A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

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:

November 5, 2004 - February 23, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according to OECD 407. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

EC Number:

460-110-3

EC Name:

A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

Cas Number:

797751-43-0

Molecular formula:

not applicable, multiconstituent substance

IUPAC Name:

2,5,8-trimethyl-5-{[(propan-2-ylidene)amino]oxy}-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene; 3-methoxy-3,6-dimethyl-2,4-dioxa-5-aza-3-silahept-5-ene; 5-methoxy-2,5,8-trimethyl-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene

Details on test material:

- Name of test material (as cited in study report): Wasox-MMAC2
- Physical state: Clear liquid.
- Lot/batch No.: 1000024854.
- Expiration date of the lot/batch: December 2005.
- Storage condition of test material: Storage under a nitrogen atmosphere at ambient temperature
- Other: Handling precautions: Exothermic reaction with water or humidity from the air.

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Fischer, F344/NHsd, SPF.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman GmbH, 0-33176 Borchen.
- Age at study initiation: about 7 weeks
- Weight at study initiation: mean males: 152-154 g; mean females: 110-112 g.
- Housing: Group caging (2 or 3 animals of one group and one sex per cage). The first 3 animals of a group and the second 2 animals of a group are housed together. Makroion cages Type IV (33 cm x 55 cm area, 20 cm height). Bedding material: Aspen wood chips.
- Diet (e.g. ad libitum): ad libitum (Altromin 1324). Exception: feed was withdrawn the day before blood sampling and was offered again immediately thereafter.
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average of 21.9 ºC (continuous control and recording).
- Humidity (%): average of 45.5 % (continuous control and recording).
- Air changes (per hr): 12 per hour
- Photoperiod (hrs dark / hrs light): artificial light from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was blended with the vehicle (corn oil). Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Altromin 1324 forte, gamma irradiated with 25kGy60Co

VEHICLE
- Justification for use and choice of vehicle (if other than water): as the test substance is known to react immediately with water, corn oil was chosen as solvent.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Purity: the water content of the vehicle was determined by titration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determinations of the test substance in some selected preparations for stability, concentration and homogeneity were performed by gas chromatography with flame ionisation detection (GC/FID). The stability, homogeneity and the actual concentration analyses were performed.

Duration of treatment / exposure:

Test duration: 28 days
Satellite groups were kept after cessation of dosing without a further administration for 15 additional days.

Frequency of treatment:

Once a day for 28 consecutive days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5 animals per sex and per dose (control (K), control satellite (KS), low dose (A), mid dose (B), high dose (C) and high dose satellite (CS)).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were derived from the results of the dose range findings study.
Groups of 5 male and 5 female rats were given doses of 100 mg or 316 mg or 1000 mg test substance per kg body weight for 7 consecutive days. Investigations: Body weights, feed consumption, animal observation, gross pathology.
Results: 2 high dosed males died early, with the death related to the test substance. Gross necropsy findings gave some indication for hepatotoxicity. Body weights of the mid and high dosed males were significantly, the corresponding female groups notably, but not significantly, reduced. Feed consumption was reduced in parallel.
Therefore 200 mg test substance per kg body weight, interpolated between low and mid dose of the dose range finding study, are chosen as high dose for the main study. 20 mg test substance per kg, i.e. 10 % of the high dose, is chosen as the low dose, where no toxic effects are anticipated. The mid dose is interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status once a day and additionally checked once a day for viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days -3, 4, 11, 18 and 25 (all animals of all groups). Days 32 and 39 (all animals of the satellite groups.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: Day -4 to 31: once a week, all animals. Days 35 and 41: satellite groups.

