Sodium polysulfide aluminosilicate with a SOD-type framework structure

Endpoint summary

Description of key information

Key studies:

- 90-day study: Test method is similar to OECD guideline 408. No GLP study.

- Combined repeated dose and reproduction / developmental screening: OECD guideline 422. GLP study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1965

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test method is similar to OECD guideline 408. No GLP study.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 21 days
- Weight at study initiation: 39-47 g
- Housing: rats of the same sex were placed in pairs in individual galvanised wire mesh cage
- Diet (e.g. ad libitum): oxoid breeding diet

ENVIRONMENTAL CONDITIONS
- Temperature: 70±1ºF
- Humidity (%): greater than 50%

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

-All drug diets were mixed in batches of 20 Kg at approximately 14 day intervals with unpelleted Oxoid breeding diet.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

91 days

Frequency of treatment:

Every day

Dose / conc.:

0 ppm

Remarks:

controls, diet only

Dose / conc.:

100 ppm

Remarks:

Doses / Concentrations:
approx. 12 mg/kg /day
Basis:
calculated as a mean from measured intakes in diet for males and females

Dose / conc.:

1 000 ppm

Remarks:

Doses / Concentrations:
approx. 115 mg/kg /day
Basis:
calculated as a mean from measured intakes in diet for males and females

Dose / conc.:

10 000 ppm

Remarks:

Doses / Concentrations:
approx. 1236 mg/kg /day
Basis:
calculated as a mean from measured intakes in diet for males and females

Dose / conc.:

100 000 ppm

Remarks:

Doses / Concentrations:
approx. 13436 mg/kg /day
Basis:
calculated as a mean from measured intakes in diet for males and females

No. of animals per sex per dose:

1 group of 20 male rats: 100 ppm / 1 group of 20 female rats: 100 ppm
1 group of 20 male rats: 1000 ppm / 1 group of 20 female rats: 1000 ppm
1 group of 20 male rats: 10000 ppm / 1 group of 20 female rats: 10000 ppm
1 group of 20 male rats: 100000 ppm / 1 group of 20 female rats: 100000 ppm

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: - During the test period the rats were observed for any abnormal behaviour and obvious signs of ill health or untoward effects which could be atributed to feeding the test material.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-The food consumption was measured daily on 8 animals in each group. The test material intake as g/kg rat/day was calculated from the food consumption and body weight data.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken for haematological examination at the start of the experiment, at monthly intervals during the treatment period and immediately before autopsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: -Blood samples were taken for biochemical analysis at the end of the treatment period

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were taken one week before the end of the treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At the end of the treatment period the rats were killed, examined pathologically, and the main organs removed, weighed and prepared for histological examination. Animals which died during the test were sent for post mortem examination. Samples of bone marrow were taken at post mortem and examined microscopically.

Statistics:

Analysis of variance.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Of the 17 animals which died during the treatment period, only 7 were suitable for post mortem due to autolytic changes. Two of the animals fed the diet containing 100000 ppm and 2 fed the diet containing 10000 ppm were found to have inflammation of the bladder.

BODY WEIGHT AND WEIGHT GAIN
Analysis of variance was carried out on the data from each week of the test, as also was Rao´s analysis using time intervals of 1 day. A Rao´s analysis using a 7 day interval was carried out at the end of the test. As the rats were places in metabolism cages during the 12th week of the test, the growth weights were only taken for analysis up to the end of the 11th week of the test.

In comparison with the controls, growth was depressed in the high dosage group (100000 ppm) in both the males and the females, beginning at the end of the first week of the test. In the other dosage groups, the male rats if anything grew rather better than the controls but the differences were not significant. The growth rates of the female rats in all these groups were the same as the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption figures for the males do not account for the difference in growth rate between the control and the 100000 ppm and 10000 ppm dose level groups.

In the first 5 weeks the food consumption of the 100000 ppm group was less than all the other groups, and this accompanied by the fact that 10 of the intake had no nutritive value could well account for the decrese in growth. Towards the end of the test period the intake of the rats at this dose level was approximately the same as that of the other groups.

The food consumption figures for the females show an increase in food consumption of the 100 ppm dose level group and a decrease of the 100000 ppm over the controls during the first half of the test. This could account for the differences in observed growth rates.

HAEMATOLOGY
-Haemoglobin: the only significant findings were in the highest dosage group ( 100000 ppm) where in both the males and the females there was a slight but significant depression.
-Red cell count: no significant effects were observed at any dose level.
- Packed cell volume: in the males a highly significant decrease was observed at the 100000 ppm dose level at the end of the test period. A similar decrease was observed in the females at this dose level but it was not statistically significant.
- Mean corpuscular volume: at the end of the test period a significant decrease was observed in the males at the 100000 ppm dose level.
- Mean corpuscular haemoglobin: at the end of the test period a significant decrease was observed in the males at the 100000 ppm dose level.
- Mean corpuscular haemoglobin concentration: no differences of significance were observed.
- Reticulocyte counts: a very significant increase was observed in the males and a significant increase in the females at the 100000 ppm dose level.
- Total leucocyte count: an increase was observed at the 100000 ppm dose level in the females in the 4th, 8th and 12th weeks.
- Differential cell count:
Neutrophils: a highly significant increase occurred in the males at the 100000 ppm dose level in the 4th and 8th weeks but no differences were observed at the end of the test period.
Lymphocytes: a highly significant difference was observed in the males in the 8th week at the 100000 ppm dose level.
Eosinophils: no differences of significance were observed.
Basophils: no differences were observed.
Monocytes: no differences of significance were observed.

