was dusted and weighed pooled and stored at -20°C until mineral analysis (calcium, copper, iron, zinc and phosphorus).

Sacrifice and pathology:

NECROPSY
Upon sacrifice 6 males and 6 females from the highest dose group and 3 males and 3 females of the remaining dose groups were selected for radiographic examination prior to necropsy.

GROSS PATHOLOGY
All macroscopic abnormalities as well as an assessment of the level of intra-abdominal fat deposition were recorded.

ORGAN WEIGHTS
The following organs were removed and weighed: adrenal glands, brain, heart, kidneys, liver, spleen and testes.

HISTOPATHOLOGY
The following tissues were taken from each rat and preserved in 10% buffered formalin unless otherwise indicated:
Brain, Cervical lymph node, Mesemeric lymph node, Pancreas, Thymus, Ovaries, Fallopian tubes, Vagina #, Uterus, Cervix, Testes #, Epididymides #, Seminal vesicles #, Eyes $, Harderian glands $, Salivary glands #, Adrenal glands, Pituitary, Sciatic nerve, Thyroids, Parathyroids, Liver, Heart, Spleen, Lungs, Larynx, Trachea, Kidneys, Aorta, Bladder, Prostate, Tongue, Caecum, Stornach, Duodenum, Ileum, Jejunum, Colon, Rectum, Oesophagus, Mammary gland (site of), Skeletal muscle, Skin #, Femur and stifle joint, Spinal cord, Sternum, Head, All gross lesions.
# preserved in Boulins fixative, $ fixed in Davidsons fluid
Tissues were processed in paraffin wax, 4 µM slices were prepared and stained with haematoxylin and eosin (H&E) for microscopic examination. In addition, a portion of liver was taken for frozen sectioning and staining with oil red O to demonstrate neutral lipid. Microscopic examination was performed on the above mentioned tissues from all decedent animals and from all animals fed 0 % and 0.2% test substance in diet, respectively. Following the initial histopathological examination, pancreas, mesenteric lymph nodes, spleen, femur and tibia were examined from all animals fed 0.05% test substance in diet.

Statistics:

