Phosphorothioic triamide, N-propyl-

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:

May 2008 - January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

July 27, 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g
- Date of production: October 01, 2007
- Physical state and appearance: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < 18 °C (TKS)
- Stability under test conditions: The stability of the test substance in the diet over a period up to 4 days was proven before the start of the study.
- Solubility and stability of the test substance in the solvent/vehicle: 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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was administered as a solution. To prepare the solution, the appropriate amount of the test substance was weighed out depending on the desired concentration. Thereafter the vehicle (drinking water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test substance preparations were prepared between every 3 - 4 days.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

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: 33 +/- 1 days
- Housing: The rats were housed together (5 animals per cage) in H-Temp (PSU) cages in a fully air-conditioned room.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water (from water bottles), ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): fully air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: drinking water

Details on route of administration:

The test substance was administered daily via the drinking water for about 28 days. Control animals received drinking water only. At the end of the administration period the animals were sacrificed after a fasting period (withdrawal of food) for at least 16 - 20 hours.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was administered as a solution. To prepare the solution, the appropriate amount of the test substance was weighed out depending on the desired concentration. Thereafter the vehicle (drinking water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test substance preparations were prepared between every 3 - 4 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): every 3 - 4 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration control analyses revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0 - 110.0 % of the nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 ppm

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION: Yes
- Time schedule: weekly over a period of 1 day and calculated as mean food consumption grams per animal and day

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 (if drinking water study): Yes
- Time schedule for examinations: weekly over a period of 4 days and calculated as mean water consumption grams per animal per day

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified

CLINICAL CHEMISTRY: Yes
- Animals fasted: Not specified

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional observational battery)
- Time schedule for examinations: at the end of the administration period
- Battery of functions tested: home cage observations, open field observations, sensorimotor tests / reflexe, motor activity assessment

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Statistics of clinical examinations
Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed as follows:
- body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- feces, rearing, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analyses using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Statistics of clinical pathology
Means, medians and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed as follows:
- clinical pathology parameters, urine volume, urine specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- urinanalysis, except color, turbidity, volume and specific gravity: Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.

Statistics of pathology
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no substance-related effects in clinical examinations were observed

Mortality:

no mortality observed

Description (incidence):

no animal died prematurely in the present study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight was statistically significantly decreased in both sexes of the high dose group (dose group 3, 5000 ppm) from day 7 till the end of the administration period with a maximum of - 14.3 % on day 28 in males and - 9.9 % on day 14 in females. The body weight was also statistically significantly decreased in males of the mid dose group (dose group 2, 1000 ppm) on day 21 and 28 (- 6.5 % on both days).
In high dose animals of either sex the body weight change was statistically significantly decreased throughout the entire administration period (up to - 54.3 % in males on day 7 and - 43.8 % in females on day 14). In mid dosed male animals the body weight change was also statistically significantly decreased on day 21 and 28 (up to - 14.6 % on day 21).
These impaired body weight data were assessed as signs of general systemic toxicity and thus adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no substance-related effects in food consumption were measured; the consumption of treated animals was comparable to controls

