Triphenylphosphine

Endpoint summary

Description of key information

Repeated oral toxicity:

Experimental test data from several species are available, suggesting that rats are relatively insensitive towards the test substance and dogs being the most sensitive species.

Under the conditions of a 90-day combined Repeated Dose Toxicity Study with a Neurotoxicity study, the oral administration of the test item in an aqueous vehicle by gavage to Wistar rats revealed adverse findings at 60 mg/kg bw/d regarding organ weights and clinical biochemistry endpoints. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 6 mg/kg bw/d [BASF, 2002].

Oral Application of the test substance suspended in an oily vehicle over 28 days caused clinical signs of neurotoxicity in rabbits with a NOAEL of <100 mg/kg bw/d [BASF, 1973] and dogs with a NOAEL of 1 mg/kg bw/d [M&T Chemicals, 1983], which were accompanied with axonal degeneration in the dog study (LOAEL 5 mg/kg bw/d).

Repeated inhalation toxicity:

Experimental test data from several species are available, suggesting that rats are relatively insensitive towards the test substance and dogs beeing the most sensitive species.

An aerosol of the test substance in xylol produced signs of neurological impairment at 28 mg/m3 in a 4-wk inhalation study in dogs which correlated with neurotoxic tissue effects (NOAEC 10 mg/m3) [M&T Chemicals, 1983].

An aerosol of approximately 2400 mg/m3 generated by nebulizing the molten test substance, however, didn’t produce neurotoxic effects in rats during a 12-day exposure but led to clinical signs of mild respiratory irritation like salivation, lacrimation, dyspnea and red ears [Waritz, 1975].

Repeated dermal toxicity:

No data available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

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.

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

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

Critical effects observed:

no

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 .

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1970 -1971

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study procedure scientifically and as screening acceptable, giving evidence of the targets of intoxication. No histopathology, no control.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Aim of the study was to obtain knowledge about the effect of the test substance given in acute non-lethal doses using a BASF-internal standard method.
- Principle of test: The subacute oral toxicity of an A) oily or B) aqueous preparation of the test substance was tested. Therefore, a 5-20% supension in olive oil or a 1-8% supension in water/0.5% CMC equivalent to doses of 100, 200, 400 mg/kg bw/d or 100, 200, 400 and 800 mg/kg bw/d was administered daily 5 times a week by gavage for a period of 28 days. After the last application the animals were observed for 28 days. At the end of the observation period the animals were examined for pathological changes.
- Parameters analysed / observed: The behvior, external characteristics and mortality of the animals were controlled daily. Once before the start of the experiment and once weekly up to its end weight was recorded and blood and urine were collected for hematology and clinical chemistry. Deaceased and surving animals were subjected to pathoanatomical examinations.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Purity: 99%

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in A) olive oil and B) 0.5% CMC

Species:

rabbit

Strain:

other: mongrel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mixed-breed rabbits from the Medical Biology Research Laboratory of BASF.
- Weight at study initiation: about 2.5 kg
- Housing: The animals were kept in individual. wire cages in air-conditioned rooms.
- Diet: ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 2°C.
- Humidity (%): 55 + 5%.
- Photoperiod (hrs dark / hrs light): day/night rhythm of 7 - 18.00 h light and 18 - 7.00 h dark.

Route of administration:

oral: gavage

Vehicle:

other: A) olive oil and B) aqueous suspension with 0.5% CMC.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days per week, once per day (max. 20 exposures)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

suspension in olive oil

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

suspension in olive oil

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

suspension in olive oil

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

aqueous suspension with 0.5% CMC

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

aqueous suspension with 0.5% CMC

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

aqueous suspension with 0.5% CMC

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

aqueous suspension with 0.5% CMC

No. of animals per sex per dose:

A total of 18 male and 9 female animals were used.
3-4 animals per dose group were used.

Control animals:

not specified

Details on study design:

screening study
- Dose selection rationale: doses which were not lethal in the acute study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: daily.

BODY WEIGHT: Yes.
- Time schedule for examinations: Once before the start of the experiment and once weekly up to its end.

The behavior, external characteristics and mortality of the animals were controlled daily. Once before the start of the experiment and once weekly up to its end weight was recorded and blood and urine were collected for hematology and clinical chemistry. Deaceased and surving animals were subjected to pathoanatomical examinations.

HAEMATOLOGY: Yes.
- Parameters: Hemoglobin (g/100 ml), Hematocrit (%), RBC (10^6/μl), WBC (10^3/μl), Differential WBC (%).
- Time schedule for collection of blood: Once before the start of the experiment and once weekly up to its end.
- Anaesthetic used for blood collection: Not specified.
- Animals fasted: Not specified.
- How many animals: all.

CLINICAL CHEMISTRY: Yes.
- Parameters: BSP test (% retention), SGPT (mU/ml), Urea (mg/100 ml).
- Time schedule for collection of blood: Once before the start of the experiment and once weekly up to its end.
- Animals fasted: Not specified.
- How many animals: all.

URINALYSIS: Yes.
- Parameters: pH, Albumin (g/100 ml), Glucose (mg/100 ml), Urobilinogen, Sediment.
- Time schedule for collection of blood: Once before the start of the experiment and once weekly up to its end.
- Animals fasted: Not specified.
- How many animals: all.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.

