Triphenylphosphine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2000 - November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz, Essenheimer Straße 144, D-55128 Mainz.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Triphenylphosphine

EC Number:

210-036-0

EC Name:

Triphenylphosphine

Cas Number:

603-35-0

Molecular formula:

C18H15P

IUPAC Name:

triphenylphosphine

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Purity: 99.82%.

SOURCE OF TEST MATERIAL
- Batch No.of test material: "Ablieferungsnummer: 01-0140".
- Date of production: May 18,1999.
- Physical state/color: Solid/white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability was given for 2 years (information of the sponsor).
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in aqua bidest with 0.5% carboxymethylcellulose was demonstrated over a period of up to 24 hours at room temperature. Ac the mixtures were administered directly after preparation, the stability was guaranteed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was pulverised, weighed depending on the dose group. Then the vehicle was filled up to the desired volume and mixed with a magnetic stirrer or highspeed sonicator (ultra turrax). During the administration the test substance peparations were kept homogenous by a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test substance was administered in aqua bidest with 0.5% carboxymethylcellulose.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female Wistar rats were supplied by Charles River GmbH, Sulzfeld, Germany.
- Animal identification: The rats were identified clearly by ear tattoo. The animals were tattooed in consecutive order after arrival and before randomization.
- Females (if applicable) nulliparous and non-pregnant: not specified.
- Age at study initiation: supplied at an age of 33-37 days (males) and 32-36 days (females).
- Weight at study initiation: 120-150g at day -7, 155-192 g at day 0.
- Housing: The rats were housed singly in type DK III stainless steel wire cages from Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 314 dustfree embedding, supplied by SSNIFF, Soest, Germany). The motor activity measurements were conducted in Polycarbonate cages with wire Covers from Ehret, Emmendingen, Germany (floor area about 800 cm2 and small amounts of absorbent material (see above). The animals were housed in a completely air-conditioned room in which a central air-conditioner ensured temperatures and relative humidity values. The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. The walls and the floor were cleaned once a week, in each case using water containing 0.1 % lncidin perfect (supplied by Henkel, Düsseldorf, Germany).
- Diet: The food used was basic maintenance diet for mouselrat, 9433 LL Meal, supplied by Eberle Nafag AG Gossau, Switzerland. Food was available ad libitum (except during motor activity measurements, functional observational batteries and during fasting periods).
- Water (e.g. ad libitum): Drinking water was available ad libitum from water bottles (except during motor activity measurements and functional observational batteries).
- Acclimation period: Only animals free from clinical signs of disease were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C.
- Humidity (%): 30 - 70%.
- Photoperiod (hrs dark / hrs light): 12 hours dark from 6:00 p.m. - 6:00 a.m.
IN-LIFE DATES: From: 28.07.2000 To: 09. an 10. 11.2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: aqua bidest with 0.5% carboxymethylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was pulverised, weighed depending on the dose group. Then the vehicle was filled up to the desired volume and mixed with a magnetic stirrer or highspeed sonicator (ultra turrax). During the administration the test substance preparations were kept homogenous by a magnetic stirrer. The test substance preparations were made daily.

VEHICLE
- Concentration in vehicle: 6, 60 and 120 mg/kg bw/d.
- Amount of vehicle (if gavage): 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in aqua bidest with 0.5% carboxymethylcellulose was demonstrated over a period of up to 24 hours at room temperature. All the mixtures were administered directly after preparation, the stability was guaranteed.

Duration of treatment / exposure:

91 daysgavage

Frequency of treatment:

once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The following dose levels were selected: 120 mg/kg bw/day as high dose level with expected toxic effects; 60 mg/kg bwlday as mid dose level; 6 mg/kg bw/day as low dose level. A factor of 10 between mid and low dose level was selected in order to assure a safe no observed adverse effect level. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Positive control:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: twice a day.
- The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further general clinical examinations were carried out daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: twice a day.

BODY WEIGHT: Yes.
- Time schedule for examinations: The body weight was determined before the first neurofunctional test in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter in weekly intervals. Body weight was also determined on the days when functional observational batteries were carried out. 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Food consumption was determined weekly. The values were calculated as grams per animal per 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.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at the end of the application period (Study day 92).
- Anaesthetic used for blood collection: No.
- Animals fasted: Yes.
- How many animals: 10 animals per test group and Sex.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at the end of the application period (Study day 92).
- Animals fasted: Yes.
- How many animals: How many animals: 10 animals per test group and Sex.

