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

Endpoint summary

Description of key information

No study with 4-NPP, di -TRIS (CAS 68189-42-4) is available to draw a conclusion on its acute toxicity properties.

Data from the analogues Tris (CAS 77-86-1) and 4-Nitrophenyl phosphate (4NPP, CAS 4264-83-9) was used on a read-across approach to fulfil this endpoint information requirement (See Read Across justification in Chapter 13).

For the components of the substance 4-NPP, di -TRIS (CAS 68189-42-4), 4-Nitrophenyl phosphate (4NPP, CAS 4264-83-9) and Tris (CAS 77-86-1), there are reliable data available. 

Acute toxicity data for Tris (CAS 77-86-1) is available from an oral gavage study with female mice. The acute oral toxicity of the substance was investigated in a GLP study according to OECD TG 425. The LD50 of the substance was determined as >5000 mg/kg bw.

Acute toxicity data for 4-NPP (CAS 4264-83-9) is available from an oral gavage study with female mice. The acute oral toxicity of the substance was investigated in a GLP study according to OECD TG 423. The LD50 of the substance was determined as >300 - 2000 mg/kg bw.

 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and the target substance have similar toxicological properties because the source substances, Tris and 4-NPP, are the only components of the target substance 4-NPP, di-TRIS. It is assumed that acute oral toxicity of the target substance 4-NPP, di-TRIS is determined by its components, Tris and 4-NPP.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source Substance 1
Trometamol, Tris
EC number: 201-064-4 | CAS number: 77-86-1

Source Substance 2
Disodium 4-nitrophenyl phosphate, 4NPP
EC number: 224-246-5 | CAS number: 4264-83-9

Target substance:
4-NPP, di -TRIS
(4-nitrophenoxy)phosphonic acid; bis(2-amino-2-(hydroxymethyl)propane-1,3-diol)
EC number 269-198-6 | CAS number: 68189-42-4

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of 4-NPP, di-TRIS's toxicological properties from Tris and 4NPP.
The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - <= 2 000 mg/kg bw

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

For the components of the substance 4-NPP, di -TRIS (CAS number 68189-42-4), 4-Nitrophenyl phosphate (4NPP, CAS 4264-83-9) and Tris (CAS 77-86-1), there are reliable data available. In aquatic media, the substance 4-NPP, di -TRIS will dissolve.
For Tris, the observed acute toxicity is >5000 mg/kg bw (LD50).
For 4NPP, the observed acute toxicity is >300 - 2000 mg/kg bw (LD50).
Consequently, it can be assumed that also the target substance 4-NPP, di -TRIS (CAS number 68189-42-4) will have acute toxicity of >300 - 2000 mg/kg bw (LD50) and is therefore classified as Category 4.

Executive summary:

For both components of the substance 4-NPP, di -TRIS (CAS number 68189-42-4), 4-Nitrophenyl phosphate (4NPP, CAS 4264-83-9) and Tris (CAS 77-86-1), there are reliable data available. In aquatic media, the substance 4-NPP, di -TRIS will dissolve. 

For Tris, the observed acute oral toxicity is >5000 mg/kg bw (LD50). Tris is therefore not classified for Acute oral Toxicity. 

For 4NPP, the observed acute oral toxicity is >300 - 2000 mg/kg bw (LD50). 4NPP was ranked into classes of the current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008 as Category 4.

Consequently, it can be assumed that also the substance 4-NPP, di -TRIS (CAS number 68189-42-4) will have acute oral toxicity of >300 - 2000 mg/kg bw (LD50) and is therefore classified as Category 4.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May - 06 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

National GLP Compliance Monitoring Authority, Department of Science & Technology, New Delhi, India; Food and Consumer Product Safety Authority (VWA), The Hague, the Netherlands; Bundesinstitut für Risikobewertung, Berlin, Germany