HISTOPATHOLOGY: No.

Statistics:

none

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A) Oily suspension (olive oil):
- 400 mg/kg: weakness of the hind legs, lateral decubitus with convulsive running movements, atonia, unsteady locomotion, tremors and anorexia were observed.
- 200 mg/kg: same symptoms as at 400 mg/kg.
- 100 mg/kg: transient anorexia and only occasional unsteadiness and careful locomotion.
------------------------------------
B) Aqueous suspension (0.5% CMC):
- 800 mg/kg: disorders in locomotion, lateral decubitus, atonia, tonic and clonic spasms with opisthotonus, tremors, unsteady locomotion and arorexia.
- 400 mg/kg: same symtoms like at 800 mg/kg but less pronounced
- 200 mg/kg: intermittent anorexia and only minor and transient signs of atonia and careful locomotion
- 100 mg/kg: were tolerated without intoxication symptoms.

Mortality:

mortality observed, treatment-related

Description (incidence):

A) Oily suspension (olive oil):
- 400 mg/kg: 3/3 animals died after 3 - 6 applications.
- 200 mg/kg: 2/4 animals died after 4 - 5 applications.
- 100 mg/kg: No mortality.
------------------------------------
B) Aqueous suspension:
- 800 mg/kg: 3/4 animals died after 4 - 9 applications.
- 400 mg/kg: 2/4 animals died after 5 - 7 applications.
- 200 mg/kg: 1/4 animals died after 13 applications
- 100 mg/kg: No mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group A as well as B, some animals showed a weight loss during repeated application of 800, 400 and 200 mg/kg.
With the exception of one animal, no weight loss occurred in group A after 20 oral applications of 100 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A) Oily suspension (olive oil):
- 400 mg/kg: A dose of 400 mg/kg x 6 revealed no changes of the hematological and biochemical parameters in the surviving animal.
- 200 mg/kg: in the two animals which survived 20 doses led to a decrease of Hb, Ht and RBC as well as an elevation of blood urea; all of these values normalized largely already during the experiment or in the follow-up period. The SGPT showed a transient tendency to a slight elevation during application of the substance, but the peak values were still within normal limits and dropped to the base level in the follow-up period.
- 100 mg/kg: decrease of Hb, Ht and RBC; the levels normalized in the follow-up period. 2/4 animals developed a slight but reversible elevation of BSP retention and SGPT also showed a tendency to increase, but the values remained within the range of physiological variation and returned to the initial level in the follow-up period. A transient elevation of blood urea was also observed.
- The differential WBC showed a shift to the left during the experiment with all doses and in some cases, this persisted at the end of the follow-up period.
------------------------------------
B) Aqueous suspension (0.5% CMC):
- 800 mg/kg: animals which survived 20 applications showed a decrease of Hb, Ht and RBC as well as elevation of SGPT and blood urea. All changes were largely reversible. No such changes were observed in the animals which had received 800 mg/kg 4-9 times.
- 400 mg/kg: moderate decrease of Hb, Ht and RBC, and SGPT was clearly elevated in one animal and blood urea in two. Except for one blood urea value, the others normalized already during the experiment or in the follow-up period.
- 200 mg/kg: slight changes of the hematological and biochemistry parameters and these were also reversible already during the experiment or in the follow-up period.
- 100 mg/kg: same as at 200 mg/kg.
All experimental groups showed a shift to the left of the differential WBC which in some cases could still be detected during the
follow-up period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine analysis revealed no effects.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A) Oily suspension (olive oil):
- Cardiac dilatation associated with venous hyperemic congestion at 400 mg/kg bw and in animals which died during exposure period after 200 mg/kg bw, no effects in surviving animals of this group as well as in rabbits of the low dose group.
------------------------------------
B) Aqueous suspension (0.5% CMC):
- The high-dosed animals showed cardiac dilatation associated with venous hyperemic congestion, in 2 cases small necrotic foci and fat deposits in the liver. Same effects at 400 mg/kg bw in animals which died during exposure period, no effects in survivors. No effects at <= 200 mg/kg bw.

Dose descriptor:

NOAEL

Remarks on result:

other: not determinable due to adverse toxic effects at the lowest tested dose

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

nervous system

Organ:

other: nervous system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

The test substance administered in the oily suspension proved to be more toxic than when given in aqueous suspension, probably due to facilitated bioavailability of the test substance in oil.

Executive summary:

The object of this study was to follow up the acute toxicity test by investigating the effect of the test substance with repeated oral application of doses which were not lethal in the acute study.

Therefore, the test substance was administered repeatedly to groups of 3-4 rabbits in the form of A) oily suspensions as well as B) aqueous suspensions in carboxymethyl cellulose (CMC) in doses of 100, 200 and 400 mg/kg bw/d or 100, 200, 400 and 800 mg/kg bw/d, respectively. All animals were intubated daily (5 times a week), a total of 20 times during an exposure period of 28 days. After the last intubation, followup observation continued for 28 days, after which the rabbits were sacrificed and autopsied. The behavior, external characteristics and mortality of the animals were controlled daily. Once before the start of the experiment and once weekly up to its end weight was recorded and blood and urine were collected for hematology and clinical chemistry. Deaceased and surving animals were subjected to pathoanatomical examinations.

