Cobalt Lithium Manganese Nickel Oxide

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:

8 May 2007 to 11 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-390-0

EC Name:

-

Cas Number:

182442-95-1

Molecular formula:

LiCoNiMnO2 with the stoechiometry of Co+Ni+Mn equal to 1 and the ranges of the elements approximately as: Li: >0.90 - <1.20 Co: >0.0 - <0.50 Ni: >0.20 - <0.98 Mn: >0.0 - <0.50 O: 2

IUPAC Name:

Cobalt Lithium Manganese Nickel Oxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Cellcore MX
- Physical state: solid
- Appearance: black powder
- Storage condition of test material: room temperature, in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD) IGS BR
- Age at study initiation: 5 - 8 weeks old
- Weight at study initiation: 135 - 171 g (males); 117 - 148 g (females)
- Housing: animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: pelleted diet, ad libitum
- Water: ad libitum access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 % (relative)
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (low intensity fluorescent lighting)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Arachis oil BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a suspension in vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 0, 3.75, 37.5 and 62.5 mg/mL
- Amount of vehicle (if gavage): The volume of test and control material administered to each animal (4 mL/kg) was based on the most recent body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test material formulation were taken weekly for analysis.

- Samples: The test material formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test material residue. The samples were then dried in an oven at approximately 105 °C then allowed to cool over silica gel in a desiccator and re-weighed.

- Homogeneity Determinations: The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.

- Verification of Test material Formulation Concentration: The test material formulations were sampled and analysed within two days of preparation. However, due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test material in the test material formulations was determined using a gravimetric technique.

The analytical method has been considered to be sufficiently accurate for the purpose of this study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 15, 150 and 250 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a 14-day repeated dose range-finding study in which rats were dosed with the test material at 0, 250, 500 and 1000 mg/kg bw/day for up to 14 days. Mortality and clinical signs of toxicity were noted in the rats dosed at 500 and 1000 mg/kg bw/day. 250 mg/kg bw/day was therefore selected as the top dose for the 28-day study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on days 4, 11, 18 and 25, all animals were observed for signs of behavioural toxicity. Observations were carried out from approximately 2 hours after doing on each occasion.
- Behavioural Assessments: Detailed individual clinical observations were performed to assess behaviour for each animal using a purpose built arena. The following parameters were observed: gait, tremors, twitches, hyper/hypothermia, skin colour, respiration, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, palpebral closure, urination, defecation, transfer arousal and tail elevation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes
Food efficiency was determined as follows:
% Food Efficiency = [Group mean bodyweight gain (g/rat/week) / Group mean food consumption (g/rat/week)] x 100

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes, except during Week 3 when water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All surviving animals
- Parameters checked included: haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), total leucocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), platelet count (PLT) and reticulocyte count (Retic)
- Additionally, prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.
- Animals fasted: No
- How many animals: All surviving animals
- Parameters checked included: urea, glucose, total protein (Tot.Prot.), albumin, albumin:globulin (A/G) ratio (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol) and total bilirubin (Bili).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional toxicity was assessed prior to the start of treatment and on days 4, 11, 18 and 25. Functional performance tests were also performed on all animals during week 4 together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately 2 hours after dosing on each occasion.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity (grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex), forelimb/hindlimb grip strength and motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: Yes
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin: adrenals, aorta (thoracic*), bone and bone marrow (femur including stifle joint, sternum)*, brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes*, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), mesenteric and cervical lymph nodes, muscle (skeletal)*, oesophagus*, ovaries, pancreas*, pituitary*, prostate, rectum, salivary glands (submaxillary)*, sciatic nerve, seminal vesicles, skin (hind limb)*, spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

With the exception of tissues marked with *, the tissues from all control and 250 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.
As there were indications of treatment-related changes, examination was extended to include similarly prepared sections of spleen kidney and bone marrow (sternum) from all animals in other treatment groups.