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: on study days -7, 22, 50 and 85.
- Dose groups that were examined: all dose groups.
- Battery of functions tested: Functional observational battery (Home cage observations, Open field observiations, Sensorimotor Tests/Reflexes), Motor activity assessment
- FOBs were performed in all animals as given in the time table, each time starting at 10 a.m.. The FOB started with passive observations, without disturbing the animals, followed by removal from the home cage and Open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. The findings were ranked according to the degree of severity, if applicable. In order to guarantee the blind status of the observer, the cages were randomly distributed in the racks at least 30 minutes prior to the examinations, and the cage labels (indicating the dose group) were turned. Thus only the animal number, but not the allocation of the animals to the different dose groups could be identified by the observer. Moreover, the examinations were carried out in randomized order. The findings and values obtained were documented by another technician knowing the identification of the animals.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals.
- Open field observations: The animals were transferred to a standard arena (50 X 50 cm with sides of 25 cm height) and observed for at least 2 minutes.
- The animals were removed from the Open field and subjected to following sensorimotor or reflex tests.
- Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The cages were cleaned prior to each use. The animals were put into the cages in a randomized order. The measurements started at about 2 p.m. and the number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

IMMUNOLOGY: No.

OTHER: Sperm examination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.

HISTOPATHOLOGY: Yes.

Other examinations:

lmmediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from scheduled animals.
The following parameters were determined:
Sperm motility
Sperm morphology
Sperm head count (cauda epididymis)
Sperm head count (testis)
Sperm motility examinations were carried out in a randomized sequence.

Statistics:

Statistics of clinical examinations, statistics of clinical pathology and statistics of pathology.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two high dose female animals showed anogenital region smeared with urine (slight) during the later phase of the study. A relationship to treatment cannot be excluded with certainty.
One low dose female animal showed loss of tail end. This was clearly incidental in nature.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight data were similar for all groups, including the controls. No substance-related findings were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar for all groups, including the controls. No substance-related findings were observed.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency was statistically significantly decreased in mid dose males on day 84. Due to the isolated occurrence and the lack of a dose-response relationship, this was clearly incidental.

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

no effects observed

Description (incidence and severity):

Water consumption was similar for all groups, including the controls. No substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period prothrombin times were significantly shortened in the plasma of the mid and high dose females. No effects related to treatment were seen in the other hematology parameters measured.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Enzyme examinations revealed reduced alanine aminotransferase and aspartate aminotransferase activities in the serum of the mid and high dose females after 3 months of test substance administration. In the serum of the low dose females the activity of
aspartate aminotransferase was also decreased at the end of the study. The decrease in aspartate aminotransferase activities was dose dependent (-29%, -33%, -38% at 6, 60 and 120 mg/kg bw), but not considered as of toxicological relevance by the study authors (increase in activities advers but decrease questionable). No treatment related changes were found in the other enzyme measurements.
Blood chemistry investigations showed decreased glucose levels in the serum of the high-dose males and increased calcium, total protein, globulin and triglyceride concentrations in the peripheral blood of the high dose females at the 3-month interval. No toxicologically relevant changes were seen in the other blood chemistry parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The treatment with the substance did not influence findings in the functional observation battery.
No significant or biologically relevant changes were observed in the motor activity assessment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean liver weights of males of the top dose showed a trend towards an increase (approx. 14%). The mean weights of liver of females of group 2 (approx. 16%) and top dose (approx. 31 %) showed also a significant increase.
A significant increase was noted for the adrenal glands of males of group 1 (approx. 28%) and group 2 (approx. 28%). In the female top dose, the mean kidney weights were significantly increased (approx. - 15%).

In the male top dose, the relative organ weights of liver were significantly increased (approx. 18%). A significant increase was noted for the liver of females of the top dose (approx. 30%). Also the relative weights of kidneys of top dose females were significantly increased (approx. 13%).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Forestomach: The macroscopic finding "Thickening of wall" was recorded for one female animal of the top dose.
Glandular stomach: The gross lesion "Erosion/ulcer" was diagnosed for one female animal of group 1, one female group 2 and for two female animals of the top dose.
Lungs: One of the male control animals showed an "Adhesion".
Kidneys: A "Pelvic dilation" was detected in one female group 1 animal.
Ureter: In the dose group 1, one female animal showed a "Dilation".
Testes: A "Focus" was found in one male top dose animal.
Thymus: One of the male control animal showed a "Reduced Organ size".

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Axonal degeneration was found in the proximal sciatic nerve in one top dose female and axonal degeneration was also seen in the sural nerve of another top dose female out of five examined female animals.
These single findings were assessed as being of spontaneous or incidental nature and not related to treatment. No other significant substance-related effects were seen after detailed examination of the central and peripheral nervous system.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In both sexes, centrilobular hepatocyte hypertrophy was observed at 60 and 120 mg/kg bw/d in both sexes. The extent and higher incidence of centrilobular hypertrophies in the dose groups is regarded to be treatment - related.