From the following statistical methods the appropriate method was chosen for analysis of live animal data, urinalysis, clinical data and organ weights:
Kruskal-Wallis, Adjusted Wilcoxon, Adjusted Satterthwaite’s approx t-test, ANOVA, Dunnett' s test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs consisted of laboured respiration, exophthalmus and nasal discharge in all treatment groups. In addition animals at the high dose group showed ataxia, piloerection, hunched posture, fur staining, excessive salivation, tremors, pale appearance, swollen abdomen, colored feces, erythema, torticollis, hyperactivity, cold feel, moribund and swellings (limbs and perineum). With exception of laboured respiration, exophthalmus and nasal discharge, no treatment related findings were seen at the lower dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Due to low physical health and reduced food intake, rats of the high dose group (1% test substance in diet) were fed with 0.5% test substance in diet from day 58 onwards. However, all rats of this dose group had to be sacrificed on day 64 due to humane reasons. A total of 8 animals were sacrificed before or on day 64. Four female and one male rat were sacrificed on humane reasons before day 58.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in mean body weight gain was seen in male and female rats fed the high dose level of the test substance during weeks 0-4 and 4-8 in comparison to the control group. No other statistically significant differences were observed (see table 1).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The animals were individually housed and the food intake was measured for each cage. A statistically significant reduction in accumulated food intake and food conversion efficiency was seen in both male and female rats at the high dose during weeks 0-4 and 4-8 in comparison to the control group. At 0.2 % test substance in diet a statistically significantly reduced food conversion efficiency was also seen in male and female during weeks 4-8, although no overall differences were seen during weeks 0-13 (see table 2).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No statistically significant differences in water consumption were seen.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related abnormalities were seen upon ophthalmoscopic examinations at study week 12.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Premature deaths: Blood was taken from 2 male and 2 female rats sacrificed prior day 64. Signs of anaemia like low haemoglobin and haematocrit as well as increased red blood cell and reticulocyte count were seen in 3 of these animals. Increased white cell counts and increased platelet count was seen observed in two animals. Bone marrow investigations revealed no abnormalities.
At termination reduced monocyte count, an increased activated partial thromboplastin time and an increased red cell size distribution width were seen in males treated with 0.2 % test substance in diet. None of these effects was considered to be of toxicological relevance. The examination of the bone marrow revealed no abnormalities.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Premature deaths: Sufficient blood for clinical chemistry analysis was obtained for 3/4 animals only. The main findings were a moderate increase in plasma urea for all three animals and increased creatine kinase activity in two animals. In addition numerous minor changes were observed; electrolyte alterations, decreased plasma sodium, increased chloride, calcium, phosphate and magnesium; increased ALT and CK, decreased plasma total protein decreased serum pseudocholinesterase; intermediate metabolites.
At termination increased plasma ALT, ATP and CK as well as reduced plasma cholesterol was seen in males treated with 0.2 % test substance in diet. The increased plasma CK and reduced total plasma cholesterol was also seen in females of this dose group. All changes were very small in absolute terms and were therefore considered to be of no toxicological relevance.
When analyzing whole blood samples taken at week 13 for mineral content no differences were seen between control and treatment groups. However, when mineral content of blood from controls (week 13) was compared with the mineral content of blood taken at day 64 from animals of the high dose group following effects were seen:
- 4 fold increased zinc levels,
- 7 fold decreased copper levels,
- 20 to 40 fold decreased iron levels.
The reduction in copper and iron levels in the presence of high levels of zinc was considered to be consistent with the development of the hypochromic microcytic type of anaemia observed in these rats.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At the 4 hour urine sampling interval no statistically significant differences were observed in urine samples collected from each group during weeks 4, 7 or 13 of the study.
At the 20 hour urine sampling interval decreased urinary calcium and magnesium level were the most consistent findings in urine collected during Weeks 4 and 7 at the mid and high dose group. Since this effect decreased by time and was not seen in the urine collected in week 13, it was concluded to be a physiological adaptation rather than a toxicological effect.
Statistically significant findings at the 4 week sampling consisted of:
- Increased refractive index for male rats fed at the high dose level
- Reduced calcium level for male and female rats fed the intermediate and high dietary levels, and female rats fed the low dietary level.
- Reduced magnesium level for male and female rats fed the high dietary level, and female rats fed the intermediate dietary level.
- Reduced creatinine and gamma-GT/creatinine excretion for male rats fed the high dietary level.
- Increased NAG activity and NAG/creatinine excretion for female rats fed the high dietary level.
- Increased AAP/creatinine excretion for female rats fed the high dietary level.
- The zinc excretion was 28 and 27 fold increased for male and female rats, respectively at the high dose level, and 4 fold increased for male rats at the intermediate dose.
Treatment related statistically significant findings at the 7 week sampling consisted of:
- Reduced chloride level for female rats at the intermediate concentration.
- Reduced calcium and magnesium level for female rats at the intermediate dose level and reduced magnesium level for females at the low concentration.
- The zinc excretion was 2 fold increased for male and female rats at the intermediate dose level, being statistically significant for males only.
At the 13 week sampling interval no statistically significant differences were seen.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant differences in organ weights or relative organ weights were seen for male and female rats fed with the test substance when compared with control rats after 13 weeks treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Decedents: at necropsy, abdominal fat was absent from all male rats and from most of the female rats. Intestinal contents were yellow. In males the testes, prostate and seminal vesicles were small and the testes were also flaccid. In female rats the uterus was smalI. Enlargement of the mesenteric Iymph nodes and slight pitting of the surface of the kidneys were noted. Fractures of the limbs were found in one male and four female rats and these were considered to be contributory to the poor health of the animals with subsequent saerifice and also related to treatment.
Terminals: at neeropsy, a dose-related reduction in the quantity of abdominal fat in male rats fed Zinc monoglycerolate was noted. Caecal contents were yellow-brown or yellow-green in rats fed 0.20% or 0.05% of Zinc monoglycerolate respectively. In addition, enlargement of the mesenteric Iymph nodes was apparent in six out of 20 male rats fed 0.20% Zinc monoglycerolate and in one male rat fed 0.05% Zinc monoglycerolate.

