N-(2-nitrophenyl)phosphoric triamide

Endpoint summary

Description of key information

28-day oral repeated dose toxicity study according to OECD guideline 407: NOAEL 30 mg/kg b.w.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

2005-07-15 - 2006-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species, Strain: rat/Wistar Crl:WI BR

Rationale:The rat is a suitable rodent species for repeated dose toxicity studies and is acceptable to regulatory authorities as an indicator of potential human hazards. Extensive background data are available for this species.

Sex: male, female (nulliparous, non-pregnant)

Supplier:Charles River Wiga GmbH, D-97320 Sulzfeld, Germany

Hygiene status upon supply: SPF
Age at start of acclimatisation: approximately 6 weeks
Acclimatisation/Randomisation: 6 (males) or 7 (females) days before start of dosing the animals were allocated randomly to treatment groups using random charts.
Mean body weight at the start of dosing: Males: 197.6g± 8.2 g (4.1%) n = 30 Females: 170.2 g± 8.6 g (5.0%) n = 30
Identification: ear notches, cage labelling showing the animal number, study number, dose, sex, time of dosing and sacrifice

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % (m/v) solution of Tylose MH 1000 in deionised water

Details on oral exposure:

Doses were administered once daily (between 6.30 and 11.00 a.m.), by oral gavage, using a metal catheter and plastic syringes, 7 days a week, for 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test item in the formulation was analytically determined shortly after the preparation and after the completion of administration of each dose group twice (in the first and in the last week of administration).

Samples of the suspensions of all three dose groups each were dissolved in methanol using an ultrasonic bath and filtered using a syringe filter 0.2 µm.

The concentrations of N-(2-Nitrophenyl)phosphoric triamide were determined by use of the HPLC with UV detection after calibrating by the external standard method.

For the investigations a Shimadzu HPLC system equipped with a LC-6A isocratic pump, a Rheodyne seven port valve Model 7125, a column oven CTO-6A, an UV-VIS spectrophotometric detector SPD-6AV and a C-R4A Chromatopac Processor was used. The experimental chromatographic conditions were the followings:

Column: Reprosil PUR 120 C18 AQ 5µm 125x3mm

Eluent: 40% Methanol / 60% Water

Elution: isocratic

Flow rate: 0.5 ml/min

Temperature: 30 °C

Sample loop volume: 20 µl

Detection: UV (254 nm)

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg body weight / day
Basis:
actual ingested

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Group 2 and 3 each comprised 5 males and 5 females and group 1 and 4 each comprised 10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels had been selected after two pre-experiments at first using doses of 1000 and 300 mg/kg body weight and second using doses of 100 and 50 mg/kg body weight in 3 male and 3 female rats each for a period of 12 or 11 (second pre-experiment) administration days.

In the main study sixty rats (30 males and 30 females) of the Wistar CiT.WI BR strain were allocated to four groups. Group 2 and 3 each comprised 5 males and 5 females and group 1 and 4 each comprised 10 males and 10 females. At the end of the 28 day treatment period 5 males and 5 females of group 1 and 4 (groups la and 4a) were maintained, undosed, for further 14 days. Three groups received the suspension of the test item in 0.5 % (m/v) solution of Tylose MI-I 1000 in deionised water by oral gavage at dose levels of 30, 100 and 300 mg/kg body weight / day. The group 1 received the vehicle 0.5 % (m/v) solution of Tylose MH 1000 in deionised water only, and served as control. Animals were dosed once daily, and treatment continued for 28 days.

Observations and examinations performed and frequency:

Clinical Observations
A detailed off-cage clinical examination was carried out daily twice (before the administration and in the afternoon).
Assessments of sensory reactivity (auditory, visual and proprioceptive stimuli), grip strength and motor activity were carried out prior to administration, in the last week of dosing and in the last week of the recovery period.

Mortality
Animals were examined daily for mortality and morbidity.

Body Weight
Body weights were recorded at the start of the study and then weekly thereafter and at the end of the study before food withdrawal.

Food Consumption
Food consumption was recorded weekly throughout the treatment and recovery period and mean daily intake for each group was calculated.

Clinical Laboratory Examinations

Urine from all animals was collected over 24 hours (immediately after the daily administration up to the next administration) in the last week of the administration period and in the last week of the 14 day recovery period.

Blood samples from all animals were taken from the retroorbital plexus under ether anaesthesia after an overnight fasting immediately prior to killing at the termination of the study one day after the last administration of the test item or, if necessary, one day after the 14 day recovery period for examinations.

