p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2017 - 14 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 December 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-nitrophenyl phosphate

EC Number:

224-246-5

EC Name:

Disodium 4-nitrophenyl phosphate

Cas Number:

4264-83-9

Molecular formula:

C6H4NO6P.2Na

IUPAC Name:

disodium 4-nitrophenyl phosphate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid

Details on test material:

Batch Nr: 11674900
Storage: 2-8°C.

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:(WI) rats
- Source: TOXI COOP ZRT. Cserkesz u. 90. 1103 Budapest, Hungary
- Hygienic level at arrival: SPF (Specific Pathogen Free)
- Hygienic level during the study: Good conventional
- Justification of strain: The Wistar rats as a rodent is one of the standard species of acute toxicity studies
- Number of animals: 3 animals/group
- Sex: Female, nulliparous and non pregnant animals
- Age of animals: Young adult rat, 9 weeks old in first, second, third and fourth step
- Body weight range at starting (first step): 212 - 218g
- Body weight range at starting (second step): 220 -224 g
- Body weight range at starting (third step): 234-252 g
- Body weight range at starting (fourth step): 235-250 g
- Acclimatization time: 12 days in first step, 13 days in second step, 14 days in third step and 15 days in fourth step


ENVIRONMENTAL CONDITIONS
- Animal health: Only healthy animals were used for the study.
- Room: 13/4
- Housing: Group caging (3 animals/cage)
- Cage type: Type III polypropylene/polycarbonate.
- Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Ventilation: above 10 air exchanges/hour by central air-condition system.
- The temperature and relative humidity were recorded daily during the study.

- Food and Water Supply: Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from municipal supply, as for human consumption from bottle ad libitum. The diet and drinking water are periodically analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in Toxi-Coop Zrt.’s archive.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Aqua purificata Ph.Hg. VIII.
- Batch number: 1707-5506
- Date of expiration: 06.01.2018
- Produced by: Parma Produkt Kft.
- Formulation: All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 200 mg/mL. Formulations were prepared just before the administration and stirred continuously during the treatment.

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg bw. The test item was applied in a concentration of 200 mg/mL and 30 mg/mL.

Dosage Preparation

CLASS METHOD
- Justification of the doses: Starting dose was selected on the basis of the available information about the test item.
The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats.
Only one animal died in the first step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. Two animals died in the second step, so treatment with 300 mg/kg bw was repeated on further three female rats.
No animal died in the third step, so treatment with 300 mg/kg bw was repeated on further three female rats. No animal died in the fourth step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met

Doses:

2000 mg/kg body weight (first and second step)
300 mg/kg body weight (third and fourth step)

No. of animals per sex per dose:

3 females per dose (first step)
3 females per dose (second step)
3 females per dose (third step)
3 females per dose (fourth step

Control animals:

no

Details on study design:

- Procedure: A single oral administration - followed by a fourteen-day observation period - was performed by gavage. The day before treatment the animals were fasted. The food but not water was withheld overnight. Animals were weighed before the application and the food was given back 3 hours after the treatment.

- Duration of the Experimental Period: 12 days in first step, 13 days in second step, 14 days in third step and 15 days in fourth step of acclimatization, treatment’s day, 14 days post-treatment observation period, necropsy on Day 0 and on Day 15.
- Frequency of observations: Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and once each day for 14 days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

- Body weight: The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.
- Necropsy: At the end of the observation period all rats were sacrificed under isofluran anaesthesia. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of its location, colour, shape and size.

Statistics:

No statistical analysis was performed.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 mg/kg bw

Based on:

test mat.

Mortality:

Altogether three animals treated with 2000 mg/kg bw single oral dose of the test item 4-Nitrophenyl phosphate disodium salt died during the study. In group 1, one rat (No.: 7491) died on the treatment day 30 minutes after the treatment. In group 2, two rats (No.: 7515, 7516) died on the treatment day 30 minutes after the treatment and 1 hour after the treatment, respectively. Deaths seemed to be consequences of systemic toxic effect of the test item.No death occurred at 300 mg/kg bw single oral dose of the test item. All female rats in step 3 and step 4 survived until the end of the 14-day observation period.