Neuropathological findings:

not examined

Description (incidence and severity):

Premature deaths
Microscopic findings considered to be of toxicological significance were observed in the pancreas, bones, incisor teeth, eyes, kidneys, spleen, skin, tongue, hard palate, oesophagus, stomach, caecum, larynx, lymphoid tissues, adrenal glands and reproductive organs. The pancreatic degeneration was considered to be contributory to the poor health of the animals. The pathological changes observed in the pancreas, bones, incisors, eyes, kidneys and spleen were considered to be related directly to the feeding of the high dietary level of test substance. A detailed description of findings upon histopathology is given in any other information on results.
At termination following treatment-related microscopic findings were noted in the pancreas, spleen, mesenteric lymph nodes, femur and tibia:
Pancreas - an increase in the incidence and severity of focal acinar degeneration and single cell necrosis was noted in male and female rats fed 0.2% test substance. A slight increased focal acinar degeneration was apparent also in rats fed 0.05% (details see Table 3).
Spleen - a decreased number of pigmented macrophages was observed in the red pulp of the spleen of male and female rats fed 0.2% test substance. This reduction was present also in male rats which received 0.05% test substance in diet (details see Table 3).
Mesenteric lymph nodes - a slight increase in the degree of histiocytosis was apparent in male rats which received 0.2% test substance in diet. This increase was neither seen in female rats from this group, nor in male or female rats fed 0.05%.
Femur and Tibia - A reduced number of trabeculae in the metaphysis of the tibia was seen in 5 males and 3 females fed with 0.2% test substance in diet. Of these rats, 4 males and 1 female had a similar reduction in the metaphysis of the femur.
The histiocytosis in the mesenteric Iymph nodes was considered to be a physiological response to the oral administration of a xenobiotic rather than to a direct toxic effect of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Mineral analysis of feces
The mean phosphorus level of 2.95% for rats fed the high dietary level of the test substance was increased when compared to control (2.44%). Zinc levels increased with increasing doses in the diet (0.24, 0.79 and 3.1 %; mean of both sexes at 0.05, 0.2 and 1% test substance in diet).

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: corresponding to 33.7 mg/kg calculated by the mean daily ingestion level

Dose descriptor:

LOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Degeneration in the pancreas/bone and a reduced number of pigmented macrophages in the spleen were the most prominent effects at 0.2% test substance in diet.

Remarks on result:

other: corresponding to 136.7 mg/kg calculated by the mean daily ingestion level

Critical effects observed:

not specified

Table 1: Body weight gain in gram
Week	Sex	Dose (% of test substance in diet)
		0	0.05	0.2	1 / 0.5%
0-4	Male	113.4	117.5	118.6	12.9***
	Female	46.1	49.5	46.3	1.5***
4-8	Male	56.4	55.5	49.1	1.8***
	Female	23.7	27.4	28.5	0.9***
8-13	Male	28.0	33.6	30.7	n.A.
	Female	19.1	19.0	18.3	n.A.
0-13	Male	199.1	205.2	197.6	n.A.
	Female	88.3	94.1	91.2	n.A.
***, p<0.001 (Dunnett’s Test); n.A.,not Applicable