Sacrifice and pathology:

Pathology

The animals were killed at the end of the treatment or recovery period, respectively, by CO2 asphyxiation.

Macroscopic Pathology

All animals were examined externally. The cranial, thoracic and abdominal cavities were opened and examined macroscopically.

Organ Weights

The following organs of all animals were weighed after trimming off fat and other contiguous tissue:

adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus. From bilateral organs both were weighed and reported separately.

Histology
Whole organs or samples of the tissues listed below of all animals were fixated and preserved in 4 percent formaldehyde.
Adrenals Sciatic nerve
Bone marrow (from sternum) Small and large intestine (including Peyer's patches)
Brain (3 sections) Spinal cord (from second cervical vertebra [Axis])
Epididymides Spleen
Heart Stomach
Kidneys Testes
Liver Thymus
Lungs Thyroid
Lymph nodes (mesenteric and submandibular) Trachea
Urinary bladder
Ovaries Uterus
Prostate

Clinical signs:

no effects observed

Mortality:

no mortality observed

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical Observations:
Apart from a discoloration of the urine (yellow or orange not any alteration of general state of well-being and behaviour was observed in any animal in the entire course of the investigation.

Body Weight and Body Weight Gain:
Neither the body weights nor the body weight gain of the animals were influenced by the administration of the test item in comparison to the vehicle control group.

Food Consumption:
The food consumption of the animals was not influenced by the administration of the test item in comparison to the vehicle control group.

Urinalysis:
The glucose content of the urine was dose dependent increased in both male and female animals. This increase was calculated as statistically significant only in the high dose group. An increase of the glucose content of the urine in the animals of the satellite group was not observed.

Haematology:
The leucocyte count was dose dependent slightly increased, statistically significant in the male animals of the high dose group. This was the reason for the determination of all haematological parameters (one determination step by the COULTER MAXM) also in the satellite groups of the male animals. Here the leucocyte count was slightly increased compared to the satellite control group, too but not calculated as statistically significant.

Clinical Biochemistry:
The activities of the alkaline phosphatase and of the aspartate aminotransferase (AST) were calculated as statistically significantly decreased in the male animals of the high dose group. The data of female animals showed the same trend without statistical significance and a slight dose dependence could be suspected. This was the reason for the determination of this parameters also in the satellite groups. Here a statistical significance was not observed.

Organ Weights:
The absolute and relative organ weights of the testes were dose dependent decreased, statistically significantly in the animals of the high dose group. These values and also the values of the middle dose group fall below the range of the historical control data of the testing facility (absolute organ weights: 1260 - 2107 mg, relative organ weights: 0.38 - 0.54
In the animals of the satellite group this statistically significant decrease was observed, too.
Dose dependent decreased absolute and relative organ weights were observed for the epididymides, too however statistically significant in the animals of the middle and high dose group. These values fall below the range of the historical control data of the testing facility, too (absolute organ weights: 227 - 427 mg, relative organ weights: 0.067 - 0.113 %). In the animals of the satellite group this decrease was observed, too however only for the left organ statistically significant.
A slight tendency of increased absolute and relative organ weights of the kidneys was observed in all animals of the high dose group, statistically significant in the male animals (absolute and relative organ weights right kidney) and in the female animals (relative organ weights left kidney). The statistically significantly increased value in males exceed the range of the historical control data of the testing facility (relative organ weights: 0.33 - 0.45 %). A slight tendency of increased absolute and relative organ weights of the kidneys was observed in male animals however not in the female animals of the satellite group.
A slight tendency of increased absolute and relative organ weights of the spleen was observed in the male animals, statistically significant in the animals of the high dose group and regarding the relative organ weights in the animals of the middle, too.
The increased absolute and relative organ weights of the spleen were observed in male animals of the satellite group, too.
This effect was not observed in the female animals.

Macroscopic Pathological Findings:
A micro-orchidia and epididymides with reduced size were observed in all male animals of the high dose group (5/5) and in 3/5 animals of the middle dose group concerning both the left and the right organ. This effect was irreversible in the course of the recovery period. These macroscopic pathological findings correspond to the decreased organ weights.The kidneys in the male animals of the high dose group were brightened and the renal pelvis was yellowish discoloured in both the left and the right organ. This effect occurred only slightly in the male animals of the middle dose group, in the female animals of the high dose group and in the male animals of the satellite group.