CLINICAL CHEMISTRY
- Blood urea: consistent results were obtained in the controls and all the treatment groups. A small but significant increase of the blood urea in the males on the higher dose level was not confirmed in the females. It is concluded that the test substance has no significant influence on the level of urea in the blood.
- Blood reducing sugar as glucose: The means were consistent in all treatment groups and there were no real differences between levels in the treated and control rats. It it concluded therefore that the chronic treatment of rats with the test substance does not affect the blood sugar levels.
- Serum inorganic phosphorus: the results were consistent in all groups. There was a slight but significant rise in this constituent at the highest dose level both in the males and females.
- Serum cholesterol: while the values were consistently higher in the females than the males there were no differences in the group values between the treated and control rats in either sex.
- Serum silica: there was not evidence that feeding of the test substance had any influence on the serum silica levels.
- Serum alkaline phosphatase: while the values were lower in females than in the males there were not significant differences between the controls and the treated groups in either sex.
- Serum transaminase: while the values were higher in the treated groups than in the controls the differences were small and of low significance.
- Serum protein and serum albumin: administration of the test substance had no influence on the serum protein and serum albumin levels.
- Serum sodium and serum potassium: there were no significant differences between the treated and control groups at all dosage levels.

URINALYSIS
- Urine volume: there was a highly significant increase in the males and a significant increase in the females at the top dosage group.
- Sodium excretion: there was a highly significant increase in sodium excretion in both the males and females at the top dosage groups.
- Potassium excretion: there was no significant effect on urinary potassium excretion at any dose level.
- Creatinine excretion: there was no significant difference between the treated and controls rats.
- Silica excretion: the levels of silica excreted in the urine were increased very significantly when feeding the test substance to rats and the increase was related to the dose.
- Qualitative tests: examination of the urines for sugars and ketones showed no difference between the control and treated groups. Deposits of siliceous material were found in the urine of a few of the animals at the two highest dose levels.

ORGAN WEIGHTS
No significant differences were observed in the following organs: adrenals, brain, gonads (ovaries, prostate and uterus), heart, pituitary, thymus, thyroid.
- Gonad - Testes: At the 1000 ppm dose level there was a significant increase in the actual weight as compared with the controls but these were not significant when the weights were expressed as a percentage of body weight.
- Kidneys: in the males there was a highly significant increase in the actual weight at the 100 ppm dose level when compared with the controls but the difference was not significant when the weights were expressed as a percentage of the body weight. In the females a significant decrease in the actual weights was observed at the 1000, 10000 and 100000 ppm levels but no difference or significance were observed when the weights were expressed as a percentage of the body weight.
- Liver: a significant increase in actual weight of the males and females was observed at the 100000 ppm dose level. These increases persisted when the weights were expressed as a percentage body weight.
- Lungs: in the females a significant increase in the actual weight was observed at the 100 and 1000 ppm dose levels and at the 100000 ppm dose level. These increases in weight still persisted when the weights were expressed as a percentage body weight. In the males a similar pattern was observed but the differences were not significant.
- Spleen: a very highly signficant increase in actual weights was observed in the males at the 100000 ppm dose level. This increase was accentuated when the weights were expressed as a percentage of the body weight. A similar pattern was observed in the females but the differences were not significant.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Bone marrow smears of animals from both the control and treated groups showed no significant differences. There were about an equivalent number of animals showing an increase in erythroid cells and in total eosinophil cells in all groups.
No pathological changes were observed at any dose level in any of the following: adrenals, brain, eye, uterus, pancreas, parathyroid, skin, thymus (males), thyroid (females).
- Bladder: inflammation of the bladder wall was observed at the 1000, 10000 and 100000 ppm dose levels, the incidence increasing with the dose. This was associated with the presence of small siliceous stones and deposits particularly at the high dose level.
- Colon: inflammation of the mucosa was a common finding in all groups including the controls. This could in part be accounted for by the nature of the compound administered and also to a worm infestation seen in the controls.
- Gonads: The incidence of these effects was of no significance.
Ovaries: one of the females on the 10000 ppm dosage level showed interstitial inflammation.
Testes: one of the control group showed atrophy and two at the 100000 ppm dosage level showed a depression of spermatogenesis.
Prostate: inflammation occurred in four of the controls, in 2 at the 100 ppm and 2 at the 100000 ppm dosage level groups.
- Heart: one of the males on the 1000 ppm dosage level and two of the females on the 100000 ppm showed a slight myocarditis. This was most likely due to an infection.
- Kidney: pyelonephritis was a common finding and occurred at all dosage levels and also in the controls. This is a common finding in laboratoy rats and is possibly due to a virus infection. It is not therefore regarded as being primarily due to the treatment of the rats with the test substance although there was evidence that it did increase in severity particularly at the high dose level.
- Liver: all the sections showed some glycogen depletion together with a slight fatty infiltration. This occurred in the controls as well as in the treated groups and was probably due to deprivation of food overnight before the rats were killed.
- Lung: sections of the lungs from the controls as well as the treated groups showed evidence of pneumonitis. This has been a constant finding in our rats and is no doubt due to an inherent infection.
- Lymph node: sinus catarrh was found in one female on the 100 ppm dose level and in one male on the 1000 ppm and one on the 100000 ppm dose level. These observations are of no significance.
- Pituitary: grannulation of the thyrotrophs was observed in one male in the control group and one on the 10000 ppm dose level.
- Small intestine: slight inflammation of the mucosa was a common finding at all dose levels including the controls. The effects were not severe and are to be expected from feeding this foreign material.
- Spleen: an increase in brown pigmented macrophages was observed in a male on the 1000 ppm dose level, in a control female and in 4 females on the 10000 pmm dose level. Vascular sclerosis was observed in a male in the 10000 ppm dose level. This was of no significance.
- Stomach: inflammation of the mucosa was observed particularly at the highest dosage level. This is to be expected following administration of this foreign material.
- Thyroid: in the males diffuse hyperplasia was observed in one on the 100, one on the 1000 and two on the 10000 ppm dose levels and in one of the females on the 1000 ppm dose level.
- Thymus: two of the females on the 100000 ppm dose level showed hyperplasia. The incidence is of no significance.
- Tongue: one of the control females showed arteriolar sclerosis.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mortality observed at 10000 and 100000 ppm.