Table 2: Accumulated food intakes in gram
Week	Sex	Dose (% of test substance in diet)
		0	0.05	0.2	1 / 0.5%
0-4	Male	563.7	566.2	575.6	409.9***
	Female	395.1	397.0	397.5	323.1***
4-8	Male	579.1	580.0	579.2	369.7***
	Female	408.5	418.3	420.3	327.3***
8-13	Male	706.7	709.1	700.4	n.A.
	Female	515.5	531.8	534.6	n.A.
0-13	Male	1849.5	1855.4	1855.3	n.A.
	Female	1319.1	1347.1	1352.3	n.A.
Food efficiency (=BW gain/Food intake)
0-4	Male	0.2	0.207	0.205	0.029***
	Female	0.115	0.123	0.116	0.005***
4-8	Male	0.098	0.094	0.084*	0.001***
	Female	0.056	0.064	0.064*	0.011***
8-13	Male	0042	0.045	0.043	n.A.
	Female	0.036	0.032	0.031	n.A.
0-13	Male	0.107	0.111	0.107	n.A.
	Female	0.067	0.070	0.067	n.A.
*, p<0.05; ***, p<0.001(Dunnett’s Test); n.A., not Applicable

Table 3: Details on main histopathological findings in pancreas and spleen
	No. of animals				Severity score
Dietary level (%)	0		0.05	0.2	0	0.05	0.2
MALES
No. with focal acinar degeneration	1	3		7	0.2	0.4	0.6
No. with pigmented macrophages	20	20		20	3.2	2.6	2.1
FEMALES
No. with focal acinar degeneration	0	1		9	0	0.1	0.5
No. with pigmented macrophages	20	20		20	3.7	3.9	3.0
A total of 20 animals were examined. Severity scores were allocated on a numerical scale of 0 to 5. Group means are calculated as the group severity total divided by the number of animals in that group with that tissue examined.

Microscopic findings in premature deaths at the high dose level considered to be of significance:

Pancreas - diffuse acinar degeneration associated with a mononuclear cell infiltration, fibroplasia and many mitotic figures was observed in all male and female rats.

Bones - in all of the rats examined, the femur, the tibia and the bones of the skull showed deposits of osteoid, mainly diffuse but occasionally focal. In addition, the cortices of the femur and tibia were thin in a number of rats and 6 fractures were identified. The epiphyses of these bones also showed distortion and thickening and there was a reduction in the number of trabeculae in the metaphysis.

Eyes - retinal atrophy was present in the eyes of all male and female rats.

Kidneys - degeneration of the tubular epithelium, sometimes associated with the presence of dilated tubules, was noted in the kidneys of all male and female rats.

Spleen - the number of pigmented macrophages observed was considered to be less than that in untreated rats of this age and strain.

Skin - hyperkeratosis was noted in all males and females.

Incisors - abnormal growth of the incisors, characterised by layers of dentine showing incomplete mineralisation, and accompanied by necrosis of the pulp, was noted in a large proportion of male and female rats.

Tongue - epithelial hyperplasia was apparent in all rats examined. In addition, 1 male rat had an ulcerated inflammatory lesion of the mucosa.

Hard palate - epithelial hyperplasia was observed in all male and female rats and an ulcerated inflammatory lesion was present in 1 female rat.

Oesophagus - hyperkeratosis of the epithelium was apparent in 8 males and 4 females.

Stomach - epithelial hyperplasia was noted in the fore stomach of 12 male and 12 females. In the glandular mucosa, erosion or ulceration of the superficial epithelium was present in 4 males and 1 female and recent haemorrhage was observed in 7 males and 2 females. In addition, all rats showed an infiltration of eosinophils, mainly in the lamina propria, and 4 male rats had foci of metaplasia characterised by the presence of Paneth cells in the glandular mucosa.

Caecum - an infiltration of eosinophils was present in the lamina propria of 16 males and 18 females. This was accompanied by single cell necrosis in 2 males and 1 female.

Larynx - an ulcerated inflammatory lesion was noted in 1 female rat.

Lymphoid tissues - lymphocytolysis was apparent in the cervical and mesenteric lymph nodes, the thymus and the spleen. In addition, a generalised depletion of lymphoid elements was noted in the mesenteric lymph nodes, thymus and spleen of a number of animals. Histiocytosis was prominent in the mesenteric lymph nodes.

Adrenal glands - vacuolar degeneration of the adrenal cortical cells occurred in 14 out of 20 male rats but was not apparent in any of the females.

