Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Endpoint summary

Description of key information

The observed "no-adverse-effect-level" of DMPPA_701-402-5 is 1000 mg/kg bw/day for males and females.

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:

1992

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: DMPPA_701-402-5 (FAT 80001/I)
Description: White solid
Batch number: EN 746916/1991
Stability of test article: Expiry date: September 1996
Stability of test article dilution: Stable for atleast 48 h in water
Instructions for test article storage: At room temperature in the dark.
Vehicle: Bi-distilled water.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4 CH-4414 Fuellinsdorf
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Housing: Individually in Makrolon type-3 cages (size: 22 x 37.5 x 15 cm) with autoclaved standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard K U b a no. 343, Batches 87/91 and 79/92 rat maintenance diet ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum.
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum.
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 42 - 67
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

First, stock solutions of the test article in bidistilled water with concentrations of 1.12 mg/ml and 1.01 mg/ml were prepared as follows: 112 mg and 101 mg of the test article was each weighed into a 100 mL volumetric flask; then, about 70 mL of bidistilled water was added and these mixtures were treated in an ultrasonic bath for dissolution. Next, the volumetric flasks were filled to volume with bidistilled water. Finally, standard solutions were prepared by respective dilution of these stock solutions with bidistilled water to yield concentrations in the range from 10.1 µg/ml to 112.0 µg/ml. These standard solutions were used to calibrate the HPLC.

Analysis of Samples: About 2 g of each test article/vehicle mixture was precisely weighed (to the third decimal place) into a 100 mL volumetric flask. These mixtures were dissolved with about 70 mL of bidistilled water by treatment in an ultrasonic bath. Subsequently, the volumetric flasks were filled to volume with bidistilled water. These sample solutions were further diluted with bidistilled water to yield a concentration within the calibration range. Finally, an aliquot (10 µL) was quantified by HPLC.

HPLC-Determination: (Operating Conditions)
Apparatus:Merck L-6200 pump, Merck L-4000 photometer, Merck D-2500 integrator, Merck AS-2000 sampling unit.
Column: Nucleosil C-18 AB; 5 µm; 125 • 4.0 mm.
Eluent: Methanol: 30mL, Bi-distilled water 970 mL.
Flow rate: 0.5 ml/min.
Temperature: Room temperature
Injection volume: 10 µL
Detection: UV 200 nm

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily, 7d/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: Control

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2: Test group

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 3: Test group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4: Test group

No. of animals per sex per dose:

10 males, 10 females (group 1 and 4)
5 males, 5 females (group 2 and 3)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats.
- Rationale for animal assignment (if not random): Randomly allocated to groups by body weight.

Positive control:

none

Observations and examinations performed and frequency:

FOOD CONSUMPTION: The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the RCC computer.

BODY WEIGHTS: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

VIABILITY / MORTALITY: Observations for viability and mortality were recorded once daily.

CLINICAL SIGNS: Signs of toxicity were assessed once dally. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

OPHTHALMOSCOPIC EXAMINATIONS
Dates:
at 4 weeks: March 27, 1992 (on all surviving animals)
at 6 weeks: April 10, 1992 (on all surviving animals of groups 1 and 4)
Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment
and a second time on the recovery individuals of groups 1 and 4 at termination of the recovery period. 10 - 90 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil).


CLINICAL INVESTIGATIONS: GENERAL
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between 07.05 and 08.30 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube. Urine was collected during the 18-hours fasting period into a specimen vial, using a metabolism cage.

Blood and urine sampling: The assays of blood and urine parameters were performed under internal laboratory quality control conditions to assure reliable test results.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were taken from all animals necropsied at termination of treatment or recovery: Adrenals; Brain; Heart; Kidneys; Liver; Ovaries; Pituitary gland; Spleen; Testes; Thyroid, Parathyroid gland.

Sacrifice and pathology:

NECROPSY:
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 20 to 25 hours, but water was provided. Necropsies were performed by experienced prosectors. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution: Adrenals; Aorta; Bone (sternum, femur); Bone marrow (sternum, femur); Brain; Cecum; Colon; Ouodenum; Epididymides; Esophagus; Eyes with optic nerve and Harderian gland; Mammary gland area; Femur including joint; Heart; Ileum; Jejunum; Kidneys; Larynx; Lacrimal gland, exorbital; Liver; Lung Infused with formalin; Lymph nodes, mandibular, mesenteric; Nasal cavity; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland, (mandibular, sublingual); Seminal vesicles; Sciatic nerve; Skeletal muscle; Skin; Spinal cord, (cervical, midthoradc, lumbar); Spleen; Stomach; Testes; Thymus; Thyroid incl. Parathyroid gland; Tongue; Trachea; Urinary bladder infused with formalin; Uterus; Vagina; Gross lesions.

HISTOPATHOLOGY
Slides of Adrenals, Heart, Kidneys, Liver, Spleen and Stomach collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to all Gross lesions. All abnormalities were described and reported.

