Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to GLP; OECD-guideline 407; and 96/54/EEC.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

EC Number:

700-457-2

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Details on test material:

- Name of test material (as cited in study report): LIMUS-Sambaydestillation
- Test-substance No.: 07/0684-1
- Physical state: solid/yellowish
- Analytical purity: 87%
- Lot/batch No.: 8712/062
- Stability under test conditions: stable over 5 days
- Storage condition of test material: < -18°C

Test animals

Species:

rat

Strain:

other: Crl:Wl (HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 42 +/- 1 days (males and females)
- Weight at study initiation: ca. 185 g (males); ca. 140 g (females)
- Housing: 5 animals per cage (H-Temp (PSU) cages, Techniplast, Hohenpeißenberg, Germany; floor area about 2065 cm2).
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): water from bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water with 1% Carboxymethycellulose) was filled up to the desired volume, subsequently mixed using a Ultraturrax. The suspension was hold homogeneous by magnetic stirrer during application. The test-substance preparations were prepared at least every 5 days.

VEHICLE
- drinking water with 1% Carboxymethylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

Homogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period. These samples also served for concentration control analyses.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Doses / concentrations

Remarks:

Doses / Concentrations:
17, 84 and 208/416 mg/kg b.w. (see details on study design)
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose level of 416 mg/kg bw/day in female animals was reduced by half (208 mg/kg bw/day) from day 16 till the end of administration period.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule / examinations
- check for moribund animals: twice daily, once daily on Saturdays, Sundays and public holidays
- clinical observations (=check for any clinically abnormal signs): daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to commencement of the study and thereafter weekly. Examined parameters: abnormal behavior during "handling", fur, skin, p osture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairement of gait, lacrimation, palpebral closure , exophthalmus, feces (appearance/consistency), urine and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: once prior to study commencement; on the das of the study start and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE
Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day. No substance-related influence on food consumption was measured.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume. No substance-related overt changes in water consumption were observed.

HAEMATOLOGY: Yes
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte, erythrocyte, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood, reticulocytes. Blood sampling performed the 29th day of the study.
- How many animals: 5 animals per test group and sex

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), y-glutamyltransferase (GGT), Sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), ccreatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL) and magnesium (MG).

URINALYSIS: Yes
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Time schedule for collection of urine: 26th day of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity and volume.
- How many animals: 5 animals per test group and sex

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: performed the 24th and 25th day of the study
- Dose groups that were examined: 5 animals per test group and sex
- Battery of functions tested (FOB): posture, tremor, convulsions, abnormal movement, impairment of gait, behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes, approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.

Motor activity assessment measurements were carried out in all animals toward the end of the administration period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; The weight of the following organs was determined: liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus, and thyroid glands
HISTOPATHOLOGY: Yes,
Examination by light microscopy and assessment of findings in all dose groups concerning: all gross lesions, liver, prostate
Examination by light microscopy and assessment of findings in the low and high dose groups concerning: brain, thyroid glands (with parathyroid glands), thymus, trachea, lungs, heart, spleen, kidney, adrenal glands, testes, epididymides, ovaries (with oviducts), uterus, vagina, seminal vesicle, stomach (forestomach and glandular stomach), duodenum and ileum, jejunum (with Peyer's plaques), cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and axillary lymph nodes), sciatic nerve, bone marrow (femur), spinal cord (cervical, thoracic and lumbar cords).

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study. Reduced general condition slight was observed in 2 female animals of dose group 3. Salivation from slight to moderate was observed in 1 male animal and in 2 female animals of dose group 3. Additionally, salivation slight was observed in 1 male animal of dose group 2. The above-mentioned findings were assessed as signs of general systemic toxicity and thus related to treatment with the test compound.

BODY WEIGHT AND WEIGHT GAIN
The body weight was decreased in both sexes of the high dose group (dose group 3, 208/416 mg/kg bw/day) throughout the whole administration period, statistically significant with a maximum of -12.4% on day 14 in males and -20.4% on day 21 in females. The body weight was also decreased in males of the mid dose group (dose group 2, 84 mg/kg bw/day) within the entire study, even though not statistically significant. These impaired body weightdata were assessed as signs of general systemic toxicity and
thus adverse.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No substance-related influence on food consumption was measured.

FOOD EFFICIENCY
The food efficiency was not clearly dose- or time-dependent influenced by the test substance. The observed deviations were inconsistent. In addition, food efficiency is a calculated parameter and observed effects are therefore only secondary and thus no direct signs of systemic toxicity.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No substance-related overt changes in water consumption were observed.


HAEMATOLOGY
At the end of the study, the total white blood cell counts (WBC) in the male rats of the 416 mg/kg bw/day dose group were increased although not statistically significantly. The higher WBCs were due to higher relative and absolute lymphocyte counts (the latter not statistically significant) at the expense of the relative neutrophil counts. In the females of the 17 mg/kg bw/day dose group, the relative monocyte counts were decreased. This decrease was isolated in the differential blood cell counts, and not dose-dependently. Therefore, it was regarded as incidental not treatment-related.