Reproductive organs - in the testes of all male rats, hypoplasia of the seminiferous tubules was noted to a varying degree and this was associated with the absence of spermatozoa or the presence of spermatogenic cells in the epididymal tubules. In addition, the prostate and seminal vesicles showed hypoplasia and scant secretion.

In all but one female rat, the uterus was hypoplastic and the vagina showed an infiltration of neutrophils. An assessment of the stage of the oestrous cycle identified a preponderance of rats in dioestrus.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Bone marrow investigated in the course of this study was harvested from animals of a 90 d repeated dose toxicity feeding study conducted according to OECD guideline 408 (ESL FT930588, IUCLID Chapter 7.5.1). Animals of this study were taken to assess the potential of the test substance to induce genotoxicity in vivo by means of a micronucleous test. The conduct of the micronucleous test was in principle similar to OECD 474. It was stated in the report that the bone marrow micronucleus test, as described in this study, is an equally sensitive alternative to an (more common) acute micronucleus test. This was validated by MacGregor et al. (Fundamental and Applied Toxicology, 14,513-522, 1990) and Henderson, L. et al. (Mutation Research, 291, 79-85, 1993) for subacute 28 d studies. However, validity for a subchronic 90d study was not given. It was further stated that Wistar rats have been used in the course of this study, while the study report of the subchronic toxicity study mentioned above was performed with Sprague-Dawley rats.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

EC Number:

700-107-9

Cas Number:

87189-25-1

Molecular formula:

(C3H6O3Zn)n

IUPAC Name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

Details on test material:

White Powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 5-6 weeks, three females treated with the positive control were 21 weeks old.
- Weight at study initiation: 100 - 200 g
No further details are given in the study report. For further details see study FT930588 in IUCLID Chapter 7.5.1.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

The bone marrow investigated in the course of the study was harvested from male and female Wistar rats fed with the test substance at 0, 0.05, 0.2 and 1 % in purified diet over a 13 week period as part of study FT930588 (see IUCLID Chapter 7.5.1). The experimental diets in the mentioned study were based on the ESL Modified AIN-76A diet and were prepared weekly.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

The animals were given free access to the test diets at all times.

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Doses / concentrations 3

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

Treatment groups: 20 (10 animals per sex per dose were investigated in the course of this study)
Positive control group: 7
Bone marrow of 10 animals of each sex was investigated at 0 and 0.2% and the bone marrow of 5 animals of each sex was investigated at 0.05% and positive control.

Control animals:

yes

Positive control(s):

Cyclophosphamide was used as positive control. The dosing solution was prepared immediately before use by diluting in 0.9% physiological saline.
Seven male and seven female rats were dosed with cyclophosphamide during the final week of the live animal phase for this study (1994-02-01 to 1994-05-06). The positive control rats were dosed by oral gavage at 20mg/kg bw/d in an application volume of 10ml/kg bw. The animals were sacrificed 24 hours after treatment and bone marrow slides were prepared. The male rats and four of the female rats were spare animals from the feeding study (see study FT930588 in IUCLID Chapter 7.5.1). Three females were from the palatability study (see study ESL FP930559 in IUCLID Chapter 7.5.1) and were four weeks older at the time of dosing with the positive control (21 weeks).

Examinations

Tissues and cell types examined:

The femoral bone marrow was examined for incidence of micronuclei in both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) together with the PCE to NCE ratio. The PCE/NCE ratio was recorded when 2000 of either PCEs or NCEs had been scored.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The dose selection was based on the results of a 14 day palatability study (ESL study FP930559).

TREATMENT AND SAMPLING TIMES
Bone marrow slides were prepared from 10 males and 10 females from each treatment group, and initially slides from 5 males and 5 females from each group were scored. Due to considerable variation in individual rat data at the 0.2% dose group further 5 males and 5 females were analysed from the 0 and 0.2 % dose. Rats of the top dose group were sacrificed during the study due to brittle bones and consequently were not assessed for micronuclei induction.