Statistics:

Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths attributed to test material toxicity. One male treated with 15 mg/kg/day was found dead pre-dose on day 28 and the cause of death was unknown following histopathological examination. There were no further unscheduled deaths during the study.
No clinically observable signs of toxicity were detected. Incidental findings of increased salivation up to ten minutes post dose were detected in animals of either sex treated with 250 and 150 mg/kg/day with an isolated incidence also observed in one 15 mg/kg/day male on day 28 only, however this was not considered to be an indication of systemic toxicity. In addition an episode of fur stained black by the test material was evident in one 250 mg/kg/day female and one 150 mg/kg/day male, with red/brown staining around the mouth noted in one 250 mg/kg/day male and staining of the test material around the mouth seen in one 150 mg/kg/day male. Again, this was considered to be incidental and not indicative of toxicity. No such effects were detected for females treated with 15 mg/kg/day.
No clinical observations were seen for the interim male animal prior to death on day 28.

BODY WEIGHT AND WEIGHT GAIN
Males treated with 250 and 150 mg/kg/day showed an overall reduction in bodyweight gain throughout the treatment period, however statistical significance was only achieved for bodyweight gain for the 250 mg/kg/day males during week 1 (p<0.01). A slight but statistically significant reduction in bodyweight gain was also detected throughout the female treatment groups in comparison to controls during week 1 (p<0.05). Recovery was evident thereafter and as such the effects in the females were considered not to represent an adverse effect. No such effect was detected for males treated with 15 mg/kg/day.

FOOD CONSUMPTION
Males treated with 250 and 150 mg/kg/day showed a reduction in dietary intake throughout the treatment period, however food efficiency was not similarly affected. No adverse effect on dietary intake was detected for females treated with 250 or 150 mg/kg/day or for animals of either sex treated with 15 mg/kg/day.

WATER CONSUMPTION
Daily visual inspection of water bottles and daily gravimetric measurements undertaken during week 3 of treatment did not reveal any inter-group differences for treated animals when compared to controls.

HAEMATOLOGY
A microcytic, hyperchromic polycythemia was evident for animals of either sex treated with 250 and 150 mg/kg/day. This was characterised by increases in haemoglobin, erythrocyte and haematocrit counts (males: p<0.01, females: p<0.05-0.01) and increases in reticulocyte counts (p<0.01). Furthermore, a reduction in mean corpuscular haemoglobin concentration was evident for males treated with 250 mg/kg/day whilst females treated at this dose level showed a reduction in mean corpuscular haemoglobin and mean corpuscular volume (p<0.05).
An increase in activated partial prothrombin time was evident for animals of either sex treated with 250 mg/kg/day (males p<0.01, females p<0.05) and a reduction in platelet count was also observed for animals of either sex treated with 250 mg/kg/day and females treated with 150 mg/kg/day, however, statistical significance was only achieved for females (p<0.01).
No such effects were detected in animals of either sex treated with 15 mg/kg/day.

CLINICAL CHEMISTRY
Increases in aspartate aminotransferase were evident for animals of either sex treated with 250 and 150 mg/kg/day, however, statistical significance was only achieved for males (p<0.05). An increase in alkaline phosphatase was also apparent for males treated with 250 mg/kg/day (p<0.01) and 150 mg/kg/day (p<0.05). Slight increases in alanine aminotransferase were also observed for males from the 250 and 150 mg/kg/day dose groups, however, statistical significance was not achieved. Furthermore, statistically significant reductions in cholesterol were evident for males treated with 250 and 150 mg/kg/day (p<0.01), with some values outside of the normally expected ranges for these parameters.
Males treated with 250 and 150 mg/kg/day showed a statistically significant reduction in total protein (p<0.01), with the effect extending into the 15 mg/kg/day male dose group (p<0.05). Corresponding reductions in plasma albumin levels were also evident throughout the male treatment groups when compared to controls (p<0.01). A statistically significant reduction in bilirubin levels (p<0.05) was also evident in males treated with 250 and 150 mg/kg/day. No such effects were evident for these parameters for treated females when compared to controls; however a reduction in calcium levels was evident throughout the female treatment groups in comparison to controls, with a number of individual values outside of the normally expected ranges for this parameter. The remaining electrolyte changes consisted of an increase in plasma chloride observed for animals of either sex treated with 250 and 150 mg/kg/day (males: p<0.05, females: p<0.01), extending into the 15 mg/kg/day female dose group (p<0.05).
Males treated with 250 and 150 mg/kg/day showed a statistically significant reduction in inorganic phosphorus levels (p<0.05). All values were within the normal range for rats of the strain and age used and in the absence of any supporting findings this is considered to be of no toxicological significance. A slight but statistically significant reduction in plasma glucose levels (p<0.05) was seen in female 250 mg/kg/day animals with one individual value below the expected normal range for rats of the strain and age used. However, in the absence of a reduction in dietary intake this finding is considered to be of no toxicological importance.

