Reaction mass of Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate and Octadec-9-en-1-yl ammonium mono- and di-butylphosphate

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:

The study was conducted between 24 November 2003 and 25 November 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101) , EPA 7 12-C-00-366 , 2000.

Deviations:

yes

Remarks:

See below

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Substance Law 1987 , Notification of Dec. 1986 by EA, MHLW and METI.

Deviations:

yes

Remarks:

See below

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Molecular formula:

C22H48NO4P - C26H56NO4P

IUPAC Name:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Constituent 2

Reference substance name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Molecular formula:

C30H64NO2PS2

IUPAC Name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Test material form:

liquid: viscous

Details on test material:

Material is a yellow viscous liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 30 males, 30 females (females were nulliparous and non-pregnant).
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: Earmark and tattoo

Animal Husbandry
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21. 0 ± 3.0°C (actual range: 18.5 - 21. 7 °C) , a relative humidity of 30-7 0% (actual range: 17 - 7 3% ) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Accommodation
Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21. 5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (Woody Clean bedding (Woody-Clean type 3/4) , Tecnilab-BMI BV, Someren, The Netherlands) provided as bedding. Results of bedding analyses for contaminants are examined and archived.

Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1 , Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.

Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived. Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036
Rationale for vehicle: Based on trial formulations performed at NOTOX.
Stability of test substance in vehicle: Stable
Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and the test substance (0.95 g/ml).
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for dosing mixture homogeneity and stability analyses were collected on the first day of test article administration. While undergoing stirring in the beaker, the following sample aliquots (5ml) were drawn for analysis: control, three aliquots (from the middle); all treatment groups, twenty-seven aliquots (3 each from the top, middle and bottom). Two samples were sent to ChevronTexaco Energy Research and Technology Company for analysis of homogeneity and stability. The remaining samples from each dose level and strata were sent to ChevronTexaco Energy Research and Technology Company for possible future analysis. The samples were shipped under ambient conditions.
Once weekly, two 5 ml aliquots were taken (middle stratum) from freshly prepared dosing mixtures, including the control group. The dosing mixture was thoroughly mixed before taking each sample. The samples were sent to ChevronTexaco Energy Research and Technology Company: one sample from each dose level was analyzed; the remaining sample was retained for possible future analysis.

Sample Handling and Shipment
Each 5 ml sample was placed in a glass vial and stored at ambient temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg bw/day : 10/sex
30 mg/kg bw/day : 5/sex
100 mg/kg bw/day : 5/sex
300 mg/kg bw/day : 10/sex

Control animals:

yes

Details on study design:

Treatment
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Method
Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.

Frequency
Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dose up to the day prior to necropsy of the Main group animals.

Dose volume
5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Recovery
14 days. However, due to the bad health of the animals treated at 300 mg/kg/day, all main and recovery animals were sacrificed for humane reasons during week 2 of treatment. Recovery animals of the control group were sacrificed as well.

Observations
Mortality/Viability At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

Clinical signs
Once daily, detailed clinical observations were made in all animals.
Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

Functional Observations
During week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets):
hearing ability (HEARING) , pupillary reflex (PUPIL L/R) , static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England) .

Ophthalmoscopic Both eyes were examined following instillation of tropicamide examinations solution (5 mg/ml): week 4 (all surviving animals)

Body weights
Treatment period: on days 1 , 8, 15, 22 and 28.

Food consumption Weekly.
Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

Examinations

Observations and examinations performed and frequency:

Clinical Laboratory Investigations
Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a. m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0. 5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml).

All surviving males and females of group 4 (main and recovery) and the recovery males and females of group 1 were sacrificed for humane reasons on 04 December 2003. Blood from these animals was collected from the aorta under isoflurane anaesthesia during necropsy.

Acetylcholinesterase Measurements
Plasma
Acetylcholinesterase activity was measured in plasma samples of the animals that were killed in extremis (collected from the aorta) and in plasma samples of animals at the planned necropsy (drawn from the retro-orbital sinus). An extra separate blood sample (Li-heparin prepared tubes, 250 ?I) was drawn from each animal for these purposes and stored at ?-75°C until analyses.

Brain
At necropsy the frontal part of the brain (a part of approx. 4 mm) was removed after weighing and stored as soon as possible at ?-75°C until analyses.

Sacrifice and pathology:

Necropsy
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:

Identification marks: not processed
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions.

Organ Weights
The following organ weights (and terminal body weight) were recorded from the surviving, animals on the scheduled day of necropsy:
Adrenal glands, Epididymides, Kidneys, Ovaries, Testes, Brain, Heart, Liver, Spleen, Thymus


Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

Histopathology
The following slides were examined by a pathologist:
all tissues and organs collected at the scheduled sacrifice from all main group animals of the control and the highest dose group;
all tissues and organs from all animals of all dose groups which died spontaneously or were sacrificed in extremis;
all gross lesions of all animals (all dose groups).