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

During numerous time intervals, food efficiency differed from controls (increased / decreased values) in all dose groups.
However, as there was no consistent trend, these deviations were considered incidental.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was decreased in high dose males as well as females (dose group 3, 5000 ppm) throughout the entire administration period, continuous escalating from start of the study to the end up to the maximum of - 34.7 % in males and - 38 % in females on day 28. In addition, water consumption was also reduced in male and female animals of the mid dose group (dose group 2, 1000 ppm) towards the end of the administration period with a maximum of - 15 % in males and - 13.8 % in females on day 28.
The impaired water consumption was assessed as related to the test substance. Due to the fact that the reduction was increasing from the start of the study till the end of administration, this finding was rather caused by an incipient intoxication than reflecting only an impaired palatability.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related adverse changes in the hematological parameters were measured.
All the end of the study, in the females of the 5000 ppm dose group the red blood cell counts (RBC) and hematocrit values were decreased, whereas the mean corpuscular hemoglobin concentration (MCHC) was increased compared to the controls. These marginal deviations in the red blood cell parameters were not changed dose-dependently, and the means of the high dose group were within the historical control ranges (RBC 7.15 - 7.97 T/L; hematocrit 0.358 - 0.410 L/L, MCHC 22.24 - 24.34 mmol/L). Therefore, these changes were regarded as non-adverse.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related adverse changes in the clinical chemistry parameters were measured.
In the male rats of the 5000 ppm dose group the inorganic phosphate levels were increased. This was the only parameter deviated in clinical chemistry in the males of this dose group. The mean inorganic phosphate level was within the historical range (1.90 - 2.63 mmol/L). Therefore, this change this change was regarded as non-adverse.
In the females the urea levels were statistically significantly decreased beginning in the 200 ppm dose group. This was the only deviated clinical chemistry parameter in the dosed female rats. There was no indication of a decreased protein synthesis or a liver failure in these rats which might be a reason for decreased urea levels in general. In addition no histopathological findings were observed in the dosed females. Therefore, according to the ECETOC Technical Report No. 85 (References), this isolated deviation is regarded as non-adverse.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes in the urinanalysis parameters were measured.
At the end of the study, in the female rats of the 1000 ppm and 5000 ppm dose groups as well as in the males in the 5000 ppm dose group, only, the urinary volume was decreased and the urinary specific gravity was increased. This is regarded as an adaptive rather than an adverse effect. Probably, the water intake of the rats was reduced, and therefore the smaller volume of excreted urine was concentrated, which reflected the normal function of the kidneys.
In the female rats of the 5000 ppm dose group the incidences of higher urobilinogen levels were increased. Again, this finding was isolated. There is no relation to the decreased urea levels or any histo-pathological correlative in the dosed females. Therefore, this deviation is regarded as non-adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Besides this, the following examinations were carried out within the FOB and have to be assessed individually:
- Home cage observations: No substance-related findings were observed.
- Open field observations: No substance-related findings were observed.
- Sensorimotor tests / reflexes: No substance-related findings were observed.
- Quantitative parameters: No substance-related findings were observed.
- Motor activity measurement: No substance-related findings were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute Organ Weights
The decrease of terminal body weights in the 5000 and 1000 ppm groups is considered to be a treatment-related effect.
All other weight changes are regarded to be incidental, of no biological relevance or related to the terminal body weight decrease.
No significant weight changes were recorded in females.

Relative Organ Weights
The thyroid weight changes are regarded to be incidental, of no biological relevance or related to the terminal body weight decrease.
The brain weight increase in the 5000 ppm group is regarded to be related to the terminal body weight decrease.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The single gross lesions noted are considered to be incidental or spontaneous in nature and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Details on results:

Regarding clinical examinations, the oral administration of the test substance in drinking water over a period of 4 weeks caused signs of general systemic toxicity, like decreased body weight as well as body weight change.
In addition water consumption was reduced in animals of both sexes of high (5000 ppm) and mid dose group (1000 ppm). Due to the fact that the reduction was increasing from the start of the study till the end of administration, this finding was rather caused by an incipient intoxication than reflecting only an impaired platability.
Concerning the clinical pathology parameters no treatment-related adverse effects were observed in the rats of both sexes.
Regarding pathology, the necropsy and histopathology examinations did not reveal any treatment-related findings.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Intake of test substance

Test group	Concentration in the drinking water (ppm)	Mean daily test substance intake (mg/kg bw/d)
		Males	Females
1	200	18.1	19.8
2	1000	86.0	98.2
3	5000	377.1	419.5

Table 2: Absolute organ weights, males

Organ	Dose groups
	200 ppm	1000 ppm	5000 ppm
Terminal body weight	98 %	94 %*	88 %**
Brain	101 %	100 %	96 %*
Liver	98 %	93 %*	90 %**

*: statistically significant (Kruskal-Wallis-H- +Wilcoxon test, two-sided, p <= 0.05)