Histological Findings:
The main finding of the histopathologieal examination was a dose dependent damage of testes (damage of the seminiferous tubules), also slight damage in the animals of the low dose group, which led in the high dose group to a complete absence of spermatozoa in the epididymides. However the epididymides of the animals of the low dose group contained the normal number of spermatozoa.

A further important finding was a dose dependent slight damage of the kidneys (flattened and partly degenerated tubular epithelium in the medulla) in both male and female animals. The male animals seem to be slightly more sensitive. The animals of the low dose group did not show a damage. A slight, however not complete reversibility of the damages could be observed after the 14 day recovery period.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

kidney

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study results show that the test item administration caused partly severe, in the course of the 14 day recovery period not completely reversible, clearly dose dependent effects. The male animals seem to be slightly more sensitive than the female animals.

Because of the discoloration of the urine, caused by the colour of the test item, and the slight damage of the seminiferous tubules in the testes observed also in the animals of the low dose group a NOEL (No Observed Effect Level) can not be declared.

However based on the normal state of the epididymides, the normal number of spermatozoa and the normal state of kidneys (according to the histopathologieal examinations) in the animals of the low dose group the dose of 30 mg/kg body weight is the NOAEL (No Observed Adverse Effect Level) after the 28 days treatment.

Executive summary:

In a 28-day oral repeated dose toxicity study according to OECD guideline 407 under GLP, rats were exposed daily to 0, 30, 100, or 300 mg N-(2-Nitrophenyl)phosphoric triamide/kg bodyweight by oral gavage followed by a 14-day recovery period.

The most pregnant toxic effect of the test item was the dose dependent damage of testes, which led in the high dose group to a complete absence of spermatozoa in the epididymides. A severe micro-orchidia and epididymides with reduced size and an alteration of testes (alteration of the seminiferous tubules) was observed in one animal of the satellite control group, too. However a complete absence of spermatozoa was not observed in this case. This shows that in single cases the normal development of testes and epididymides can be disturbed without any visible reason. But the test item administration at the high dose group disturbed the normal development of testes and epididymides completely. It can be assumed that the test item is quickly excreted via kidneys in case of intake of not damaging doses. However considering the slight damage of the kidneys in the present study and the results of the pre-experiments a damage of kidneys has to be feared in the course of concentration of the primary urine in the renal tubules if the concentration of test item is to high. According to the findings the testes/epididymides and the kidneys can be assumed as the target organs of the test item. The increased organ weight of the spleen shows that the total organism (presumable via immune system) is involved in the reaction to the test item administration.

Because of the discoloration of the urine, caused by the colour of the test item, and the slight damage of the seminiferous tubules in the testes observed also in the animals of the low dose group a NOEL (No Observed Effect Level) can not be declared. However based on the normal state of the epididymides, the normal number of spermatozoa and the normal state of kidneys (according to the histopathologieal examinations) in the animals of the low dose group the dose of 30 mg/kg body weight is the NOAEL (No Observed Adverse Effect Level) after the 28 days treatment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

4

Species:

rat

Quality of whole database:

Only one study is available.

System:

urinary

Organ:

kidney

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

In a 28-day oral repeated dose toxicity study according to OECD guideline 407 under GLP, rats were exposed daily to 0, 30, 100, or 300 mg N-(2-Nitrophenyl)phosphoric triamide/kg bodyweight by oral gavage followed by a 14-day recovery period.

The most pregnant toxic effect of the test item was the dose dependent damage of testes, which led in the high dose group to a complete absence of spermatozoa in the epididymides. A severe micro-orchidia and epididymides with reduced size and an alteration of testes (alteration of the seminiferous tubules) was observed in one animal of the satellite control group, too. However a complete absence of spermatozoa was not observed in this case. This shows that in single cases the normal development of testes and epididymides can be disturbed without any visible reason. But the test item administration at the high dose group disturbed the normal development of testes and epididymides completely. It can be assumed that the test item is quickly excreted via kidneys in case of intake of not damaging doses. However considering the slight damage of the kidneys in the present study and the results of the pre-experiments a damage of kidneys has to be feared in the course of concentration of the primary urine in the renal tubules if the concentration of test item is to high. According to the findings the testes/epididymides and the kidneys can be assumed as the target organs of the test item. The increased organ weight of the spleen shows that the total organism (presumable via immune system) is involved in the reaction to the test item administration.