FOOD EFFICIENCY:
- Food consumption: Determined per cage in weekly intervals in all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken on Day 29 from all animals of groups K, A, Band C and of all animals of groups KS and CS on Day 42.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters determined: red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC), mean cell volume (MCV), platelet count (PLT), differential white blood cell count (% of the different cell species), prathrambin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken on Day 29 from all animals of groups K, A, Band C and of all animals of groups KS and CS on Day 42.
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters determined: alanine aminotransferase (ALT, GPT), albumin (ALB), alkaline phosphatase (AP), aspartate aminotransferase (AST, GOT), calcium (Ca), cholesterol (CHOL), creatinine (CREA), gamma-glutamyltransferase (GGT), glucose (GLU), potassium (K), sodium (Na), total protein (TP), urea (UREA)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necropsy on Day 29 (groups K, A, B, C) and on Day 42 (groups KS and CS).
HISTOPATHOLOGY: Yes: gross lesions, tissue masses or tumours, adrenal glands, aorta*, brain, caecum*, coagulating glands*, colon, duodenum(preparedas "SwissRoll"), epididymides*, eyes*, femur bone with joint*, heart, ileum (prepared as "Swiss Roll"), jejunum (prepared as "Swiss Roll"), kidneys, lacrimal glands (extraorbital)*, liver, lungs, Iymphnodes(mandibular, mesenteric), oesophagus*, ovaries, pancreas *, pituitary*, prostate, rectum*, salivary glands*, seminal vesicles *, sciatic nerve, skeletal muscle (thigh)*, skin and mammary gland*, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid and parathyroid glands, trachea, urinary bladder, uterus.
*: not included into routine histopathology of groups K anc C.
ORGAN WEIGHTS: Yes: adrenal glands (both together), brain, epididymides (both together), heart, liver, kidneys (both together), spleen, testes (bothtogether), thymus.

Statistics:

Analysis of variance followed by the Scheffé-test, t-test and H-test of Kruskal and Wallis followed by the test of Nemenyi, Chi2-test and Fisher's exact test were used.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All animals were normal any time. There were no test substance related effects noted.

Mortality:

no mortality observed

Description (incidence):

No test substance related death occurred during the study. All animals survived until their scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
The body weight gain of the mid, the high and the high dosed satellite group males was significantly lower than that of the control group in the first week of dosing (Days 1-8).
The body weight gain of the high dosed satellite group females was significantly higher than that of the corresponding control group in the last week of the recovery period (Days 35-41).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the males, the high dosed and the high dosed satellite group had a slightly lower feed consumption during the dosing period, followed by a slightly higher feed consumption in the recovery period, both when compared with the controls. The differences were not markedly pronounced and are given only borderline relevance. There were no note worthy differences or dose related trends noted in the feed consumption of the females. No statistical evaluation of the results is useful due to group caging.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Results included below (see table 1)
All significant differences in haematology are attributed to the test substance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Results included below (see table 2)
All significant differences in clinical biochemistry are attributed to the test substance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver and spleen weight changes at the end of the dosing as well as the end of the recovery period are attributed to the test substance. No interpretation can be given for the other organ weight changes at the end of the recovery period. A test substance relation is considered as highly unlikely, but cannot be excluded with certainty.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In all high dosed animals a large spleen was noted. This alteration is attributed to the test substance. Isolated cases of a dilated lumen of the uterus or small testes are considered as spontaneous alterations without relation to the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The presence of extramedullary haematopoiesis in the spleens of all high dosed and almost all mid dosed animals of both sexes is related to the test substance. A minor pronounced effect, similar to that mentioned before, is hypercellularity in the bone marrow, noted in a few animals without gaining statistical significance. All other histopathological findings are not related to the test substance and considered as spontaneous alterations without toxicological relevance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Stability of the test substance: The three major components of the test substance were found to vary extremely in their stability in the preparations. Only one component was sufficiently stable for 4 h. As there was no alternative available, the poor stability had to be accepted.

Concentration and homogeneity: The instability of the test substance, causing deviations in concentration and homogeneity, had to be accepted as to the lack of alternatives. The weighing in procedure of the test substance was checked with special care to ascertain, that - in spite of problems with the stability - the amount of test substance weighed in for the preparations, was correct.

Water content of the vehicle: The water content of the vehicle was found to be 0.03 %.