NEUROBEHAVIOUR
There were no treatment-related changes in the behavioural parameters measured. All intergroup differences in urination, defecation and transfer arousal scores were considered to be a result of a normal variation for rats of the strain and age used and were of no toxicological significance. There were no toxicologically significant changes in the functional performance parameters measured. A statistically significant reduction in forelimb grip strength was seen throughout the female treatment groups in comparison to controls (p<0.01). The reduction was observed in only one out of three tests performed on this parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, this finding is considered to be of no toxicological significance. There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and ages used, and were of no toxicological importance.

ORGAN WEIGHTS
There were no toxicologically significant changes detected. A slight but statistically significant reduction in adrenal weight (both absolute and relative to terminal bodyweight) was observed in males treated with 250 mg/kg/day (p<0.05). All individual values were within the normal ranges for rats of the strain and age used and in the absence of any histopathological correlates this finding is considered to be of no toxicological importance. No such effects were detected in females treated with 250 mg/kg/day or animals of either sex treated with 150 and 15 mg/kg/day.

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected at terminal kill. Findings were confined to a dark, enlarged and malformed spleen seen in one 150 mg/kg/day male and hydronephrosis of the right kidney evident for one control female. These were isolated and considered to be low incident findings in laboratory maintained animals. The interim death 15 mg/kg/day male displayed reddened lungs, ileum and a dark liver. In isolation and in the absence of any histopathological correlates there is insufficient evidence to attribute the death of the animal to the effect of the test material.
No such effects were observed in animals of either sex treated with 250 mg/kg/day or females treated with 150 and 15 mg/kg/day.

HISTOPATHOLOGY
The following treatment-related changes were observed:
SPLEEN: A greater incidence of higher grades of severity of extramedullary haemopoiesis was seen in animals of either sex treated with 250 mg/kg/day and males and possibly also for females treated with 150 mg/kg/day.
BONE MARROW: A lower incidence of higher grades of severity of adipose infiltration, indicative of increased cellularity of the marrow, was seen as a consequence of treatment in animals of either sex treated with 250 mg/kg/day together with females and possibly males treated with 150 mg/kg/day.
KIDNEY: Basophilia of tubules of the inner cortex was seen in males treated with 250 and 150 mg/kg/day. Associated karyomegaly of tubular cells was also seen in two 250 mg/kg/day males and one 150 mg/kg/day male.
No such effects were detected for animals of either sex treated with 15 mg/kg/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced food consumption and bodyweight gain, polycythaemia and associated splenic pathology, perturbations in clincial chemistry, kidney pathology.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Group 1 = 0 mg/kg/day (control); Group 2 = 15 mg/kg/day; Group 3 = 150 mg/kg/day; Group 4 = 250 mg/kg/day.