Repeated applications of the test substance produced hyperexcitability, convulsions with subsequent atonia, lataral decubitus and unsteady locomotion from 100 mg/kg or 200 mg/kg bw/day onwards, when the oily or aqueous vehicle was used, respectively. Mortality was observed from 200 mg/kg onwards with both vehicles. The degree of the symptoms was dose-dependent.

The test substance appeared to be absorbed more readily from the oily phase than from the aqueous phase in the subacute experiment similar to the acute studies.

Repeated doses of the test substance led to a transient deterioration of the red blood cell count as well as of liver and kidney function, and sometimes also of the differential white blood cell count.

However, these changes were reversible even during the study or during the four-week followup period.

After doses of 800 and 400 mg/kg, the animals which died showed signs of heart enlargement and in several cases, fine necrotic foci and fatty degeneration of the liver.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable test method based on scientific principles with detailed documentation, only 2 animals per dose group

Principles of method if other than guideline:

Procedure: Evaluation of effects produced by oral gavage. This study was conducted to assess the oral toxicity of the test substance when administered via gastric intubation in a corn oil vehicle to male and female dogs. Four dose levels and two different durations of treatment were used in the study; the specific test regimes were as follows: 2 dogs (1 male, 1 female) per group received 20 doses over four weeks at 1, 5, 10 and 20 milligrams test substance per kilogram (mg/kg); 2 doses were administered to 2 dogs (l male, 1 female) per group at concentrations of 1, 5 and 20 mg/kg. These shorter duration treatments were followed by a four week recovery period. A concurrent vehicle control group (1 male, 1 female) received 20 doses of corn oil, the volume was comparable to that given the test substance treated animals.
Each batch of treated corn oil was prepared fresh daily and the level of test substance in the vehicle was determined by gas chromatographic and/or high pressure liquid chromatographic methods.
Body weight measurements, physical observations and neurological examinations were performed pre-test and weekly throughout the study. Complete gross postmortem examinations were performed and selected sections of nervous system tissue were preserved from all animals for hisopathological evaluation.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Purity: 94.9%

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 7 months initiation of dosing.
- Weight at study initiation: males: 11.2 kg, females 9.3 kg.
- Housing: individually in metal grid cages.
- Identification: Each dog was assigned a unique identification number which appeared on an ear tag and on the animals cage. In addition, each animal bore an ear tattoo with a USDA number.
- Diet: standard laboratory diet (approx. 400 g/day).
- Water: city tap water ad libitum.
- Acclimation period: Yes, minimum 4 weeks (4 - 5 months at receipt and 6 - 7 months at initiation of dosing).
- Immunization: against distempter, hepatitis and leptospirosis by supplier.
- Parasites: fecal examination conducted during pretest an all animals during the equilibration period.

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Appropriate amounts of the test substance were dissolved in Mazola corn oil.
- Dosing solutions were prepared daily. Individual doses were adjusted on the basis of the most recent weekly body weight data.
- Dosing volume: 1 ml/kg across all groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling: 10 ml samples were taken from each dosing solution at least once a week for analytical verification of dosing solution concentrations.
Analysis of the daily dosing solutions of the test substance in corn oil was performed using a gas chromatograph.

Duration of treatment / exposure:

20 doses during 4 weeks exposure period

Frequency of treatment:

2 - 5 days/week (totally 20 doses)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

No. of animals per sex per dose:

1

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Animals were observed twice daily for incidence of abnormal signs.
- Observations of any unusual conditions were recorded when made.
- Full lphysical and neurological assessments were performed weekly.
- Individual body weights were recorded pretest and weekly during exposure and/or recovery period.

Sacrifice and pathology:

- Tissues fixed: brain, sciatic nerve with muscle, spinal cord
- Fixative: all tissues - 10% neutral buffered formalin.
- Histopathology: slides of tissue sections were prepared and examined microscopically from all animals; sections of all tissues were prepared with H&E stain and luxol fast blue stain.
- Sciatic nerve (right and left): 1 longitudinal secion (on each); 2 transverse sections (on each).
- Cervical spinal cord: 1 longitudinal section; 2 transverse sections
- Lumbar spinal cord: 1 longitudinal section; 2 transverse sections

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No physical signs of toxicity but neurological observations revealed the following signs: At 5, 10, and 20 mg/kg (20 exposures), male dogs showed indications of neurological impairment: failure in the extensor postural thrust reflex with tendency to knuckle over, unsteadiness and weakness in the hind limbs, failure of the patellar reflexes. Indications of similar neurological lesions were found in two female dogs having received 20 doses of 1 and 2 doses of 5 mg/kg, but not at the higher doses. The effects noted in male dogs were considered treatment-related as no such signs were observed in the controls and the other groups. The cause of the effects in the low-dosed female dogs was unclear.