**: statistically significant (Kruskal-Wallis-H- +Wilxocon test, two-sided, p <= 0.01)

Table 3: Relative organ weights, males

Organ	Dose groups
	200 ppm	1000 ppm	5000 ppm
Thyroid glands	112 %	127 %*	115 %*

*: statistically significant (Kruskal-Wallis-H- +Wilcoxon test, two-sided, p <= 0.05)

Table 4: Relative organ weights, females

Organ	Dose groups
	200 ppm	1000 ppm	5000 ppm
Brain	100 %	102 %	110 %*

*: statistically significant (Kruskal-Wallis-H- +Wilcoxon test, two-sided, p <= 0.05)

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of the test substance in drinking water over a period of 4 weeks caused signs of general systemic toxicity, like decreased water consumption and reduced body weight as well as body weight change. At least water consumption was stoll affected in animals of both sexes treated with 1000 ppm.
No signs of toxicity were found during clinicochemical and hematological examinations, at urinanalyses as well as in pathology.
Therefore, the no observed adverse effect level (NOAEL), under the conditions of the present study, was 200 ppm in Wistar rats of either sex (18.1 mg/kg bw/day in males; 19.8 mg/kg bw/day in females).

Executive summary:

The test substance was administered to groups of 5 male and 5 female Wistar rats via the drinking water gavage at dose levels of 0 (group 0), 200 (group 1), 1000 (group 2) and 5000 (group 3) ppm over a period of 4 weeks.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and measurement of motor activity was carried out after about 4 weeks of treatment in males and females. Clinicochemical and hematological examinations as well as urinanalyses were performed towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations.

The various analyses

- demonstrated the stability of the test substance preparations over a period of up to 4 days at room temperature,

- showed the homogenous distribution of the test article in the drinking water,

- showed the correctness of the prepared concentrations.

The following test substance related adverse findings were noted:

5000 ppm (377.1 mg/kg bw/day in males; 419.5 mg/kg bw/day in females)

Clinical examinations

- Water consumption was decreased in both sexes during the whole study, up to - 34.7 % in male animals on day 28 and - 38 % in female animals on day 28

- Body weight was statistically significantly decreased in both sexes from day 7 till the end of the study, up to - 14.3 % on day 28 in male animals and - 9.9 % on day 14 in female animals

- Body weight change was statistically significantly decreased in both sexes through the whole study, up to - 54.3 % in male animals on day 7 and - 43.8 % in female animals on day 14

Clinical pathology

- No substance-related adverse findings were obtained

Pathology

- No substance-related adverse findings were obtained

1000 ppm (86.0 mg/kg bw/day in males; 98.2 mg/kg bw/day in females)

Clinical examinations

- Water consumption was decreased in both sexes towards the end of the administration period with a maximum of - 15 % in males and - 13.8 % in females on day 28

- Body weight was statistically significantly decreased in male animals on day 21 and 28 (- 6.5 % on both days)

- Body weight change was statistically significantly decreased in male animals on day 21 and 28 (up to - 14.6 % on day 21)

Clinical pathology

- No substance-related adverse findings were obtained

Pathology

- No substance-related adverse findings were obtained

200 ppm (18.1 mg/kg bw/day in males; 19.8 mg/kg bw/day in females)

Clinical examinations

- No substance-related adverse findings were obtained

Clinical pathology

- No substance-related adverse findings were obtained

Pathology

- No substance-related adverse findings were obtained

In conclusion, the oral administration of the test substance in drinking water over a period of 4 weeks caused signs of general systemic toxicity, like decreased water consumption and reduced body weight as well as body weight change. At least water consumption was still affected in animals of both sexes treated with 1000 ppm. No signs of toxicity were found during clinicochemical and hematological examinations, at urinanalyses as well as in pathology. Therefore, the no observed adverse effect level (NOAEL), under the conditions of the present study, was 200 ppm in Wistar rats of either sex (18.1 mg/kg bw/day in males; 19.8 mg/kg bw/day in females).