Based on the treatment related morphologic changes, duodenum, jejunum, ileum, spleen and mesenteric lymph nodes were also examined from all rats of the 30 mg/kg/day groups. All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Statistics:

Interpretation
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test 1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test 3 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test4 was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis were performed on motor activity data and histopathology findings.

Results and discussion

Results of examinations

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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Observations
Mortality
Two females receiving 300 mg/kg/day (no's 52 and 58) were found dead on study day 11.
Furthermore, two males (no's 21 and 30) and one female (no. 59) were killed in extremis on day 10. Remaining males and females treated at 300 mg/kg/day (main and recovery groups, ) were sacrificed for humane reasons on day 11. The control recovery groups were sacrificed on that same day for scientific reasons.
Findings in these animals consisted of emaciation and several clinical signs pointing at a poor condition. Furthermore, body weights of these animals were (severely) depressed.


Clinical Signs
Males treated at 300 mg/kg/day showed hunched posture, breathing rales, piloerection, salivation, chromodacryorrhoea, lean appearance and squeaking before sacrifice/death. High dose females showed lethargy, hunched posture, uncoordinated movements, rales, piloerection, yellow discolouration of the skin, salivation, chromodacryorrhoea, pale ears, dehydration, lean appearance, ptosis, squeaking and hypothermia. During the last treatment week chromodacryorrhoea was also found in two females at 100 mg/kg/day.
Incidental findings included alopecia and scabs. These findings are commonly noted in rats of this age and strain and housed and treated under the conditions in this study and considered of no toxicological significance.


Functional Observations
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with 434-280-4, when compared to control animals.
Variations noted in motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of the low or high sensors, and/or supportive clinical signs. Therefore, they were considered of no toxicological relevance.


Ophthalmoscopic Examinations
There were no ophthalmoscopic findings in week 4 that were attributed to treatment with OLOA 289M. Focal corneal opacity, observed in one male at 30 mg/kg/day was considered of no toxicological relevance due to its incidental occurrence.


Body Weights
At the end of the first treatment week, reduced body weight gain or body weight loss was noted in the high dose males and females. Body weight gain of males at 100 mg/kg/day was also lowered during the study.
No body weight effects were noted in females at 100 mg/kg/day and in males and females treated at 30 mg/kg/day.


Food Consumption
Absolute and relative food consumption was lowered in rats of both sexes at 300 mg/kg/day and in males at 100 mg/kg/day. Food consumption before or after allowance for body weight was comparable between animals at 30 (both sexes) and 100 mg/kg/day (females) and the control animals.


Clinical Laboratory Investigations
Haematology
In intercurrent deaths (high dose animals), a number of changes were noted in the when compared to the controls:
• Higher white and red blood cell values (males and females)
• Higher neutrophil counts (males and females)
• Higher monocyte counts (males)
• Lower eosinophil counts (females)
• Lower lymphocyte counts (males and females)
• Higher haemoglobin values (females)
• Higher haematocrit values (females)
• Lower mean corpuscular volume (males)
• Lower mean corpuscular haemoglobin (males and females)
• Lower mean corpuscular haemoglobin concentration (females)
• Higher number of platelets (males and females)
• Higher red cell distribution width (males and females)
• Higher partial thromboplastin time (males and females).
Higher white blood cell counts (leucocytosis) were also noted in males (not statistically significant) and females at 100 mg/kg/day and in females at 30 mg/kg/day, and a dose-related trend was present. Moreover, higher neutrophil counts (neutrophilia) were noted as well at 30 mg/kg/day (males) and 100 mg/kg/day (both sexes), but without statistical significance.
Number of platelets was statistically significantly increased in males at 100 mg/kg/day.
Moreover, in females at 100 mg/kg/day, higher red blood cell counts were found. In females of the mid dose group haematocrit and red cell distribution width was elevated and mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and partial thromboplastin time were lowered. Furthermore, in females at 30 mg/kg/day, higher haematocrit values were found.

Clinical Biochemistry
In intercurrent deaths (high dose) , the majority of the parameters in both sexes were changed with statistical significance when compared to the controls:
• Higher alanine aminotransferase values
• Lower alkaline phosphatase values
• Lower bilirubin concentration (only statistically significant in males)
• Higher urea concentration (not statistically significant in males)
• Lower creatinine, glucose and cholesterol values
• Lower triglycerides and sodium concentration (not statistically significant in females)
• Higher potassium concentration (only in females)
• Lower calcium concentration
• Lower inorganic phosphorus concentration (only statistically significant in males)
• Lower values of total protein, albumin and albumin/globulin ratio
At the end of treatment gamma glutamyl transferase was lowered in females at 30 and 100 mg/kg/day, but absent at 300 mg/kg/day. Furthermore, glucose concentration was decreased and triglycerides and potassium values were increased in females at 100 mg/kg/day. In the absence of findings at higher dose levels, these effects were considered not to be toxicologically significant.