Mortality:

no mortality observed

Description (incidence):

All animals survived.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight development was normal.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related post-mortem findings.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

The sections prepared from male and female dogs after 20 exposures to 10 or 20 mg/kg bw revealed mild to moderate generalised vacuolative axonal lesion in the ventral and lateral funicule of the spinal cord. No such findings in the sciatic nerve.
Only isolated damaged axons were observed in other groups including controls. Also in a histopathological reevaluation compound related axonal degeneration was evident only at 10 and 20 mg/kg bw.

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects at this dose

Dose descriptor:

LOAEL

Effect level:

5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

System:

nervous system

Organ:

other: nervous system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Executive summary:

This study was conducted to assess the oral toxicity of the test substance when administered via gastric intubation in a corn oil vehicle to male and female dogs.Four dose levels and two different durations of treatment were used in the study; the specific test regimes were as follows: 2 dogs (1 male, 1 female) per group received 20 doses over four weeks at 1, 5, 10 and 20 milligrams test substance per kilogram (mg/kg); 2 doses were administered to 2 dogs (l male, 1 female) per group at concentrations of 1, 5 and 20 mg/kg. These shorter duration treatments were followed by a four week recovery period. A concurrent vehicle control group (1 male, 1 female) received 20 doses of corn oil, the volume was comparable to that given the test substance treated animals.

Each batch of treated corn oil was prepared fresh daily and the level of test substance in the vehicle was determined by gas chromatographic and/or high pressure liquid chromatographic methods.

Body weight measurements, physical observations and neurological examinations were performed pre-test and weekly throughout the study. Complete gross postmortem examinations were performed and selected sections of nervous system tissue were preserved from all animals for hisopathological evaluation.

All animals survived the duration of the study. Signs of neurological impairment were noted in male dogs receiving 20x 5, 10 or 20 mg/kg test substance doses. Similar findings were noted in two female dogs which received 1 or 5 mg/kg doses for twenty or two treatments, respectively; however, these findings were not noted in female dogs receiving either two or twenty treatments at higher dose levels.

Microscopic pathology evaluations (H&E, and luxol fast blue stains) revealed mild to moderate generalized vacuolative axonal lesions in the ventral and lateral funicle of the spinal cord at both sexes of dogs receiving twenty doses of 20 mg/kg. Similar lesions, although minimal and focal in nature, were noted in spinal cord sections in dogs receiving 1 or 10 mg/kg test substance. In

the group receiving 1 mg/kg (2 or 20 doses) only females exhibited this change. These lesions were not seen in any dog receiving only corn oil.

Evaluations of body weight, general physical observations (non-neurological) and gross postmortem results were considered unremarkable.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subacute

Species:

dog

System:

nervous system

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable test method based on scientific principles with detailed documentation, only 2 animals per dose group

Principles of method if other than guideline:

This study was conducted to assess the inhalation toxicity of the test substance when administered as a respirable aerosol in a xylene vehicle to male and female beagle dogs. Four atmospheric concentration levels and two different durations of treatment were used in the study; the specific test regimes were as follows: two dogs (1 male, 1 female) per group received twenty 6-hour exposures over four weeks at exposure levels of 0.5, 3.2. 9.7 and 28 milligrams test substance per meter cubed (mg/m3); two 6-hour exposures were administered to two dogs (1 male, l female) per group at concentrations of 0.3, 3.6 and 24 mg/m3 test substance. These shorter duration treatments were followed by a four week recovery period. A concurrent vehicle control group (1 male, 1 female dog) received twenty 6-hour exposures to150 parts per million (v/v in air) of xylene, a level of solvent which was comparable to that in each test exposure chamber. Chambers were monitored analytically for test substance levels at least 3 times/chamber/day. Particle size distribution were determined periodically throughout the study. Body weight measurements, physical observations and neurological examinations were performed pre-test and weekly throughout the study. Complete gross postmortem examinations were performed and selected sections of nervous system tissue were preserved from all test animals for histopathological evaluation.

GLP compliance:

not specified

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

body weight 9.6-13.2 kg in males and 8.2-10.4 kg in females

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Details on inhalation exposure:

- Analytical concentrations of 0, 0.5, 3.2, 9.7, 28 mg/m3 (as aerosol in xylene, aerosol particles 0.5 um) or for only 2 times to 0.5, 3.2 or 28 mg/m3 (post exposure observation period 30 days).
- Whole-body exposure using chambers of 10 m3 which received an aerosol produced from the test substance dissolved in xylene (complete air change in the chamber every 3.3 minutes).
- The concentration of xylene was 150 ppm or less in all chambers (solvent not specified, isomer mixture??).
- Chambers were monitored analytically for test substance levels 3x/d per chamber and particle-size distribution periodically.
- The count medium diameter of the aerosol particles was found to be 0.5 um or less, i.e. respirable.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

5 weeks

Frequency of treatment:

3 - 5 days/week, 6 hours/day (20 exposures)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

as aerosol in xylene

Dose / conc.:

3.2 mg/m³ air (nominal)

Remarks:

as aerosol in xylene

Dose / conc.:

9.7 mg/m³ air (nominal)

Remarks:

as aerosol in xylene

Dose / conc.:

28 mg/m³ air (nominal)

Remarks:

as aerosol in xylene

No. of animals per sex per dose:

1

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Body weight determined once weekly. Animal observation twice daily, full physical and neurological assessment once weekly.

