2,4,6-tri-n-propyl-2,4,6-trioxo-1,3,5,2,4,6-trioxatriphosphorinane

Endpoint summary

Description of key information

A subacute oral repeated dose toxicity study in rats revealed a NOAEL of 62.5 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-08-24 to 1995-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: HOE CG 0501 0D ZD00 0001
- Expiration date of the lot/batch: June 1996
- Purity test date: 1995-03-06

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness at room temperature in a fume cupboard
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test compound was dissolved homogeneously in the vehicle by means of a magnetic stirrer.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat has proved to be a suitable species for subacute oral toxicity testing with many different substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst Aktiengesellschaft, Kastengrund, SPF breeding colony
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males about 130 g, females about 123 g
- Housing: in fully air-conditioned rooms in makrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff® R/M-H (V 1534) ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 50 +/-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
After each measurement of the body weight, the calculation of the application volume was repeated.

VEHICLE
- Concentration in vehicle: 0, 3.125, 12.5, 50 %

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 62.5 mg/kg bw/day
Male: 5 animals at 250 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 62.5 mg/kg bw/day
Female: 5 animals at 250 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose range finding study 3 male and 3 female Wistar rats received the test substance at the dose of 1000 mg/kg body weight per day over a period of 14 days. Addtionally, a control group (3 males / 3 females) was treated with the vehicle (DMSO) alone. No signs of clinical symptoms were observed in both groups. Development of body weight was not impaired. The animals killed at the end of the observation period showed no macroscopically visible changes.
Based on these results the substance was tested in the present study at the dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The behaviour and general health condition of the animals were observed once daily. The animals were examined weekly for neurological disturbances, opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were determined before the start of the study and then twice weekly throughout the study.

FOOD CONSUMPTION:
Food consumption was determined continuously (2 times per week). The values on the printouts refer to the intervals between one measurement and the next. They are converted to the food consumption per 100 g body weight over a 24 hour period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 50-100 mg Ketamin / kg body weight)
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Animals fasted: No
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After exsanguination, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes

Statistics:

The following parameters were compared statistically with the control group values at the level of significance p = 0.05:
Body weights at the designated measurement times
Haematological data
Clinical chemistry parameters
Urine analysis (Volume, pH-value and specific weight)
Absolute organ weights and organ to body weight ratios
Evaluation was performed by Pharma Informatics with the aid of a program package for the evaluation of toxicological studies. The calculation methods used are referred to on the computer printouts.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In all dose groups increased salivation occurred sporadically after application and is therefore considered to be a local reaction due to the administration of the acid test compound.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality occurred in four females of the 1000 mg/kg b.w. group. The animals were found dead on days 12 (two animals), 27 and 28 of the study. The animals found dead on days 27 and 28 showed gasping, respiratory sounds, swollen abdomen, bristled coat, stilted gait and poor general condition on days 26 or 27 of the study.
One male animal of the 1000 mg/kg b.w. group showed gasping, respiratory sounds, bristled coat, stilted gait and poor general condition from day 12 of the study. This animal was killed for animal welfare reasons on day 14 of the study.
The mortality of the four females and one male was not compound-related but due to technical error.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Final value: Haematological examinations revealed statistically significant decreases in erythrocyte counts, haemoglobin and haematocrit values as well as increases in reticulocyte and thrombocyte counts in both sexes from the high dose group. Additionally, haematocrit values were statistically significantly decreased and thrombocyte counts statistically significantly increased in females from the intermediate dose group.
Recovery value: Statistical evaluation revealed no differences.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Final value: Statistical evaluation revealed increases in cholesterol values in males from the intermediate dose group. Females from the intermediate dose group showed decreases in chlorid values and females from the low dose group exhibited statistically significant increases in triglyceride values as well as decreases in inorganic phosphorus values.
In all cases there was no dose dependency. Therefore, a compound-related effect is not evident.
Recovery value: The aspartate aminotransferase value was increased to a statistically significant degree in high dose males. The values were within the physiological range of rats. Furthermore, no changes were detected in females. Therefore, this finding is considered not to be compound-related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Specific weight of urine was increased to a statistically significant degree in males from the low dose group. There was no dose dependency and the control values were relatively low. Therefore, a compound-related effect is not evident.
Examination of the sediment did not reveal any abnormalities.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Final value: Statistical evaluation revealed increases in absolute and relative spleen weights in males and females from the high dose group. Additionally, absolute heart and kidneys weights were increased in females from the high dose group.
Recovery value: Relative heart and spleen weights were increased to a statistically significant degree in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically visible changes were observed in the forestomach in both sexes from the high dose group. These changes included attachement to the liver, brown discolouration, hyperkeratosis, and nodular changes. Some of these changes (attachement to the liver) were still detectable after the recovery period. Dilatation of the kidney pelvis was found in male and female animals of all dose groups including control and therefore was not considered to be compound related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In terminally killed animals of the high dose group histopathological examination showed extramyeloic hemopoiesis in the spleen which was also evident to a less extent in the high dose recovery group. Additionally, ulceration of the fore- and glandular stomach accompanied by inflammatory cell reaction was found in the high dose group. Two rats of the high dose recovery group also showed ulceration of the glandular stomach. No histopathological changes were detected in the other dose groups.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