DETAILS OF SLIDE PREPARATION
The bone marrow was transferred from the femur to a clean glass slide by the use of a moistened paint brush and slides were prepared by carefully stroking the slide a few times with the brush. Two bone marrow smears were prepared from each femur. The smears were air dried, fixed in methanol and stained using a Feulgen staining procedure. All slides were dried and mounted with DPX.

METHOD OF ANALYSIS
One slide prepared from the femur of each test animal was examined by light microscopy. From each slide the incidence of micronuclei in both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined together with the PCE to NCE ratio. The PCE/NCE ratio was recorded when either 2000 of either PCEs or NCEs had been scored.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Rats of the high dose group had to be sacrificed for humane reasons. The PCE/NCE-Ratio was, however, not affected by the treatment.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initially, the mean number of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of females treated with 0.2% test substance was doubled over the control value for the females (1.4 vs. 3.8, see table 2). Since, the individual rat data varied considerably at this dose level another five males and five females from the control group and the 0.2% dose group were analysed. The doubling of MPEC over the control value was not seen in the second assessment (combined results 2.4 vs. 3.6) and the doubling of MPECs seen in the first analysis was considered to be due to the low incidence of micronuclei in the first five control females. A summary of results is given in table 1.
The mean number of micronucleated PCEs in bone marrow of animals treated with the positive control was 25.4 and 18.6 for females and males, respectively.
The top dose assessed for micronuclei was limited due to systemic toxicity. Animals of the high dose group were sacrificed due to humane reasons (reported in ESLstudy FP930588). No deaths occurred in the lower dose groups.
The PCE/NCE-ratio indicated no evidence of cytotoxicity.

Any other information on results incl. tables

Table 1: Summary of Results
Dose	PCE/NCE (Mean)		MPCE incidence (%)		MNCE incidence (%)
	Male	Female	Male	Female	Male	Female
0 %	0.99	0.99	0.11	0.12	0.01	0.01
0.05 %	0.98	1.13	0.14	0.10	0.01	0.00
0.2 %	1.00	0.99	0.12	0.18	0.01	0.01
Positive Control	0.9	1.0	1.27	0.93	0.01	0.01

Table 2: Details on Analysis 1 and 2 of animals fed with 0 and 0.2% of test substance in the diet
	PCE/NCE-Ratio		MPCEs			N (NCE)
Analysis	I	II	I		II	I	II
0 % (5 Males)	0.84	1.07	4		3	2000	1875
	0.88	1.12	1		2	2000	1788
	1.05	0.93	0		2	1903	2000
	1.05	0.89	2		4	1898	2000
	1.01	1.10	2		2	1990	1812
Mean	0.97	1.02	1.8		2.6	1958.2	1895
Analysis	I	II	I		II	I	II
0 % (5 Females)	0.95	0.89	2		4	2000	2000
	1.28	0.87	2		3	1567	2000
	1.11	1.00	0		3	1802	1992
	1.17	0.83	1		4	1707	2000
	1.02	0.78	2		3	1960	2000
Mean	1.11	0.87	1.4		3.4	1807.2	1998.4
	PCE/NCE-Ratio		MPCEs			N (NCE)
Analysis	I	II	I	II		I	II
0.2 % (5 Males)	1.06	0.92	1	4		1889	2000
	0.78	0.97	3	2		2000	2000
	1.19	0.88	5	3		1675	2000
	1.12	1.08	1	2		1780	1846
	0.98	0.99	2	1		2000	2000
Mean	1.03	0.97	2.4	2.4		1868.8	1969.2
Analysis	I	II	I	II		I	II
0.2% (5 Females)	1.35	0.96	6	4		1481	2000
	0.83	1.04	4	3		2000	1928
	1.17	0.85	2	5		1709	2000
	0.88	0.89	1	3		2000	2000
	1.00	0.94	6	2		2000	2000
Mean	1.05	0.95	3.8	3.4		1838	1992.4