Table 1: Group Mean Body Weight Gains (g)

Group (Sex)	Base weight week 1 (g)	Mean increase in body weight between weeks (g)				Absolute gain (g)	% Gain
		1-2	2-3	3-4	4-5	Weeks 1-5	Weeks 1-5
1 (M)	156.8	64.6	64.4	49.2	28.8	207.0	132.4
2 (M)	154.4	66.2	62.8	51.0	36.3	205.8	138.7
3 (M)	152.6	55.4	56.6	37.2	25.0	174.2	114.1
4 (M)	153.2	44.4**	59.0	39.4	26.6	169.4	110.9
1 (F)	132.6	35.8	20.2	20.2	12.8	89.0	67.3
2 (F)	140.4	27.2*	26.6	17.0	12.0	82.8	59.0
3 (F)	133.0	26.0*	23.8	16.4	10.6	76.8	57.8
4 (F)	135.4	27.2*	25.2	21.0	12.8	86.2	64.0

* p < 0.05

** p < 0.01

Table 2: Group Mean Haematological Values

Group (Sex)	Hb (g/dL)	RBC (10¹²/L)	Hct (%)	MCH (pg)	MCV (fl)	MCHC (g/dL)	WBC (10⁹/L)	Neut (10⁹ /L)	Lymph (10⁹ /L)	Mono (10⁹ /L)	Eos (10⁹ /L)	Bas (10⁹ /L)	CT (s)	PLT (10⁹ /L)	APTT (s)	Retics (%)
1 (M)	14.82	7.528	44.72	19.72	59.44	33.18	11.98	1.350	10.608	0.000 n	0.024	0.000 n	24.10	864.2	16.06	7.28
2 (M)	15.03	7.318	45.38	20.53	62.05	33.08	10.50	1.418	9.023	0.000 n	0.058	0.000 n	24.23	803.3	15.15	7.88
3 (M)	17.04**	8.690**	52.20**	19.62	60.02	32.72	11.68	2.392	9.266	0.000 n	0.024	0.000 n	26.68	823.0	16.56	10.40**
4 (M)	16.88**	9.092**	52.12**	18.62	57.46	32.34**	13.30	1.354	11.914	0.000 n	0.032	0.000 n	27.00	779.8	17.78**	9.38**
1 (F)	14.26	7.234	42.34	19.74	58.58	33.68	9.22	1.176	8.010	0.000 n	0.034	0.000 n	25.72	901.4	15.26	6.02
2 (F)	14.64	7.414	43.22	19.76	58.30	33.88	9.58	0.878	8.650	0.000 n	0.056	0.000 n	28.48	788.6	15.94	6.60
3 (F)	15.86*	8.034*	47.32	19.76	58.84	33.56	7.30	0.926	6.330	0.000 n	0.044	0.000 n	26.52	671.0**	15.70	8.52**
4 (F)	16.36*	8.852**	49.32*	18.54*	55.40*	33.48	9.18	1.550	7.602	0.000 n	0.032	0.000 n	24.12	600.6**	16.84*	9.54**

* p < 0.05

** p < 0.01

n = Data not appropriate for statistical analysis

Hb = Haemoglobin; RBC = Total erythrocyte count; Hct = Haematocrit (Hct); MCH = Mean Corpuscular Haemoglobin; MCV = Mean Corpuscular Volume; MCHC = Mean Corpuscular Haemoglobin Concentration; WBC = Total leucocyte count; Neut = Neutrophils; Lymph = Lymphocytes; Mono = Monocytes; Eos = Eosinophils; Bas = Basophils; CT = Prothrombin time; PLT = Platelet count; APTT = Activated partial thromboplastin time; Retics = Reticulocyte count.