other: decrease in haematocrit values, increase thrombocyte counts

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

For details on the microscopic findings, please refer to the attached document.

Conclusions:

Based on the absence of adverse effects a NOAEL of 62.5 mg/kg bw/day was revealed under the test concitions chosen.

Executive summary:

Groups of male and female Wistar rats received the test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for 28 days. On day 29 five males and fives females from each group were killed and necropsied. The remaining animals of the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly.

Haematological examinations and clinical chemistry were carried out at the termination of the study and after the recovery period.

Urine analysis was carried out at the termination of the study.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and clinical chemistry data, urine data (pH-value, volume, specific weight), absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared with the controls.

The mortality of four females and one male was not compound-related but due to technical error. Increased salivation after application occurred sporadically in all dose groups from day 15 (low dose group), day 12 (intermediate dose group), and day 8 (high dose group) up to day 28 of the study.

The test substance caused signs of anaemia as shown by decreases in erythrocyte counts, haemoglobin and haematocrit values, increases in reticulocyte and thrombocyte counts, enlargement of the spleen, and reactive extramyeloic hemopoiesis as well as gastric ulceration in male and female Wistar rats when administered 28 times during 29 days at the dose level of 1000 mg/kg body weight. These changes had a marked tendency to reverse as seen in the recovery group.

At the dose level of 250 mg/kg body weight per day there were still decreases in haematocrit values and increases in thrombocyte counts in females.

No compound-related adverse effects were observed after repeated administration of the test substance at the dose level of 62.5 mg/kg body weight per day.

In all dose groups increased salivation occurred sporadically after application and is therefore considered to be a local reaction due to the administration of the acid test compound.

With regard to the present study the 'No Observed Adverse Effect Level' (NOAEL) for systemic effects is 62.5 mg/kg body weight per day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

62.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline-conform study, appropriate for assessment

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

oral route:

Groups of male and female Wistar rats received the test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for 28 days. On day 29 five males and fives females from each group were killed and necropsied. The remaining animals of the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly.

Haematological examinations and clinical chemistry were carried out at the termination of the study and after the recovery period.

Urine analysis was carried out at the termination of the study.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and clinical chemistry data, urine data (pH-value, volume, specific weight), absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared with the controls.

The mortality of four females and one male was not compound-related but due to technical error. Increased salivation after application occurred sporadically in all dose groups from day 15 (low dose group), day 12 (intermediate dose group), and day 8 (high dose group) up to day 28 of the study.

The test substance caused signs of anaemia as shown by decreases in erythrocyte counts, haemoglobin and haematocrit values, increases in reticulocyte and thrombocyte counts, enlargement of the spleen, and reactive extramyeloic hemopoiesis as well as gastric ulceration in male and female Wistar rats when administered 28 times during 29 days at the dose level of 1000 mg/kg body weight. These changes had a marked tendency to reverse as seen in the recovery group.

At the dose level of 250 mg/kg body weight per day there were still decreases in haematocrit values and increases in thrombocyte counts in females.

No compound-related adverse effects were observed after repeated administration of the test substance at the dose level of 62.5 mg/kg body weight per day.

In all dose groups increased salivation occurred sporadically after application and is therefore considered to be a local reaction due to the administration of the acid test compound.

With regard to the present study the 'No Observed Adverse Effect Level' (NOAEL) for systemic effects is 62.5 mg/kg body weight per day.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.