Remarks on result:

other: Equivalent to a NOAEL = 115 mg/kg /day (calculated as a mean from measured intakes in diet for males and females)

Key result

Critical effects observed:

not specified

Conclusions:

The NOAEL is considered to be 1000 ppm (equivalent to 115 mg/kg bw/day calculated as a mean from the measured intakes in diet for males and females).

Executive summary:

The aim of the study was to assess the subchronic oral toxicity of the test material following a repeted dose oral administration of the test material mixed in the diet at four dose levels (100, 1000, 10000 and 100000 ppm) to groups of 20 male and female rats (a total of 200) during 91 days. The test procedure used was equivalent to the OECD guideline 408. The NOAEL is considered to be 1000 ppm (equivalent to 115 mg/kg bw/day calculated as a mean from the measured intakes in diet for males and females).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline 422. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: Males (354-427g), Females (208-272g)
- Housing: stainless steel wire mesh cage / polycarbonate cage
- Water (e.g. ad libitum): water after filtration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day (lights at 7.00am, off at 7 pm)

Route of administration:

oral: gavage

Vehicle:

other: 1.0% w/v methylcellulose

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

42 Days for males
14 Days before mating to day 4 of lactation for females
42 Days for the satellite group for recovery of females

Frequency of treatment:

Once per day during the morning

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

12 male and 12 female animals per group
5 male and 5 female animals per recovery group (control and high dose only)

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (appearance, behaviour, mortality)
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before and after treatment, once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period and at the end of recovery period.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters: red blood cell count (electrical resistance detection method), hemoglobin amount (lauryl sulfate) by multi-item automatic hemocytometer (E-4000, Sysmex) Sodium-hemoglobin method), hematocrit value (pulse detection method), average red blood cell volume (MCV), average red blood cell pigment content (MCH), average red blood cell pigment concentration (MCHC, above, calculated value), white blood cell count and platelet count (above, The electric resistance detection method was used, and smears were prepared, and reticulocyte count (Brilliant cresyl blue staining and microscopic examination) and leucocyte percentage (May-Giemsa staining and microscopic examination), prothrombin time (Quick one-step method), blood coagulation automatic measurement device (KC-10A, Amerung Corporation, USA) Thromboplastin time (APTT, eladinic acid activation method).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period and at the end of recovery period.
- Animals fasted: Yes
- How many animals: all
- Parameters: total protein (Biuret method), albumin (BCG method), A / G ratio (calculated value) by a biochemical automatic analyzer (JCA-BM type 8 cleaner, JEOL), blood sugar, total cholesterol, triglyceride (above, enzymatic method), total bilirubin (diazo method), urea nitrogen (Urease-UV method), creatinine (Jaffe), AST, ALT, ALP (above, JSCC method), g − GTP (SSCC method), LDH (SFBC method), cholinesterase (BTC-DNTB method), calcium (OCPC method) and inorganic phosphorus (enzyme method), and with automatic electrolyte analyzer (NAKL-132, Toa Denpa Kogyo) Sodium, potassium and chlorine (above, ion electrode method).

URINALYSIS: Yes (males)
- Time schedule: On day 40 of administration and during second week of recovery.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters: appearance (Martistics, Bayer, Sankyo), qualitative examination of pH, occult blood, protein, sugar, ketone body, bilirubin and urobilinogen and examination of sediment (URI-CELL fluid, stained with Cambridge chemical product and microscopic examination). Urine volume, specific gravity (refractometer, Elma optics), and sodium and potassium (electrolytic analyzer, NAKL-132, Toa Denpa Kogyo) were measured for the urine obtained after 18 hours of storage.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: sensory activity on the last day of administration, for females once during the lactation period, and at the end of the recovery period; motor activity and grip strength on day 41 for males, once during lactation for females, and on the last day of adminstration and on the 13th day of recovery for animals in the recovery group.
- Battery of functions tested: sensory activity / grip strength / motor activity
- Sensory reflex test (males and females in the recovery group, on the final administration day, once during the lactation period, on the final administration day and at the end of the recovery period): auditory response, visual response, tactile response, ear reflex, pain response, pupillary reflex, ipsilateral flexor reflex, eyelid reflex and righting reflex were examined.
- Landing opening width, grip strength and spontaneous activity measurement (males exercise on day 41, females once during lactation, males and females in recovery groupon the final administration day and on day 13 of the recovery period): motor activity of males (30 and 60 min) and females (30 min; 60 min for females in the recovery group) (motor activity measuring device , SUPERMEX, Muromachi Machine), grip strength of the forelimbs and hindlimbs (Grip strength measuring device for rat and mouse, MK-380R / FR, Muromachi Machine) and landing leg width were measured.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes

HISTOPATHOLOGY: Yes

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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

Histopathological findings: neoplastic:

no effects observed

Details on results:

HISTOPATHOLOGY:
In the 1000 mg/kg group: Histopathological examination revealed slight squamous hyperplasia at the limiting ridge of the forestomach in both sexes. This change demonstrated reversibility or a tendency for reversibility in the male recovery group and the female satellite group.

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathology changes observed at 1000 mg/kg bw/day (forestomach)

Critical effects observed:

not specified

Table . Mortality rate of male and female rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Dose (mg/kg)	0	100	300	1000
No. males examined	12	12	12	12
No. males that died	0	0	0	0
No. females examined	17	12	12	17
No. females that died	0	0	0	0
Mortality (%)	0	0	0	0

Table . Incidence of detailed clinical signs in the FOB (Functional Observation Batteries) of male rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

		Before administration				Week 6 administration				Week 2 recovery
Items	Dose (mg/kg)	0	100	300	1000	0	100	300	1000	0	1000
Males	No. animals	12	12	12	12	12	12	12	12	5	5
Reactivity on removal from the cage	Normal	12	12	12	12	12	12	12	12	5	5
Reactivity on handling	Normal	12	12	12	12	12	12	12	12	5	5
Muscle tone	Normal	12	12	12	12	12	12	12	12	5	5
Skin	Normal	12	12	12	12	12	12	12	12	5	5
Fur	Normal	12	12	12	12	12	12	12	12	5	5
Piloerection	Not detected	12	12	12	12	12	12	12	12	5	5
Eye discharge	Not detected	12	12	12	12	12	12	12	12	5	5
Palpebral closure	Not detected	12	12	12	12	12	12	12	12	5	5
Exophthalmos	Not detected	12	12	12	12	12	12	12	12	5	5
Lacrimation	Not detected	12	12	12	12	12	12	12	12	5	5
Smudge around mouth-nose	Not detected	12	12	12	12	12	12	12	12	5	5
Salivation	Not detected	12	12	12	12	12	12	12	12	5	5
Blotted fur in the lower abdomen with urine	Not detected	12	12	12	12	12	12	12	12	5	5
Blotted fur around anus with feces	Not detected	12	12	12	12	12	12	12	12	5	5
Vocalization	Not detected	11	11	11	10	12	12	11	10	4	4
	Temporarily in handling	1	1	1	2	0	0	1	2	1	1
Breathing	Normal	12	12	12	12	12	12	12	12	5	5
Body position	Normal	12	12	12	12	12	12	12	12	5	5
Convulsion	Not detected	12	12	12	12	12	12	12	12	5	5
Tremor	Not detected	12	12	12	12	12	12	12	12	5	5
Exploration	Normal	12	12	12	12	12	12	12	12	5	5
Alertness	Normal	12	12	12	12	12	12	12	12	5	5
Locomotor activity	Normal	12	12	12	12	12	12	12	12	5	5
Walk	Normal	12	12	12	12	12	12	12	12	5	5
Abnormal behaviour	Normal	12	12	12	12	12	12	12	12	5	5
Stereotypy	Not detected	12	12	12	12	12	12	12	12	5	5
Failure of consciousness	Not detected	12	12	12	12	12	12	12	12	5	5
Limb tone	Normal	12	12	12	12	12	12	12	12	5	5
Urination	Not detected or 1	11	12	12	12	11	12	12	12	4	5
	2 or more	1	0	0	0	0	0	0	0	1	0
	Color: pale yellow	2/2	2/2	2/2	-	2/2	-	2/2	1/1	2/2	1/1
Defecation	Not detected or 1	12	12	12	12	12	12	12	12	5	5
	2 or more	0	0	0	0	0	0	0	0	0	0
	Color: pale yellow	1/1	-	1/1	1/1	2/2	1/1	-	-	1/1	-

Table . Incidence of detailed clinical signs in the FOB (Functional Observation Batteries) of female rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