Clinical signs:

other: Symptoms observed on the treatment day between 30 min and 4 h after treatment: In group 1 treated with 2000 mg/kg bw dose clinical sign of reaction comprised of decreased activity (11 cases of 39 observations), tonic convulsion (1/39), prone position (6

Gross pathology:

Three rats (No.: 7491, 7515, 7516) treated with 2000 mg/kg bw dose of the test item spontaneously died during the study. Three animals (No.: 7492, 7493, 7517) of the same dose and all animals of the 300 mg/kg bw dose survived until the scheduled necropsy on Day 15.
An external necropsy finding as yellowish discharge around the mouth was observed in animal No.: 7516 of group 2. This alteration could not be related to the test item toxic effect. The animal regurgitated some test item, probably .
Internal necropsy finding as dark liver was observed in animal No.: 7515 of group 2. Strangulated spleen was recorded in animal No.: 7517 of group 2. These alterations could not be related to the test item toxic effect, but was regarded an individual variation.
Slight hydrometra was recorded in animal No.: 7517 of group 2. It is a physiological finding and connected to the cycle of the animal.
Internal necropsy finding as pale kidneys and swollen spleen was observed in animal No.: 7500 of group 3. These alterations could not be related to the test item toxic effect, but was regarded an individual variation.
No pathological changes were found related to the effect of the test item during the macroscopic examination of survivor animals.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

No death occurred after the single 300 mg/kg bw by oral dose of 4-Nitrophenyl phosphate disodium salt.
The method used, was not intended for the precise calculation of a precise LD50 value.
The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as Category 4
The test item was ranked into classes of the current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008 as Category 4
In conclusion, the LD50 of the test item 4-Nitrophenyl phosphate disodium salt (CAS No 4264-83-9) is between 300 mg/kg bw and 2000 mg/kg bodyweight by oral route in the rat. The GHS and CLP category is 4.

Executive summary:

General Information:

All criteria for the validity of the performed experiments have been met.

The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. Only one animal died in the first step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. Two animals died in the second step, so treatment with 300 mg/kg bw was repeated on further three female rats. No animal died in the third step, so treatment with 300 mg/kg bw was repeated on further three female rats. No animal died in the fourth step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met.

Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on the treatment day and 15th day after the treatment.

Lethality, Clinical Symptoms and Body weight:

In 2000 mg/kg bw 4-Nitrophenyl phosphate disodium salt (CAS No 4264-83-9) dose group, three rats died on the treatment day between 30 minutes and 1 hour after the treatment. Three rats survived until the end of the 14-day observation period in the same dose group.

All rats treated with 300 mg/kg bw dose of test item survived until the end of the 14-day observation period.

In the first step, CNS symptoms (decreased activity, tonic convulsion, vocalisation), disturbances of autonomic functions (tachycardia, dyspnoea), disturbances of coordination (prone position, incoordination, abnormal gait), decreased righting reflex and decreased muscular tension (body-, abdominal- and grip and limb tone) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment.

In the second step, CNS symptoms (decreased activity, tonic convulsion), disturbances of autonomic functions (tachycardia, dyspnoea), disturbances of coordination (prone position, incoordination, abnormal gait), decreased righting reflex and decreased muscular tension (body-, abdominal- and grip and limb tone) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment.

In the third and fourth step, no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of experimental animals were normal.

A body weight loss was observed in two females of group 1(2000 mg/kg bw) on the second week. This body weight loss was not strong and the body weight of animal exceeded the original body weight by the end of study, thus it can be evaluated as an individual variation without toxicological meaning.

The mean body weight of 300 mg/kg bw groups corresponded to their species and age throughout the study.

Gross Pathology:

Three animals treated with 2000 mg/kg bw dose died spontaneously during the study. Three animals of the same dose and six animals of the 300 mg/kg dose were sacrificed as scheduled during the study. All organs of all experimental animals treated with 2000 mg/kg bw or 300 mg/kg bw dose proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow the calculation of a precise LD50 value.

The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as below:

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	GHS category
300	0/6	between 300 and 2000	4

The test item was ranked into classes of the current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008 as below:

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	CLP category
300	0/6	between 300 and 2000	4

In conclusion, the LD50 of the test item 4-Nitrophenyl phosphate disodium salt (CAS No No 4264-83-9) ) is between 300 mg/kg and 2000 mg/kg bodyweight by oral route in the rat.