Because of the discoloration of the urine, caused by the colour of the test item, and the slight damage of the seminiferous tubules in the testes observed also in the animals of the low dose group a NOEL (No Observed Effect Level) can not be declared. However based on the normal state of the epididymides, the normal number of spermatozoa and the normal state of kidneys (according to the histopathologieal examinations) in the animals of the low dose group the dose of 30 mg/kg body weight is the NOAEL (No Observed Adverse Effect Level) after the 28 days treatment.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The key study is GLP-compliant and has Klimisch score 1.

Justification for classification or non-classification

In a 28-day oral repeated dose toxicity study according to OECD guideline 407 under GLP, rats were exposed daily to 0, 30, 100, or 300 mg N-(2-Nitrophenyl)phosphoric triamide/kg bodyweight by oral gavage followed by a 14-day recovery period.

The most pregnant toxic effect of the test item was the dose dependent damage of testes, which led in the high dose group to a complete absence of spermatozoa in the epididymides. A severe micro-orchidia and epididymides with reduced size and an alteration of testes (alteration of the seminiferous tubules) was observed in one animal of the satellite control group, too. However a complete absence of spermatozoa was not observed in this case. This shows that in single cases the normal development of testes and epididymides can be disturbed without any visible reason. But the test item administration at the high dose group disturbed the normal development of testes and epididymides completely. It can be assumed that the test item is quickly excreted via kidneys in case of intake of not damaging doses. However considering the slight damage of the kidneys in the present study and the results of the pre-experiments a damage of kidneys has to be feared in the course of concentration of the primary urine in the renal tubules if the concentration of test item is to high. According to the findings the testes/epididymides and the kidneys can be assumed as the target organs of the test item. The increased organ weight of the spleen shows that the total organism (presumable via immune system) is involved in the reaction to the test item administration.

Because of the discoloration of the urine, caused by the colour of the test item, and the slight damage of the seminiferous tubules in the testes observed also in the animals of the low dose group a NOEL (No Observed Effect Level) can not be declared. However based on the normal state of the epididymides, the normal number of spermatozoa and the normal state of kidneys (according to the histopathologieal examinations) in the animals of the low dose group the dose of 30 mg/kg body weight is the NOAEL (No Observed Adverse Effect Level) after the 28 days treatment.

According to the CLH report, Proposal for Harmonised Classification and Labelling prepared by the Environment Agency Austria for the substance N-(2-nitrophenyl)phosphoric triamide (see also: https://echa.europa.eu/documents/10162/d7f86e94-5fbe-d717-5066-8a117a448d15), a classification of the substance with STOT RE2 is proposed based on the available study results from the 28-day repeated dose toxicity study (citation):

“According to CLP Regulation Annex I: 3.9.1.1. significant health effects that impair the function, which are both reversible and irreversible, immediate or delayed should be considered for STOT RE classification. Results demonstrate that the function of the kidney is severely impaired, since there is a statistically significant change in the glucose content at the highest dose group and there have been erythrocytes detected in the urine in treated animals, the latter effect was not reversible. Although the study authors ranked the histopathological findings (e.g. partial degeneration of the tubular epithelium in the renal medulla and cortex) as not severe, the sum of the observed findings leads to significant alteration of kidney function, indicating injured kidneys.

The results clearly indicate that the function and morphology of the kidney (macroscopic and histopathological observations, increased organ weight) have toxicologically significantly changed and thus Annex I, 3.9.1.3. of CLP Regulation is fulfilled.

Based on the study results a classification for specific organ toxicity (repeated exposure) is proposed according to definitions laid down in CLP Regulation (Annex I,: 3.9.1.1 to Annex I,: 3.9.1.3, and further specified in Annex I, 3.9.2.7.3.).

Since the observed adverse effects in the 28 day toxicity study are above the guidance values for STOT RE 1 classification (≤ 30 mg/kg bw/day) classification, no classification into this hazard category is proposed.

The guidance values for STOT RE 2 classification (oral 28 day toxicity studies (rat)) are according to CLP Regulation between 30 and 300 mg/kg bw/day. Thus, based on the severity of the detected effect at 300 mg/kg bw/day classification with STOT RE 2 (H373, kidney) is proposed.”

Therefore, a classification with STOT RE 2 is assigned.

No 90-day oral repeated dose toxicity study is proposed. According to Annex IX, column 2, the test does not need to be carried out if data from a 28-day study are available which suggest that the substance should be classified on the basis of the observed severe toxic effects in terms of specific target organ toxicity on repeated exposure (STOT RE).