		Before administration				Week 6 administration				Week 2 recovery
Items	Dose (mg/kg)	0	100	300	1000	0	100	300	1000	0	1000
Females	No. animals	12	12	12	12	12	12	12	12	5	5
Reactivity on removal from the cage	Normal	12	12	12	12	12	12	12	12	5	5
Reactivity on handling	Normal	12	12	12	12	12	12	12	12	5	5
Muscle tone	Normal	12	12	12	12	12	12	12	12	5	5
Skin	Normal	12	12	12	12	12	12	12	12	5	5
Fur	Normal	12	12	12	12	12	12	12	12	5	5
Piloerection	Not detected	12	12	12	12	12	12	12	12	5	5
Eye discharge	Not detected	12	12	12	12	12	12	12	12	5	5
Palpebral closure	Not detected	12	12	12	12	12	12	12	12	5	5
Exophthalmos	Not detected	12	12	12	12	12	12	12	12	5	5
Lacrimation	Not detected	12	12	12	12	12	12	12	12	5	5
Smudge around mouth-nose	Not detected	12	12	12	12	12	12	12	12	5	5
Salivation	Not detected	12	12	12	12	12	12	12	12	5	5
Blotted fur in the lower abdomen with urine	Not detected	12	12	12	12	12	12	12	12	5	5
Blotted fur around anus with feces	Not detected	12	12	12	12	12	12	12	12	5	5
Vocalization	Not detected	12	10	12	11	11	11	12	12	5	5
	Temporarily in handling	0	2	0	1	1	1	0	0
Breathing	Normal	12	12	12	12	12	12	12	12	5	5
Body position	Normal	12	12	12	12	12	12	12	12	5	5
Convulsion	Not detected	12	12	12	12	12	12	12	12	5	5
Tremor	Not detected	12	12	12	12	12	12	12	12	5	5
Exploration	Normal	12	12	12	12	12	12	12	12	5	5
Alertness	Normal	12	12	12	12	12	12	12	12	5	5
Locomotor activity	Normal	12	12	12	12	12	12	12	12	5	5
Walk	Normal	12	12	12	12	12	12	12	12	5	5
Abnormal behaviour	Normal	12	12	12	12	12	12	12	12	5	5
Stereotypy	Not detected	12	12	12	12	12	12	12	12	5	5
Failure of consciousness	Not detected	12	12	12	12	12	12	12	12	5	5
Limb tone	Normal	12	12	12	12	12	12	12	12	5	5
Urination	Not detected or 1	12	12	12	12	12	12	12	12	5	5
	2 or more	0	0	0	0	0	0	0	0	0	0
	Color: pale yellow	1/1	1/1	5/5	1/1	2/2	2/2	1/1	-	1/1	-
Defecation	Not detected or 1	12	12	12	12	12	12	12	12	5	5
	2 or more	0	0	0	0	0	0	0	0	0	0
	Color: pale yellow	-	-	-	-	-	-	-	-	-	-

Table . Incidence of responses in the sensory/reflex funcion test of rats treated orally with the test item in the combined repeated odse toxicity study with the reproduction/developmental toxicity screening test.

	Period	Week 6 of administartion				Week 2 recovery
Item.	Dose (mg/kg)	0	100	300	1000	0	1000
Males	No. examined	5	5	5	5	5	5
Hearing reaction	Normal	5	5	5	5	5	5
Eye sight reaction	Normal	5	5	5	5	5	5
Sense of touch reaction	Normal	5	5	5	5	5	5
Pain reaction	Normal	5	5	5	5	5	5
Pupil reflex	Normal	5	5	5	5	5	5
Pinna reflex	Normal	5	5	5	5	5	5
Ipsilateral flexor reflex	Normal	5	5	5	5	5	5
Eyelid reflex	Normal	5	5	5	5	5	5
Righting reflex	Normal	5	5	5	5	5	5
Females	No. examined	5	5	5	5	5	5
Hearing reaction	Normal	5	5	5	5	5	5
Eye sight reaction	Normal	5	5	5	5	5	5
Sense of touch reaction	Normal	5	5	5	5	5	5
Pain reaction	Normal	5	5	5	5	5	5
Pupil reflex	Normal	5	5	5	5	5	5
Pinna reflex	Normal	5	5	5	5	5	5
Ipsilateral flexor reflex	Normal	5	5	5	5	5	5
Eyelid reflex	Normal	5	5	5	5	5	5
Righting reflex	Normal	5	5	5	5	5	5

Table . Mean value of landing foot splay, grip strength and motor activity of male rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental test.

Male rats				Grip strength (g)		Motor activity (counts)
Period	Dose (mg/kg)	No. animals	Landing foot splay (cm)	Forelimb	Hindlimb	0-30 min	0-60 min
week 6	0	5	10.4 ± 2.6	695 ± 235	328 ± 73	8248 ± 379	10437 ± 2172
administration	1000	5	10.4 ± 2.1	630 ± 247	378 ± 60	8901* ± 434	12211 ± 1750
week 2	0	5	10.3 ± 1.9	446 ± 122	421 ± 155	6877 ± 1305	8522 ± 2499
recovery	1000	5	9.9 ± 2.0	644* ± 150	423 ± 55	7233 ± 1849	8133 ± 2270

*Significantly different from control at 5% level of probability.

Table . Mean value of landing foot splay, grip strength and motor activity of female rats of the satellite group treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental test.

Female rats				Grip strength (g)		Motor activity (counts)
Period	Dose (mg/kg)	No. animals	Landing foot splay (cm)	Forelimb	Hindlimb	0-30 min	0-60 min
week 6	0	5	8.1 ± 1.9	485 ± 155	217 ± 34	8769 ± 1060	2310 ± 3290
administration	1000	5	6.7 ± 2.4	663 ± 176	268 ± 78	8882 ± 942	13688 ± 2628
week 2	0	5	7.5 ± 2.6	410 ± 90	295 ± 88	7982 ± 878	10789 ± 3946
recovery	1000	5	6.2 ± 1.3	631** ± 98	340 ± 49	7629 ± 637	10897 ± 2840

**Significantly different from control at 1% level of probability.

Table . Body weights of male rats treated orally with the test item.