Sacrifice and pathology:

Dogs were sacrificed and necropsy performed. Histopathologic examination was focussed on the spinal cord and sciatic nerve.

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 28 mg/m3 (20 exposures), both dogs showed indications of neurological impairment: failure in the extensor postural thrust reflex with tendency to knuckle over, unsteadiness and weakness in the hind limbs. (This effect was already noted in one male dog having received only two exposures.) The locomotor reflex in the male dog appeared also unsteady and uncoordinated (ataxia).
At 10 mg/m3 erratic placement of limbs on week 1 and 4 was observed in one male dog.
In one male receiving only 2 exposures of 3 mg/m3 some limb placement incoordination throughout the study was seen.
Only the effects at the highest dose were considered by the authors to be treatment-related (in other dose groups effects not persistent, no severe signs, animals were only exposed twice).

Mortality:

no mortality observed

Description (incidence):

All animals survived.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight development was normal.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

The sections prepared from the high-dosed dogs revealed moderate generalised vacuolative degeneration of axons in the ventral and lateral funicule of both the cervical and lumbar spinal cord. No such findings in the sciatic nerve. No such lesions were noted in the control animals having received xylene alone.
The above mentioned neuronal lesions were seen in the longitudinal sections, recognised with both H&E and luxol fast blue stains, characterised by multiple dilated or vacuolated axon sheaths with accumulations of scattered eosinophilic debris and occasional dark rounded bits of what appeared to have been nuclear chromatin.
Similar damages in the lower dose groups, but marginal in nature, were not considered to correlate with test substance exposure since in a histopathological reevaluation also historical control animals showed some axonal lesions. Sporadic degeneration is an expected finding in the spinal cord of "normal" animals.

Dose descriptor:

NOAEC

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse effects at this dose

Dose descriptor:

LOAEC

Effect level:

28 mg/m³ air

Sex:

male/female

Basis for effect level:

clinical signs

Critical effects observed:

not specified

Executive summary:

This study was conducted to assess the inhalation toxicity of the test substance when administered as a respirable aerosol in a xylene vehicle to male and female beagle dogs.

 

Four atmospheric concentration levels and two different durations of treatment were used in the study; the specific test regimes were as follows: two dogs (1 male, 1 female) per group received twenty 6-hour exposures over four weeks at exposure levels of 0.5, 3.2. 9.7 and 28 milligrams test substance per meter cubed (mg/m³); two 6-hour exposures were administered to two dogs (1 male, 1 female) per group at concentrations of 0.3, 3.6 and 24 mg/m³ test substance. These shorter duration treatments were followed by a four week recovery period. A concurrent vehicle control group (1 male, 1 female dog) received twenty 6-hour exposures to 150 parts per million (v/v in air) of xylene, a level of solvent which was comparable to that in each test exposure chamber.

Chambers were monitored analytically for test substance levels at least 3 times/chamber/day. Particle size distribution were determined periodically throughout the study. Body weight measurements, physical observations and neurological examinations were performed pre-test and weekly throughout the study. Complete gross postmortem examinations were performed and selected sections of nervous system tissue were preserved from all test animals for histopathological evaluation.

 

All animals survived the duration of the study. Signs of neurological impairment were noted during the study in male dogs receiving 2 exposures at 24 mg/m³ test substance or 20 exposures at 28 mg/m³ test substance and in the female dogs receiving 20 exposures at 28 mg/m³test substance.

Histopathological examinations"(H&E, and luxol fast blue stains) revealed moderate generalized vacuolative degeneration of the ventral and lateral funicle of the cervical and lumbar spinal cord of the dogs which received twenty exposures to 28 mg/m³ test substance. Similar lesions, although minimal and focal in nature were noted in spinal cord sections of dogs receiving 0.5 or 3.2 mg/m³test substance. In the lowest treatment group animals, this lesion was observed in one male following 20 exposures and in·one female following only two exposures. These lesions were not seen in any vehicle control dog. Evaluations of body weight, general physical observations (non-neurological) and gross postmortem results were considered unremarkable.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subacute

Species:

dog

System:

nervous system

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable test method based on scientific principles without detailed documentation

Qualifier:

no guideline followed

Principles of method if other than guideline:

Accumulation of sufficient animal toxicity data to form a basis for an evaluation of the safety.

GLP compliance:

no

Species:

rat

Strain:

other: CHR-CD

Sex:

male

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Details on inhalation exposure:

- Whole-body exposure in 18-l glass bell jars; six animals per test group.
- The atmospheric concentration was generated by nebulizing molten test substance (maintained at 90 - 115°C under dry nitrogen) and diluting the enriched nitrogen stream with oxygen to a 20-% oxygen content immediately prior to entering the exposure chamber.
- Post-observation period: 14 days

Duration of treatment / exposure:

12 days

Frequency of treatment:

4 hours per day; 10 exposures from day 1 - 5 and day 8 - 12

Dose / conc.:

2 400 mg/m³ air

Remarks:

(9.32 uM/l) (approx. 1/5 LC50)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

Concentrations of the test substance were analysed prior and after exposure by IR spectrocopy (limit of detection 1.9 umol) and did not indicate decomposition. During exposure the test substance was analysed at least hourly.