Test type:

up-and-down procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: inhouse bred (Outbred), Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, India
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 177.3-180.3 g (range)
- Fasting period before study: yes, overnight (16-18 hours)
- Housing: the rats were housed individually in standard polysulfone cages (approximately L 425 mm x B 266 mm x H 175 mm) with stainless steel top grill, with facilities for pelleted food and water; steam sterilised corn cob bedding was used and changed along with the cage twice weekly
- Diet: Ssniff rats/mice pellet food, manitenance meal (Ssniff Spezialdiaten GmbH, Soest, Gemany), ad libitum
- Water: deep bore-well water passed through activated charcoal filter and exposed to UV-rays in Aquaguard on-line water filter-cum-purifier (Eureka Forbes Ltd., Mumbai, India), ad libitum
- Acclimation period: 5-10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 58-68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 May 2011 To: 1, 3 and 6 June 2011

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 20 mL/kg bw
- Justification for choice of vehicle: the test substance forms a visibly homogenous solution in water

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bw (3.5-3.6 mL absolute volume)

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

3, dosed stepwise with a 2-day interval, according to the up-and-down procedure

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: the animals were observed for mortality and clinical signs 1/2, 1, 2, 3 and 4 hours after administration on Day 1, and daily thereafter throughout the study period; the animals were weighed on Day 1 (prior to administration), 8 and 15
- Necropsy of survivors performed: yes, external surfaces of the body, all orifices, tissues and organs of the thoracic and abdominal cavities were examined, and all gross findings were recorded

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Mortality:

There was no mortality during the study period.

Clinical signs:

other: No clinical signs were observed during the study period.

Gross pathology:

Necropsy and histopathological examination revealed no substance-related findings.

Table 1: Mortality and clinical signs during the study period

 

Dose [mg/kg bw]	Toxicological results*	Duration of clinical signs	Time of death	Mortality (%)
Females
5000	0/0/3	---	---	0
LD50 > 5000 mg/kg bw

                                                                                           

* first number = number of dead animals                                 

 second number = number of animals with clinical signs         

 third number = number of animals used                               

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified
DSD: not classified

Reference 3

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17 December 2001

Deviations:

no

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

> 300 - <= 2 000 mg/kg bw

Justification for classification or non-classification

Acute toxicity: oral

Source substance 1 | Trometamol, Tris | EC number: 201-064-4 | CAS number: 77-86-1

The acute oral toxicity of 2 -amino-2 -(hydroxymethyl)-1,3 -propanediol (trometamol) was assessed in a study performed according to the up-and-down procedure (OECD 425) in rats administered 5000 mg/kg bw (Mohan Kumar, 2011). Single animals were administered the limit dose stepwise, up to a total of 3 rats. There was no mortality, no clinical signs were observed during the 14-day observation period and no effects on the body weight were noted. There were no substance-related findings of the histopathological examination. The LD50 is considered to be > 5000 mg/kg bw.

According to the results of this study the substance is not classified for acute toxicity according to CLP-Regulation (EC) No 1272/2008. 

In addition, several pre-guideline studies with rats (Giroux and Beaulaton, 1961), mice (Giroux and Beaulaton, 1961 and Rubenkoenig, 1955) and rabbits (Machle, 1940) are available confirming the low systemic acute oral toxicity of trometamol.

Source substance 2 | Disodium 4-nitrophenyl phosphate, 4NPP | EC number: 224-246-5 | CAS number: 4264-83-9

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.

The method used, was not intended for the precise calculation of a precise LD50 value. 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 CLP category is 4.

Conclusion on classification
The acute oral toxicity classification of 4-NPP, di -TRIS (CAS 68189-42-4) is currently not harmonised according to the CLP Regulation (EC) 1272/2008. Based on available acute toxicity data for the analogues Tris (CAS 77-86-1) and 4NPP (CAS 4264-83-9), the LD50 of the substance was determined as >300 - 2000 mg/kg bw.

Based on the available acute oral toxicity data for Tris (CAS 77-86-1) and 4NPP (CAS 4264-83-9), according to the read across justification (see chapter 13) the target substance 4-NPP, di -TRIS (CAS 68189-42-4) should be classified for acute oral toxicity, Category 4, following Annex I, table 3.1.1 of the CLP regulation (acute oral toxicity >300 - 2000 mg/kg bw).