Dose

Days of administration (n = 12)

Gain

(Days of recovery) (n = 5)

Gain

(mg/kg)

1

7

14

21

28

35

42

1-42

42 (0)

49 (7)

56 (14)

42-56

0

382 ± 16

405 ± 18

430 ± 22

452 ± 23

474 ± 30

497 ± 31

514 ± 36

131 ± 27

526 ± 28

540 ± 35

548 ± 32

22 ± 5

100

381 ± 17

403 ± 18

423 ± 23

441 ± 24

464 ± 27

481 ± 29

497 ± 32

116 ± 24

300

382 ± 21

405 ± 22

425 ± 28

445 ± 30

445 ± 30

485 ± 33

491 ± 37

108 ± 24

1000

384 ± 15

411 ± 18

430 ± 21

454 ± 23

454 ± 23

498 ± 27

510 ± 31

126 ± 22

508 ± 44

523 ± 46

528 ± 48

19 ± 3

Table . Body weights of female rats treated orally with the test item (n=12).

Dose

Days of premating

Gain

Days of pregnancy

Gain

Days of lactation

Gain

(mg/kg)

1

7

14

1-14

0

7

14

20

0-20

0

4

0-4

0

236 ± 17

147 ± 18

258 ± 21

22 ± 8

263 ± 25

299 ± 29

338 ± 30

423 ± 32

160 ± 10

313 ± 31

340 ± 34

38 ± 10

100

239 ± 14

149 ± 11

264 ± 17

25 ± 9

269 ± 19

310 ± 20

348 ± 23

431 ± 29

162 ± 16

325 ± 24

348 ± 28

24 ± 7

300

238 ± 15

151 ± 18

266 ± 21

28 ± 9

275 ± 23

313 ± 24

355 ± 27

449 ± 35

174 ± 15

343 ± 27

360 ± 35

17 ± 15

1000

236 ± 13

149 ± 16

261 ± 19

25 ± 10

269 ± 20

304 ± 20

344 ± 25

426 ± 33

158 ± 20

331 ± 29

351 ± 32

20 ± 7

Table 13. Body weights of female rats in the satellite group treated orally with the test item (n=5).

Dose

Days of administration

Gain

(Days of recovery)

Gain

(mg/kg)

1

7

14

21

28

35

42

1-42

42 (0)

49 (7)

56 (14)

42-56

0

244 ± 16

256 ± 13

272 ± 12

283 ± 15

288 ± 19

303 ± 14

311 ± 15

67 ± 11

311 ± 15

323 ± 18

323 ± 17

12 ± 4

1000

239 ± 10

256 ± 22

272 ± 20

287 ± 26

299 ± 37

303 ± 33

305 ± 29

66 ± 22

305 ± 29

313 ± 28

314 ± 32

9 ± 7

Table . Hematological findings of rats treated orally with the test item.