Observations and examinations performed and frequency:

Comprehensive macroscopic and microscopic examination was carried out on varies organs including lung, liver, spleen, kidney, reproductive organs, lymph nodes and others.

Sacrifice and pathology:

Three test and three control animals each were sacrificed immediately after exposure period and 14 d after the last exposure for gross and histopathologic examination.

Statistics:

no data

Description (incidence and severity):

Clinical signs were typical of mild respiratory irritation: salivation, lacrimation, dyspnea, red ears.

Description (incidence and severity):

Exposed animals showed slight retardation in body weight at exposure day 10-12, returned to normal thereafter but did not catch up with the final mean weight of the control (difference <10%).

Description (incidence and severity):

No treatment-related macroscopical or histopathological findings.

Description (incidence and severity):

Brown discoloration of the fur during the 2nd week of exposure was noted, but cleared promptly after cessation of treatment.

Dose descriptor:

NOAEC

Sex:

male

Remarks on result:

other: A NOAEC was not identified

Dose descriptor:

LOAEC

Effect level:

2 400 mg/m³ air

Sex:

male

Basis for effect level:

other: unspecified

Critical effects observed:

no

Executive summary:

A subacute 12-day repeated inhalation toxicity study was conducted using male rats. Concentrations of 0 or approx. 2400 mg/mg3 were generated by nebulizing molten test substance. Clinical signs were typical of mild respiratory irritation: salivation, lacrimation, dyspnea, red ears. No test substance-related macroscopical or histopathological findings were seen. A NOAEC was not identified; the LOAEC was found to be 2400 mg/m3 for male rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2 400 mg/m³

Study duration:

subacute

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated oral toxicity:

Experimental test data from several species are available, suggesting that rats are relatively insensitive towards the test substance and dogs being the most sensitive species. Therefore, the endpoint conclusion is based on the experimental data on dogs. Moreover, the vehicle had an influence on the toxicity of the test substance since oily suspensions produced adverse effects at lower concentrations than aqueous suspensions, presumably due to higher bioavailability.

BASF (2002) reported a 3-month neurotoxicity gavage study according to OECD guideline 408 testing aqueous suspension of the test substance with 0.5% CMC. Male and female rats were given doses of 6, 60 or 120 mg/kg. 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. 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. The NOAEL was found to be 6 mg/kg and the LOAEL was found to be 60 mg/kg for male and female rats.

Male and female rabbits were used in a gavage screening study over 4 weeks with doses of 100, 200, 400 or 800 mg/kg as aqueous suspension with 0.5% CMC or doses of 100, 200 and 400 mg/kg as suspension in olive oil (BASF,1973). Repeated applications of the test substance produced signs of neurological impairment like hyperexcitability, convulsions with subsequent atonia, lataral decubitus and unsteady locomotion from 100 mg/kg or 200 mg/kg bw/day onwards, when the oily or aqueous vehicle was used, respectively. Mortality was observed from 200 mg/kg onwards with both vehicles. The degree of the symptoms was dose-dependent and generally more prominent with the oil-based preparation than with the aqueous suspension. In line with the results from the acute toxicity studies, the test substance appeared to be absorbed more readily from the oily phase than from the aqueous phase in this subacute experiment. Repeated doses of the test substance led to a transient deterioration of the red blood cell count as well as of liver and kidney function, and sometimes also of the differential white blood cell count. However, these changes were reversible even during the study or during the four-week followup period. After doses of 800 and 400 mg/kg of both vehicle groups and of 200 mg/kg with the oil-based vehicle, the animals which died showed signs of cardiac dilatation associated with venous hyperemic congestion in several cases, fine necrotic foci and fatty degeneration of the liver. No histopathological examinations were conducted in this study. A NOAEL was not identified; the LOAEL for male and female animals was found to be 100 mg/kg based on clinical signs.

M&T Chemicals Inc. (1983) reported a 4-week oral toxicity study using dogs in which the test substance was applied in corn oil. Doses used were 1, 5, 10 or 20 mg/kg bw/day. All animals survived; body weight development was normal. No physical signs of toxicity but neurological observations revealed the following signs: At 5, 10, and 20 mg/kg (20 exposures), male dogs showed indications of neurological impairment: failure in the extensor postural thrust reflex with tendency to knuckle over, unsteadiness and weakness in the hind limbs, failure of the patellar reflexes. Similar signs of neurological impairment were found in two female dogs having received 20 doses of 1 mg/kg and 2 doses of 5 mg/kg, but not at the higher doses. The effects noted in male dogs were considered treatment-related as no such signs were observed in the controls and the other groups. The cause of the effects in the low-dosed female dogs was unclear. Macroscopic pathology: No treatment-related post-mortem findings. Histopathology: The sections prepared from male and female dogs after 20 exposures to 10 or 20 mg/kg bw revealed mild to moderate generalised vacuolative axonal lesion in the ventral and lateral funicule of the spinal cord. No such findings in the sciatic nerve. Only isolated damaged axons were observed in other groups including controls. In a histopathological reevaluation compound related axonal degeneration was evident only at 10 and 20 mg/kg bw. The NOAEL was 1 mg/kg bw/day for male and female dogs (based on clinical signs of ataxia/impairment of reflexes).