At the end of treatment alkaline phosphatase was statistically significantly decreased in males at 30 mg/kg/day and chloride was statistically significantly increased in males at 100 mg/kg/day.

Due to the lack of similar changes at higher levels and/or in the absence of a dose-relationship, these differences were not considered to be toxicologically relevant.

Acetylcholinesterase measurements in plasma and brain
Plasma
AChE values in plasma in the intercurrents deaths showed an inhibition of 51 % in males and an inhibition of 69% in females. At the planned sacrifice inhibition of acetylcholinesterase in plasma was 9% and 46% in males and females, respectively at 100 mg/kg/day. At 30 mg/kg/day inhibition was 5% and 12% in males and females, respectively.

Brain
Percentages AChE inhibition in brain was -4% and 2% at 100 mg/kg/day and 10% and 7 % at 30 mg/kg/day in males and females, respectively.

Pathology
Macroscopic Examination
The following findings were noted in the unscheduled deaths at 300 mg/kg/day:
• 3 Males and 8 females were noted as emaciated
• Irregular surface in the stomach in 4 males and 3 females
• Reduced size was noted for prostate (6 males) , seminal vesicles (9 males) , spleen (1 male and 3 females) and thymus (5 males and 7 females)
• Enlarged mesenteric lymph node in 4 males and 5 females
At terminal sacrifice, discolouration of the mesenteric lymph node was noted in two rats treated at 100 mg/kg/day (one male and one female).
Other macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls and included diaphragmatic hernia in the liver, an accessory liver, papillary process grown together with left lobe, enlarged papillary process, dark red discolouration of papillary process, pelvic dilation of the kidneys, nodules on the epididymides, cavity formation in the brain, uterus containing fluid, hemorrhagic contents of the stomach, gastro-intestinal tract containing watery fluid, dark red discolouration of the testes, cysts on ovaries and nodules on clitoral gland. These findings are occasionally seen among rats used in these types of study and in the absence of correlated microscopic findings they were
considered changes of no toxicological significance.

Organ Weights
In males at 100 mg/kg/day, lower terminal body weights were found, accompanied by lower absolute weights of heart, liver, kidneys and testes. Relative brain weights were increased and absolute and relative thymus weights were decreased in these males. All of these deviations were attributed to lower terminal body weights.
No changes between absolute and relative organ weights of treated and control females were noted and no changes were noted between males at 30 mg/kg/day and control males.


Microscopic Examination
Primary treatment related findings were recorded in the mesenteric lymph node, small intestine, spleen, stomach and thymus.
In the mesenteric lymph node histiocytosis of a minimal to moderate degree was seen in fifteen rats at 300 mg/kg/day and at a minimal degree in one control rat. At terminal sacrifice this finding was noted at a minimal to moderate degree in all ten animals treated at 100 mg/kg/day.
At 300 mg/kg/day this finding was accompanied by a minimal to moderate degree of granulolymphocytic inflammation in thirteen cases.
In the small intestine, foamy macrophages in the villi at minimal to moderate degree were noted in most high dose rats (unscheduled deaths) in all segments and in several rats at 100 mg/kg/day at terminal sacrifice. In the high dose rats this alteration was accompanied in some individuals by minimal to moderate granulolymphocytic inflammation.
In the spleen animals treated at 300 mg/kg/day there was a reduction in haemopoiesis (primarily erythropoiesis) and in females there were minimal to moderate degrees of lymphoid atrophy.
Squamous hyperplasia of the forestomach of a minimal to moderate degree was recorded in ten high dose animals.
Increased lymphocytolysis of a minimal to moderate degree was noted in the thymus of five 300 mg/kg/day rats and slight to severe lymphoid atrophy in fifteen.
Treatment related findings considered secondary to reduced body weight gain in the high dose rats were seen in the form of atrophy or immaturity in the prostate, seminal vesicles and uterus.
The above microscopic findings were the histological correlates to the macroscopic findings noted at necropsy in these organs.
The remainder of the microscopic findings were within the range of background pathology encountered in Wistar rats of this age and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analysis of Dose Preparations

Samples of 434 -280 -4 in propylene glycol suspensions were analyzed at Chevron Texaco Energy Technology Company to verify their homogeneity and nominal concentrations. A direct dilution procedure was employed to prepare the samples for elemental analysis by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Homogeneity of OLOA 289M in propylene glycol was established. The nominal concentrations of all dosing suspensions were verified by the analytical data.