 

 

Table1: Haematology

 

parameter (sex)	low dose	mid dose	high dose	high dose after recovery
red blood cell count (males)	-	↓	↓	↓
red blood cell count (females)	-	↓	↓	-
haemoglobin concentration (males)	-	↓	↓	-
haemoglobin concentration (females)	-	↓	↓	↑
haematocrit (males)	-	-	↓	-
haematocrit (females)	-	↓	↓	-
mean corpuscular haemoglobin, pg (males)	-	-	↑	↑
mean corpuscular haemoglobin, pg (females)	-	↑	↑	↑
mean corpuscular haemoglobin, g/dL (males)	-	↓	↓	-
mean corpuscular haemoglobin, g/dL (females)	-	-	↓	-
mean cell volume (males)	-	-	↑	↑
mean cell volume (females)	-	↑	↑	↑
white blood cell count (males)	-	-	↑	-
white blood cell count (females)	-	-	↑	-
platelet count (males)	-	↑	↑	↑
platelet count (females)	-	↑	↑	-
lymphocytes, % (males)	-	-	-	↑
neutrophils, % (males)	-	-	-	↓
lymphocytes, n (males)	-	-	↑	↑
lymphocytes, n (females)	-	-	↑	-
polychromatic erythrocytes %(males)	-	-	↑	-
polychromatic erythrocytes %(females)	-	-	↑	-

 

 Table2: Clinical chemistry

 

parameter (sex)	low dose	mid dose	high dose	high dose after recovery
albumin (males)	-	-	↓	-
glucose (males)	-	-	↓	-
total protein (males)	-	-	↓	-
total protein (females)	-	-	↓	↑
gamma glutamyl transferase (females)	-	-	↑	-
urea (males)	-	-	-	↑
urea (females)	-	-	-	↑

 

Key to the symbols:

-: no significant difference to the contral,

↑: significant increase, ↓: significant decrease

Applicant's summary and conclusion

Conclusions:

The No-observed-effect-level of the test substance was at 20 mg per kg body weight and day in both sexes.

Executive summary:

The Repeated dose 28 -day oral toxicity study in rodents for the test substance Wasox-MMAC2 was performed according to OECD Guideline 407 in F344 rats. The test material was administered blended with corn oil, given orally by gavage to 3 groups of 5 male and 5 female rats, once a day for 28 consecutive days. The dose used were 20, 63, and 200 mg/kg/day (groups A, B and C). A negative control group (group C, treated with the vehicle) was included. In addition, 2 groups of 5 males and 5 females each (i.e. one high dose satellite group CS and one control satellite group KS) were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration in an attempt to observe the reversibility or persistence of test substance induced lesions. Clinical signs, functional observations, body weights, feed consumption, haematology, clinical biochemistry, necropsy with gross pathological examination, organ weights and histopathological examinations were performed within the study. One major effect of Wasox MMAC2 can be derived from several results of the present study. The test substance causes damage to mature erythrocytes in the peripheral blood, resulting in alterations in virtually all erythrocyte-related parameters at the haematological examination. In parallel to this damage there is a marked repair by ongoing by an increased new production of young erythrocytes, notable in an increased proportion of polychromatic erythrocytes in the peripheral blood and also in the presence of haematopoiesis in the spleen, inducing even a significant weight increase of this organ, and - less pronounced - a hypercellularity of the bone marrow. The repair was efficient enough to allow the animals to live without physical behavioural abnormalities or body weight effects. There was no indication for a negative effect to the blood cell production in the bone marrow found. A second - minor expressed - effect may be derived from the clinical biochemistry data. Lowered plasma albumin and total protein, lowered glucose (males) and elevated GGT values (females) and a slightly increased liver weight may be due to a borderline hepatic involvement. All indications for hepatotoxicity are only poorly pronounced. Some haematological changes in neutrophil and Iymphocyte counts remain without a clear interpretation, but may be secondary to the erythrocyte alterations. Effects were similarly noted in males and females at the same doses, no sex difference in the response to the test substance can be derived from the results. Effects were only partly reversible during the recovery period, though there was a clear tendency towards a return to normal. The No-observed-effect-level of Wasox-MMAC2 was at 20 mg per kg body weight and day in both sexes.