	After administration period				After recovery period
Dose (mg/kg) - Male	0	100	300	1000	0	1000
No. of animals	5	5	5	5	5	5
RBC (10^4/µL)	846 ± 52	834 ± 44	820 ± 46	835 ± 25	880 ± 31	915 ± 20
Hemoglobin (g/dL)	15.9 ± 0.5	14.5 ± 0.8**	14.8 ± 0.8*	14.8 ± 0.3*	15.4 ± 0.3	16.1 ± 0.3*
Hematocrit (%)	46.0 ± 1.8	42.9 ± 1.9	43 ± 1.7	43.8 ± 1.4	45.8 ± 1.2	47.0 ± 1.6
MCV (fL)	55 ± 2	52 ± 2*	53 ± 1	53 ± 2	52 ± 1	51 ± 1
MCH (pg)	18.8 ± 0.7	17.4 ± 0.7**	18.0 ± 0.3	17.7v0.3*	17.5 ± 0.4	17.6 ± 0.2
MCHC (%)	34.6 ± 0.4	33.8 ± 0.8	34.1 ± 0.8	33.8 ± 1	33.7 ± 0.7	34.2 ± 0.6
Reticulocyte (%o)	29 ± 8	28 ± 4	26 ± 6	28 ± 5	24 ± 3	26 ± 4
Platelet (10^4/µL)	121 ± 14	115 ± 12	139 ± 20	140 ± 13	131 ± 19	136 ± 11
PT (sec)	13.3 ± 0.5	13.2 ± 0.5	13.7 ± 0.7	13.1 ± 0.5	13.3 ± 0.8	13.3 ± 0.5
APTT (sec)	23.0 ± 1.8	22.0 ± 1.5	22.8 ± 0.8	22.1 ± 1.6	24.1 ± 1.1	24.7 ± 1.0
WBC(10^2/µL)	92 ± 26	59 ± 10*	58 ± 13*	60 ± 10*	84 ± 12	84 ± 25
Differential leukocyte counts (%)
Basophil	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Eosinophil	1 ± 1	1 ± 1	2 ± 2	1 ± 0	2 ± 1	0 ± 1*
Neutrophil
Stab form	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Segmented	16 ± 6	26 ± 13	19 ± 7	14 ± 2	22 ± 7	17 ± 4
Lymphocyte	80 ± 5	70 ± 15	75 ± 8	82 ± 2	72 ± 5	79 ± 3
Monocyte	3 ± 1	3 ± 2	4 ± 3	3 ± 1	4 ± 2	4 ± 1
Other	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Dose (mg/kg) - Female	0	100	300	1000	0	1000
No. of animals	5	5	5	5	5	5
RBC (10^4/µL)	698 ± 46	710 ± 33	681 ± 44	693 ± 45	821 ± 30	850 ± 17
Hemoglobin (g/dL)	13.4 ± 0.8	13.6 ± 0.2	12.9 ± 0.6	12.6 ± 1.2	15.6 ± 0.5	15.9 ± 0.5
Hematocrit (%)	39.9 ± 2.1	39.9 ± 1.0	38.3 ± 1.5	37.1 ± 1.2	45.0 ± 1.5	45.8 ± 1.6
MCV (fL)	57 ± 1	56 ± 2	56 ± 2	54 ± 4	55 ± 1	54 ± 2
MCH (pg)	19.2 ± 0.4	19.2 ± 0.8	18.9 ± 0.6	18.2 ± 1.3	19.1 ± 0.7	18.7 ± 0.6
MCHC (%)	33.5 ± 0.7	34.1 ± 0.7	33.6 ± 0.7	33.9 ± 0.6	34.8 ± 1.0	34.8 ± 0.5
Reticulocyte (%o)	65 ± 7	67 ± 8	59 ± 3	58 ± 9	25 ± 6	23 ± 5
Platelet (10^4/µL)	173 ± 21	169 ± 18	171 ± 41	183 ± 40	129 ± 9	136 ± 6
PT (sec)	13.9 ± 0.3	13.6 ± 0.8	13.7 ± 0.5	13.7 ± 0.4	12.9 ± 0.2	12.9 ± 0.4
APTT (sec)	18.9 ± 1.2	19.2 ± 1.9	17.9 ± 2.7	18.7 ± 0.9	19.1 ± 1.9	18.1 ± 0.9
WBC(10^2/µL)	86 ± 8	79 ± 22	71 ± 14	65 ± 7	47 ± 23	44 ± 14
Differential leukocyte counts (%)
Basophil	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Eosinophil	0 ± 0	0 ± 1	1 ± 1	1 ± 2	2 ± 2	1 ± 1
Neutrophil
Stab form	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Segmented	26 ± 7	26 ± 15	21 ± 10	20 ± 5	14 ± 3	17 ± 6
Lymphocyte	71 ± 6	70 ± 14	76 ± 11	76 ± 8	82 ± 3	80 ± 6
Monocyte	3 ± 2	3 ± 3	1 ± 2	3 ± 2	2 ± 1	1 ± 1
Other	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0

Significantly different from controls (*p < 0.05; **p < 0.01).

Table . Blood biochemical findings of rats treated orally with the test item.