Table 3: Group Mean Blood Chemical Values

Group (Sex)	Urea (mg/dL)	Glucose (mg/dL)	Tot. prot. (g/dL)	Albumin (g/dL)	A/G ratio	Na^+ (mmol/L)	K^+ (mmol/L)	Cl^- (mmol/L)	Ca^2+ (mmol/L)	P (mmol/L)	ASAT (IU/L)	ALAT (IU/L)	AP (IU/L)	Creat (mg/dL)	Chol (mg/dL)	Bili (mg/dL)
1 (M)	27.6	164.6	7.122	4.320	1.542	147.6	4.556	99.8	2.462	2.580	81.4	48.8	325.0	0.734	68.2	0.260
2 (M)	31.0	166.5	6.765*	4.063**	1.533	146.3	4.648	100.0	2.485	2.650	94.0	49.0	355.3	0.708	67.5	0.235
3 (M)	24.6	165.4	6.610**	4.030**	1.564	144.8	4.450	104.4*	2.262	2.300*	110.0*	56.6	429.0*	0.742	51.0**	0.128*
4 (M)	23.8	152.0	6.556**	4.040**	1.614	146.2	4.598	104.2*	2.312	2.360*	109.4*	56.4	488.4**	0.742	46.6**	0.144*
1 (F)	29.0	158.8	6.570	4.190	1.772	148.6	4.416	102.0	2.694	1.980	84.4	45.6	232.4	0.806	59.6	0.078
2 (F)	32.4	160.0	6.652	4.338	1.876	149.4	4.238	105.0*	2.532*	2.020	82.8	39.8	259.0	0.834	59.2	0.042
3 (F)	31.4	155.6	6.758	4.336	1.804	147.4	4.280	107.2**	2.376**	2.020	91.4	44.2	337.2	0.804	53.6	0.084
4 (F)	27.2	141.8*	6.928	4.336	1.686	148.4	4.494	109.2**	2.422**	1.920	120.6	47.2	277.0	0.818	63.4	0.082

* p < 0.05

** p < 0.01

Tot.Prot. = Total protein; Albumin/Globulin (A/G) ratio = albumin / (total protein - albumin); Na+ = Sodium; K+ = Potassium; Cl- = Chloride; Ca2+ = Calcium; P = Inorganic phosphorus; ASAT = Aspartate aminotransferase; ALAT = Alanine aminotransferase; AP = Alkaline phosphatase; Creat = Creatinine; Chol = Cholesterol; Bili = Total bilirubin.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level was considered to be 15 mg/kg bw/day.

Executive summary:

The repeated dose toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 407 and EU Method B.7.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for up to 28 consecutive days, at dose levels of 15, 150 and 250 mg/kg bw/day in Arachis oil BP. A control group of five male and five female rats was dosed with vehicle alone.

Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths attributed to test material toxicity and no clinically observable signs of toxicity were detected. There were no treatment-related changes in the functional performance or behavioural parameters measured.

Males treated with 250 and 150 mg/kg/day showed a reduction in bodyweight gain throughout the treatment period. A reduction in bodyweight gain was also detected throughout the female treatment groups during week 1 only. No such effect was detected for males treated with 15 mg/kg/day.

Males treated with 250 and 150 mg/kg/day showed a reduction in dietary intake throughout the treatment period, in comparison to controls. No adverse effects on dietary intake were detected for females treated with 250 or 150 mg/kg/day or for animals of either sex treated with 15 mg/kg/day. No adverse effect on water consumption was detected for treated animals in comparison to controls.

Microcytic, hyperchromic, polycythemia was detected in animals of either sex treated with 250 and 150 mg/kg/day. No such effects were detected in animals of either sex treated with 15 mg/kg/day.

A reduction in albumin levels was observed for treated males when compared to controls, together with reductions in total protein, bilirubin and cholesterol levels observed in the 250 and 150 mg/kg/day males. In addition, a reduction in calcium levels was evident throughout the female treatment groups. Furthermore animals of either sex treated with 250 and 150 mg/kg/day and female 15 mg/kg/day animals showed an increase in chloride levels with an increase in aspartate aminotransferase levels also detected in animals of either sex treated with 250 and 150 mg/kg/day. In addition, an increase in alkaline phosphatase and alanine aminotransferase levels was also evident in the 250 and 150 mg/kg/day males.

There were no toxicologically significant changes detected in organ weights and no treatment-related macroscopic abnormalities were detected at terminal kill. Histological examination of the tissues revealed treatment-related changes to the spleen, bone marrow and kidney.

The study revealed that administration of the test material to rats for a period of 28 days at dose levels of up to 250 mg/kg bw/day resulted in toxicologically significant effects at 250 and 150 mg/kg bw/day. The effects seen at 15 mg/kg bw/day were considered not to represent an adverse effect and therefore, the No Observed Adverse Effect Level was considered to be 15 mg/kg bw/day.