CLINICAL CHEMISTRY
No treatment-related, adverse changes were measured in the clinical chemistry parameters.
In the male rats of the 416 mg/kg bw/day group the globulin concentrations were decreased, and in the female animals of the same dose group the creatinin levels were decreased. These deviations were the only ones among the clinical chemistry parameters in the dosed rats of the respective sex. Moreover, in the males of the high dose group no relation of the decreased globulin values to the higher WBCs can be established. Therefore, the changes in the clincal chemistry parameters were regarded as incidental.

URINALYSIS
No treatment-related, adverse changes were measured in the urinalyses parameters.

ORGAN WEIGHTS
When compared to controls, the following mean absolute weights were significantly decreased: Term. body weight (in male and female animals of the high dose group, both); spleen (in male and female animals of the high dose group). All other mean absolute weight parameters did not show significant differences when compared to the control group.
When compared to controls, the mean relative weights of following organs were significantly increased: adrenal glands, brain, heart, kidneys,liver and spleen in female animals of the high dose group; brain in male animals of the high dose group. All other mean relative weight parameters did not show significant differences when compared to the control group.
The decreased absolute spleen weight in males of group 3, the increased relative brain weights in males and females of group 3, and the relative weights of adrenal glands, kidneys, heart, liver and spleen in females of group 3 are related to the significantly decreased body weights in these animals.

GROSS PATHOLOGY
There were no substance-related gross lesions.

HISTOPATHOLOGY: NON-NEOPLASTIC
Prostate: Focal lymphoid infiltrates were observed in the prostate of one control male, of one male in group 1, three males of group 2 and four males of group 3. The occurence of lymphoid cell infiltrates in the prostate is considered incidental. All other findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

ca. 17 other: mg/kg bw/day (by gavage)

Sex:

male

2

Dose descriptor:

NOAEL

Effect level:

ca. 84 other: mg/kg bw/day (by gavage)

Sex:

female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Asolute organ weights

When compared to controls (=100%), the following mean absolute weights were significantly decreased (printed in bolt):

	Male animals			Female animals
Dose group	1	2	3	1	2	3
Term. body weight	100%	94%	90%**	100%	100%	83%**
Spleen	96%	87%	81%*
* : p≤ 0.05 **: p ≤ 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group.

Relative organ weight

	Male animals			Female animals
Dose group	1	2	3	1	2	3
Adrenalglands				103%	106%	135%*
Brain	100%	103%	111%**	99%	99%	115%**
Heart				96%	101%	117%**
Kidneys				102%	106%	125%**
Liver				104%	107%	127%**
Spleen				99%	99%	135%*
* : p≤ 0.05 **: p ≤ 0.01

Body weight data (males and females)

	Body weight g Day 0 D	Body weight g Day 7 D	Body weight g Day 14 D	Body weight g Day 21 D	Body weight g Day 28 D
Male, 0 mg/kg bw
Mean	185.9	225.6	259.2	290.3	304.2
N	5	5	5	5	5
% dev
Male, 17 mg/kg bw
Mean	187.5	225.6	254.9	289.2	304.4
N	5	5	5	5	5
% dev	0.8	0.0	-1.6	-0.4	0.1
Male, 84 mg/kg bw
Mean	187.8	223.3	253.2	277.8	287.1
N	5	5	5	5	5
% dev	1.0	-1.0	-2.3	-4.3	-5.6
Male, 416 mg/kg bw
Mean	184.8	219.8	227.1**	270.1*	280.6*
N	5	5	5	5	5
% dev	-0.6	-2.6	-12.4	-7.0	-7.8
Key: D = Dunnett‘s test, Two-sided; * p≤ 0.05; ** p ≤ 0.01; Experimental Unit = Animal

	Body weight g Day 0 D	Body weight g Day 7 D	Body weight g Day 14 D	Body weight g Day 21 D	Body weight g Day 28 D
Female, 0 mg/kg bw
Mean	143.1	158.8	168.8	184.2	193.9
N	5	5	5	5	5
% dev
Female, 17 mg/kg bw
Mean	142.6	160.3	172.2	187.2	195.8
N	5	5	5	5	5
% dev	-0.3	0.9	2.0	1.6	1.0
Female, 84 mg/kg bw
Mean	139.9	155.9	172.5	184.9	196.6
N	5	5	5	5	5
% dev	-2.2	-1.8	2.2	0.4	1.4
Female, 416 mg/kg bw
Mean	142.3	153.9	142.9**	146.6**	165.6**
N	5	5	5	5	5
% dev	-0.6	-3.1	-15.4	-20.4	-14.6
Key: D = Dunnett‘s test, Two-sided; * p≤ 0.05; ** p ≤ 0.01; Experimental Unit = Animal

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL), under the conditions of the present study, was 17 mg/kg body weight/day in male and 84 mg/kg body weight/day in female Wistar rats. A classification according to Directive 67/548/EEC or GHS (UN) criteria is not warranted.

Executive summary:

In conclusion, the oral administration of LIMUS-Sambaydestillation by gavage over a period of 4 weeks caused signs of general systemic toxicity obtained in high dose animals of either sex (in males 416 mg/kg body weight/day throughout the entire study; in females 416 mg/kg body weight/day from day 0-15 and 208 mg/kg body weight/day from day 16 until the end of the administration period). In mid dose males (84 mg/kg bw/day) some of these findings were still observed. Target organs could not be identified, because the pathological evaluation did not reveal any substance-related adverse findings.