	After administration period				After recovery period
Dose (mg/kg) - Male	0	100	300	1000	0	1000
No. of animals	5	5	5	5	5	5
LDH (IU/L)	420 ± 85	435 ± 111	489 ± 133	515 ± 171	319 ± 185	301 ± 116
AST (IU/L)	86 ± 9	84 ± 9	93 ± 18	85 ± 8	74 ± 11	80 ± 9
ALT (IU/L)	45 ± 3	45 ± 5	46 ± 11	40 ± 5	37 ± 3	42 ± 5
ALP (IU/L)	451 ± 97	433 ± 90	448 ± 88	483 ± 112	362 ± 68	292 ± 48
γ-GTP (IU/L)	0.51 ± 0.13	0.58 ± 0.09	0.58 ± 0.17	0.66 ± 0.28	0.75 ± 0.36	0.72 ± 0.53
ChE (IU/L)	47 ± 14	54 ± 14	46 ± 8	54 ± 18	41 ± 9	54 ± 16
T.protein (g/dL)	6.08 ± 0.20	5.75 ± 0.31	5.71 ± 0.52	5.86 ± 0.15	6.14 ± 0.26	6.19 ± 0.29
Albumin (g/dL)	3.02 ± 0.03	2.76 ± 0.23	2.73 ± 0.24	2.94 ± 0.15	2.98 ± 0.11	2.95 ± 0.19
A/G ratio	0.99 ± 0.88	0.92 ± 0.06	0.92 ± 0.04	1.01 ± 0.06	0.95 ± 0.05	0.91 ± 0.06
T.cholesterol (mg/dL)	69 ± 11	64 ± 6	72 ± 16	64 ± 8	60 ± 13	59 ± 17
Triglyceride (mg/dL)	54 ± 27	53 ± 22	42 ± 24	41 ± 3	39 ± 22	49 ± 27
Glucose (mg/dL)	152 ± 12	158 ± 10	149 ± 10	146 ± 11	165 ± 13	161 ± 28
BUN (mg/dL)	14.6 ± 1.3	14.8 ± 1.3	15.1 ± 1.2	14.1 ± 1.6	14.5 ± 1.7	15.5 ± 1.3
Creatinine (mg/dL)	0.47 ± 0.04	0.43 ± 0.05	0.43 ± 0.02	0.44 ± 0.02	0.44 ± 0.04	0.44 ± 0.01
T.bilirubin (mg/dL)	0.30 ± 0.06	0.29 ± 0.04	0.28 ± 0.04	0.29 ± 0.03	0.30 ± 0.04	0.27 ± 0.07
Ca (mg/dL)	9.7 ± 0.2	9.4 ± 0.2	9.3 ± 0.4	9.5 ± 0.4	10.0 ± 0.3	9.7 ± 0.3
I.phosphorus (mg/dL)	6.9 ± 0.5	7.0 ± 0.2	7.0 ± 0.6	7.0 ± 0.6	6.4 ± 0.3	6.6 ± 0.4
Na (mEq/L)	145 ± 0	144 ± 1*	145 ± 0	145 ± 0	146 ± 1	146 ± 2
K (mEq/L)	4.54 ± 0.12	4.58 ± 0.29	4.77 ± 0.36	4.77 ± 0.36	4.83 ± 0.26	4.94 ± 0.40
Cl (mEq/L)	107 ± 0	106 ± 1	107 ± 1	106 ± 1	105 ± 1	105 ± 2
Dose (mg/kg) - Female	0	100	300	1000	0	1000
No. of animals	5	5	5	5	5	5
LDH (IU/L)	353 ± 168	342 ± 145	327 ± 124	459 ± 131	394 ± 282	396 ± 142
AST (IU/L)	85 ± 13	92 ± 13	88 ± 12	96 ± 30	151 ± 165	99 ± 60
ALT (IU/L)	65 ± 14	69 ± 8	63 ± 14	63 ± 19	56 ± 32	51 ± 44
ALP (IU/L)	346 ± 145	318 ± 130	431 ± 255	276 ± 67	164 ± 47	165 ± 22
γ-GTP (IU/L)	0.82 ± 0.24	0.75 ± 0.27	0.73 ± 0.18	0.64 ± 0.11	1.11 ± 0.53	0.94 ± 0.24
ChE (IU/L)	286 ± 65	317 ± 99	226 ± 45	282 ± 81	520 ± 117	525 ± 114
T.protein (g/dL)	6.19 ± 0.44	6.15 ± 0.13	5.87 ± 0.09	6.07 ± 0.22	6.77 ± 0.23	6.49 ± 0.25
Albumin (g/dL)	3.20 ± 0.40	3.29 ± 0.16	2.97 ± 0.08	3.11 ± 0.24	3.88 ± 0.22	3.53 ± 0.22*
A/G ratio	1.07 ± 0.14	1.15 ± 0.08	1.02 ± 0.03	1.05 ± 0.12	1.35 ± 0.11	1.19 ± 0.12
T.cholesterol (mg/dL)	77 ± 4	80 ± 19	72 ± 12	74 ± 12	101 ± 13	89 ± 26
Triglyceride (mg/dL)	105 ± 52	131 ± 28	95 ± 30	126 ± 57	24 ± 3	19 ± 4
Glucose (mg/dL)	147 ± 13	136 ± 15	146 ± 14	132 ± 8	125 ± 11	126 ± 12
BUN (mg/dL)	23.6 ± 4.5	24.1 ± 4.1	23.6 ± 2.5	23.8 ± 2.2	15.9 ± 2.4	16.1 ± 2.5
Creatinine (mg/dL)	0.50 ± 0.05	0.48 ± 0.03	0.51 ± 0.03	0.50 ± 0.01	0.47 ± 0.04	0.48 ± 0.02
T.bilirubin (mg/dL)	0.25 ± 0.04	0.24 ± 0.02	0.24 ± 0.02	0.24 ± 0.05	0.26 ± 0.03	0.25 ± 0.03
Ca (mg/dL)	10.3 ± 0.4	10.2 ± 0.4	10.2 ± 0.2	9.9 ± 0.2	10.1 ± 0.4	9.7 ± 0.1
I.phosphorus (mg/dL)	8.1 ± 0.8	7.9 ± 0.8	8.8 ± 0.5	7.5 ± 1.1	5.7 ± 0.3	5.7 ± 0.4
Na (mEq/L)	146 ± 2	146 ± 2	145 ± 2	146 ± 1	146 ± 1	147 ± 1
K (mEq/L)	5.04 ± 0.17	4.79 ± 0.15	5.12 ± 0.33	4.9 ± 0.30	5.06 ± 0.46	4.49 ± 0.29*
Cl (mEq/L)	103 ± 2	103 ± 2	104 ± 2	105 ± 2	106 ± 1	108 ± 1

Significantly different from controls (*p < 0.05; **p < 0.01).

Conclusions:

The NOAEL(s) for repeated dose toxicity is considered to be 300 mg/kg/day for parental animals of both sexes.

Executive summary:

The aim of the test was to study the oral toxicity in rats (12 males, 12 females) in a combined repeated dose toxicity and reproductive toxicity study at doses of 100, 300 and 1000 mg/kg/day. The test was performed according to OECD Test Guideline 422. In the 1000 mg/kg group, the histopathological examination revealed slight squamous hyperplasia at the limiting ridge of the forestomach in both sexes. This change demonstrated reversibility or a tendency for reversibility in the male recovery group and the female satellite group.

The NOAEL(s) for repeated dose toxicity is considered to be 300 mg/kg/day for parental animals of both sexes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

115 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Two studies are available, one is GLP compliant with Klimisch score = 1, and the other is of Klimisch score = 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The study with the longest duration (90-days) and  lowest NOAEL was selected.

Justification for classification or non-classification

Oral repeated dose toxicity (90 -day study): NOAEL= 115 mg/kg bw/day. The substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No 1272/2008.