Description (incidence and severity):

Sperm analysis: Slightly decreased spermatid counts were found in the testes of the high dose males at the end of the study. In the other sperm parameters no treatment-related effects were seen.

Details on results:

Food consumption, water consumption, and body weight data were similar for all groups, including the controls. The treatment with the substance did not influence findings in the functional observation battery. No significant or biologically relevant changes were observed in the motor activity assessment. Absolute adrenal weight of male animals in the low- and mid-dose groups was increased, but without dose-dependency, and no increase was seen in high-dose animals. The effect was therefore considered as of no biological relevance. The only finding at 6 mg/kg bw/day was a significantly decrease in aspartate aminotransferase activity in females. The decrease in aspartate aminotransferase activities was dose dependent (-29%, -33%, -38% at 6, 60 and 120 mg/kg bw), but not considered as of toxicological relevance by the study authors (increase in activities advers but decrease questionable). In females 60 mg/kg bw/day induced a decrease in plasma prothrombin time, a statistically significant increase in absolute liver weights (+16%) and a slight increase in relative liver weights (+11%, not significant), and, in both sexes, centrilobular hepatocyte hypertrophy. At 120 mg/kg bw/day, increased levels of calcium, total protein, globulin and triglycerides were found in peripheral blood of females, decreased serum glucose levels in males and a shortened plasma prothrombin time in females. Liver weights were statistically significant increased in males (+14% absolute weight, +18% relative weight) and in females (+31%, absolute weight, s.s., +30% relative weight). The observed changes concerning the liver and in clinical chemistry are indicative of liver enzyme induction. Kidney weights were statistically significant increased in females (+15% absolute weight, +13% relative weight), and centrilobular hypertrophy of hepatocytes was found in both sexes. Axonal degeneration was found in the proximal sciatic nerve in one top dose female and axonal degeneration was also seen in the sural nerve of another top dose female out of five examined female animals. These single findings were assessed as being of spontaneous or incidental nature and not related to treatment. No other significant substance-related effects were seen after detailed examination of the central and peripheral nervous system. The NOAEL of this study was at 6 mg/kg bw/day, critical effects at the LOAEL (60 mg/kg bw) were a decrease in plasma prothrombin time and a slight increase in liver weights in females, and, in both sexes, centrilobular hepatocyte hypertrophy.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

6 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects at this dose

2

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance caused changes associated with induction of the microsomal enzyme system in the liver, increased liver weights and centrilobular hypertrophy of hepatocytes. Moreover, decreases in alanine and aspartate aminotransferase activities were seen. This fall in alanine and aspartate aminotransferase is considered not to be an adverse toxic effect. No signs of neurotoxicity were observed.

Executive summary:

The test substance was administered to groups of 10 male and 10 female Wistar rats by gavage at dose levels of 0 (vehicle control), 6, 60 and 120 mg/kg body weight/day (mg/kg bw/day) for 3 months. The vehicle used was aqua bidest with 0.5% carboxymethylcellulose, and the administration volume was 5 ml/kg bw.

Food consumption was determined once a week. Body weight was determined once a week and on the days when functional observational batteries were performed. A check of the general state of health was made at least daily. The animals were observed at least twice a day and palpated once a week. Functional observational batteries and motor activity measurements were carried out in 10 animals per sex and group on days -7, 22, 50 and 85. Clinicochemical, hematological examinations and sperm analyses were carried out towards the end of the administration period. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. The remaining animals were subjected to gross-pathological assessment, followed by histopathological examinations.

The following substance-related findings were observed:

120 mg/kg body weight:

• two high dose female animals showed an anogenital region smeared with urine (slight) during the later phase of the study (slight) during the later phase of the study
• increased calcium, total protein, globulins and triglycerides in the females
• decreased glucose in the males
• shortened prothrombin times in the females
• decreased alanine aminotransferase and aspartate aminotransferase in the females
• increased liver weights in both sexes
• centrilobular hypertrophy of hepatocytes in both sexes

60 mg/kg body weight:

• shortened prothrombin times in the females
• decreased alanine aminotransferase and aspartate aminotransferase in the females
• increased liver weights in females
• centrilobular hypertrophy of hepatocytes in both sexes

6 mg/kg body weight:

• decreased aspartate aminotransferase in the females

In conclusion, the oral administration of the test substance caused changes associated with induction of the microsomal enzyme system in the liver, increased liver weights and centrilobular hypertrophy of hepatocytes. Moreover, decreases in alanine and aspartate aminotransferase activities were seen. This fall in alanine and aspartate aminotransferase is considered not to be an adverse toxic effect. Thus, the no observed adverse effect level (NOAEL) is 6 mg/kg body weight. No signs of neurotoxicity were observed .