BASF (1952) conducted a study on cats in which one animal received 300 mg/kg daily, another animal received 100 mg/kg daily (5 times per week). A daily oral dose of 300 mg/kg bw caused no symptoms during the first 5 days of application. Severe clinical signs were observed from day 8 onwards: impaired balance, ataxia, paralysis of hindlegs, followed by occasional convulsions and apathy. The animal died on day 12 after 7 applications. Another animal receiving daily 100 mg/kg bw was sacrificed in severely moribund condition on day 11 of the study after 7 applications. Neurological effects like excitability, ataxia, unusual posture of the hind limbs and emesis were seen already after the first dose. Macroscopic and microscopic pathology revealed no clear test substance related findings, in particular no significant lesions of the CNS.

 

The test substance in oil was orally applied to 2 dogs (BASF 1952). Both dogs received 4 doses of 300 mg/kg followed by 5 doses 500 mg/kg during an exposure period of 2 weeks. After the last exposure dogs were observed for clinical sings and mortalities for up to 2 month. Both dogs were examined for gross pathological and histopathological changes. Clinical symptoms such as severe tonic-clonic convulsions, spastic gait observed 2.5 h after the first oral dose in both dogs were reversible within 24 hours. Same symptoms seen in one animal after the 2nd application.  As the animals appeared to get adapted to the compound, especially showing no inappetence, weight loss and no marked hyperexcitability anymore, the dose was increased after the 4th treatment from 300 to 500 mg/kg. Paresis was observed in both dogs after the animals had received a total of 4 x 300 mg/kg and 4 x 500 mg/kg. Paresis and weakness of the hind limbs were still present 16 days after the last dosing. In one animal that was observed up to two month after the last dosing paresis and weakness of the hind limbs were still present. Macroscopic and microscopic pathology (only one animal) revealed no clear test substance related findings, in particular no histopathological lesions were evident in the CNS including cerebrum, pons region and spinal cord.

 

The test substance was administered to 6 dogs (3 males, 3 females) by gastric intubation at a does level of 25 mg/kg/day for 21 consecutive days (TSCA, 1981). A comparable control group of 6 dogs (3 males, 3 females) received the equivalent amount of corn oil alone. Following the three week dosing period, all animals were observed for an additional 4 weeks prior to sacrifice. Three of the six animals dosed with the test substance exhibited clinical signs of neurological impairment following 14 days of treatment. Only limited signs of improvement occurred during the 4 week recovery period. Microscopic evaluation of tissues from the nervous system revealed minimal to severe myelin degeneration in the central nervous system of all test substance-treated dogs. At this lower dose level, the peripheral nerves were not affected. At the higher dose levels in earlier work, both central and peripheral nerves were damaged by the test substance.

 

In a repeated oral toxicity study six dogs were dosed at 300 mg/kg test substance for 14 days (TSCA, 1981). After the first day, because of toxicity, the dosage was reduced to 50 mg/kg and continued for the remaining 13 days. One dog died one day after the single dose of 300 mg/kg. The remaining dogs exhibited signs of convulsions and tremors similar to the signs noted above for the single dose of 300 mg/kg. The five surviving dogs on 50 mg/kg showed recovery from the initial signs two days after the first dose. Beginning on Day 13-14, these dogs exhibited evidence of front and/or hind limb weakness with inability to stand erect for normal periods of time. These signs persisted throughout the remainder of the study. There was no evidence of cholinesterase inhibition in plasma or brain samples from these animals. Microscopic examination of the brain, spinal cord and sciatic nerves revealed morphologic evidence of injury characterized by multifocal swelling and degeneration of axonal fibers.

In a 4 -day toxicity study (TSCA, 1995), 4 hens per group were exposed to 500, 2000 and 5000 mg/kg bw/d. The test substance administration resulted in signs of ataxia and paralysis in the hens which progressed to death at high doses. There were no clinical signs of intoxication after a single dose of 5000 mg/kg. The only effect noted after the first 3 doses was progressive weight loss, with clinical signs developing on day 4 and later. Neither brain neurotoxic esterase (NTE) nor brain and plasma acetylcholinesterase were inhibited following a single oral dose. No histopathological data were reported.

Repeated inhalation toxicity:

Experimental test data from several species are available, suggesting that rats are relatively insensitive towards the test substance and dogs being the most sensitive species. Therefore, the endpoint conclusion is based on the experimental data on dogs.

M&T Chemicals Inc. (1983) reported a 4-week inhalation study using dogs. One male and one female animal were exposed to concentrations of 0.5, 3.2, 9.7 or 28 mg/m3 test substance administered as an aerosol in xylene. All animals survived. Body weight development was normal. At 28 mg/m3 the dogs showed indications of neurological impairment: failure in the extensor postural thrust reflex with tendency to knuckle over, unsteadiness and weakness in the hind limbs. At 10 mg/m3 erratic placement of limbs was observed in one male dog in week 1 and 4. In one male receiving only 2 exposures of 3 mg/m3 limb placement incoordination was seen throughout the study. Only the effects at the highest dose were considered to be treatment-related by the authors, since the effects were neither persistent nor severe in the other dose groups. No treatment-related post-mortem findings were observed. The sections prepared from the high-dosed dogs revealed moderate generalised vacuolative degeneration of axons in the ventral and lateral funicule of both the cervical and lumbar spinal cord. No such findings were seen in the sciatic nerve. No such lesions were noted in the control animals having received xylene alone. The above mentioned neuronal lesions were seen in the longitudinal sections, recognised with both H&E and luxol fast blue stains, characterised by multiple dilated or vacuolated axon sheaths with accumulations of scattered eosinophilic debris and occasional dark rounded bits of what appeared to have been nuclear chromatin. Similar damages were seen in the lower dose groups, however, they were marginal in nature, and were not considered to correlate with exposure since in a histopathological reevaluation also historical control animals showed some axonal lesions. Sporadic degeneration is an expected finding in the spinal cord of "normal" animals. The NOAEC was found to be 10 mg/m3 for both sexes (based on the correlation between clinical signs of ataxia and neurotoxic tissue effects in the highest dose group).

Waritz et al. (1975) reported a 12-day study using male rats. Concentrations of 0 or approx. 2400 mg/mg3 were generated by nebulizing molten test substance. Clinical signs were typical of mild respiratory irritation: salivation, lacrimation, dyspnea, red ears. No test substance-related macroscopical or histopathological findings were seen. A NOAEC was not identified; the LOAEC was found to be 2400 mg/m3 for male rats.

BASF (1952) reported a 8 -day inhalation study on different test species. A test substance-saturated atmosphere generated by guiding an air stream of 1000 l/h over molten test substance (maintained at 90 °C) was directed in a 400-l glass chamber harbouring 1 cat, 1 rabbit, 4 rats and 10 mice. The average concentration of the test substance in the atmosphere was calculated to be 3000 - 3500 mg/m3 based on the decrease of test substance from the storage container and from the specific airation rate. Since part of the vapour condensed in the pipes prior to entering the exposure chamber, the concentration range given is considered to be the maximum possible concentration. The animals were whole-body exposed for 1x 7h and 3x 6h during a time period of 8 days. During exposure and subsequent 8 day observation period the animals were inspected for clinical signs of intoxication and mortality. No macroscopic and microscopic examination was carried out. No clinical signs of intoxication clinical or mortality were observed in rats, rabbits or cats during exposure and subsequent 8 day observation period. Five out of 10 mice died 1 - 3 days after the last exposure without evident signs of intoxication.

In a 4-week inhalation study (TSCA, 1981) four dogs (2 males, 2 females) were exposed to integrated mean concentrations of 18.0 or 94.5 mg/m3 of the test substance for 6 hours/day (5 days/week). A similar constituted group was exposed for the same period to xylene vapor and served as controls. After treatment, all 12 dogs were observed for a 4 week recovery period prior to necropsy. Visual evidence from locomotor dysfunctions were seen at the high level only from the second week of treatment on. This was neither progressive during the exposure period nor recuperable in the recovery period. In an effort to determine if peak excursions seen at the high level were the cause of some of these effects, 2 dogs were exposed to the test substance for 6 hours/day for 2 consecutive days followed by a 2 day observation period. The levels for each day were 216 mg/m3 and 162 mg/m3, respectively. These dogs did not show any reaction to treatment or neurological impairment during the exposure or observation periods. These two studies indicate that both cumulative exposure to 18 .0 and 94 .8 mg/m3 with excursions to 41 and 228 mg/m3 respectively on isolated occasions, and two exposures to 162 and 216 mg/m3, did elicit micropathological changes in the central nervous system.

 

Repeated dermal toxicity:

No studies/information available.

Repeated dose toxicity - other routes:

BASF (1952) repoted a study in which a suspension of the test substance in olive oil equivalent to a dose of 300 mg/kg was applied daily (5 injection per week, max. 1 injection per day) intramuscular to one rabbit. The dosing was paused from day 14 on due to severe clinical symptoms. A dose of 300 mg/kg was lethal after 15 days (9 injections). The animal showed ataxia and paralysis of the hind limbs from day 10 on and apathy and atony of the whole body at day13. Before death, lateral positioning and labored breathing was observed. Macroscopic and microscopic pathology revealed no characteristic substance-related findings: cachexia, cardiac flaccidity, collapsed/condensed lungs in one case, no significant lesions of the CNS.

 

In another study of BASF (1952), a suspension of 10-% test substance in olive oil equivalent to a dose of 50 mg/kg was applied daily intramuscular to one cat for 3 days. A dose of 50 mg/kg was lethal at day 4 (3 injections). The animal showed ataxia, inappetence and lateral positioning at day 2. Clonic-tonic seizures and occasional screaming was observed at day 3. Macroscopic and microscopic pathology revealed no characteristic substance-related findings.

 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Clinical signs of neurological impairment and neuropathological effects were seen in the oral and inhalation studies depending on the species and the vehicle used. Based on a worst case consideration, the substance is considered to be classified for repeated dose toxicity STOT RE Cat 1 under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.