Other examinations:

FOOD CONSUMPTION: The food consumption is calculated per rat individual and per food consumption interval. It expresses the average food consumed per animal and per day over the food consumption interval.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights, all ratios and clinical laboratory data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnetttest (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The Fisher's exact test for 2x2 tables was applied to the overall spontaneous mortality data and for the overall ophthalmoscopy data.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were round-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed in any animal throughout the entire study period.

Mortality:

no mortality observed

Description (incidence):

No death occurred throughout the entire study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of male rats at 1000 mg/kg was slightly, but statistically significantly higher from treatment day 1 to 8, when compared with the corresponding controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of male rats at 1000 mg/kg was slightly, but statistically significantly higher from treatment day 22 to 28, when compared with the corresponding controls.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of male rats at 1000 mg/kg was slightly, but statistically significant higher from treatment day 22 to 28, when compared with the values of the corresponding controls.

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected on ophthalmoscopic examination in any animal.

Haematological findings:

no effects observed

Description (incidence and severity):

The assessment of hematological data included no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The assessment of clinical bio data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. All differences in the results of the clinical laboratory data were considered to be incidental and of normal biological variation for rats of this strain and age.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In female rats of group 2 (50 mg/kg) the heart weights as well as heart to body weight ratio were slightly, but statistically significantly higher when compared to the corresponding controls. In female rats of group 3 (200 mg/kg) a slightly, but significantly increased pituitary weight and pituitary to body weight ratio were noted. After termination of the recovery period a significantly higher spleen to brain weight ratio and a lower thyroid to body weight ratio were recorded in male rats of the high dose group (1000 mg/kg). No corresponding findings were observed in the females of the same group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related findings were detected during the necropsy.
The following findings are regarded as common:
kidneys: pelvic dilation, gravel in pelvis, cys
stomach/intestine: discolorations or red foci of the mucosa
thymus: discolorations and reddish or red foci
lymph nodes: discolorations and red foci
seminal vesicles: discoloration.
ovaries: discoloration.
uterus: discoloration.
exorbital lacrimal gland: red foci

The following findings are regarded as uncommon:
body cavities: black and firm nodule of the pancreatic tissue (animal no. 3, group 1)
testes: reduced in size, d=13x8 mm (animal no. 23, group 4)
Liver: diaphragmatic hernia and grey white foci (animal no. 52, group 4 ) .

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The following, common microscopic findings correspond to some but not to all detected gross lesions:
foci/discolorations: congestion
pelvic dilation of kidneys: pelvic dilation
dilation of uterus: horn dilation (proestrus).
The nodule of the pancreatic adipose tissue of animal no. 3 was found to be an hemal lymph node. The testes of male no. 23 were hypoplastic with Sertoli's cells lining the tubules.
Corresponding to the diaphragmatic hernia of the liver of animal no. 52 was an hepatodiaphragmatic nodule which showed a slight, focal cholangiofibrosis and linear nuclear chromatin pattern. These nodules are a not uncommon spontaneous abnormality in rats.
All findings recorded are within the normal range of background alterations commonly observed in rats of this strain and at these ages.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

mortality

Critical effects observed:

not specified

Conclusions:

The no-observed-adverse-effect-level (NOAEL) was set at 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The test article DMPPA_701-402-5 was administered orally by gavage, once daily at doses of 50, 200, and 1000 mg/kg/day for 28 consecutive days to rats of both sexes. This test was conducted in accordance with OECD TG 407 and EEC Directive 84/449 B.7 in a GLP compliant laboratory. The control animals received the vehicle (double-distilled water) only. After termination of the treatment period five rats per sex of the vehicle control group and at 1000 mg/kg/day were observed for a further 15-day treatment-free period. No death occurred throughout the entire study period and no clinical signs of toxicity were noted. The slightly, but statistically significantly higher food consumption during week 4 of treatment and body weight gain during week 1 in male rats at 1000 mg/kg are considered to be of coincidental origin. The assessment of hematological, clinical biochemical and urinalysis data indicated no changes of toxicological significance. No abnormalities were detected on ophthalmoscopic examination. The determination of organ weights and calculation of organ to body and organ to brain weight ratios revealed minor, but statistically significant deviations from the corresponding controls. Since no correlation with results of clinical laboratory investigations and pathological examinations could be established these deviations are considered to be of coincidental origin without any toxicological significance. There were no treatment-related macroscopic alterations detected at necropsy. The microscopic examination revealed no morphological evidence of toxicity produced by the treatment with DMPPA_701-402-5. Based on the results obtained in this study, the "no-adverse-effect-level" of DMPPA_701-402-5 is 1000 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Repeated dose toxicity, oral:

One fully reliable study is available (Brunke, 2010, Repeated dose toxicity: oral) conducted according to OECD 407 and GLP (28 d, oral gavage, doses: 50, 200 and 1000 mg/kg/ d, Wistar rats). No death occurred throughout the entire study period and no clinical signs of toxicity were noted. The slightly, but statistically significantly higher food consumption during week 4 of treatment and body weight gain during week 1 in male rats at 1000 mg/kg are considered to be of coincidental origin. The assessment of haematological, clinical biochemical and urinalysis data indicated no changes of toxicological significance. No abnormalities were detected on ophthalmoscopic examination. The determination of organ weights and calculation of organ to body and organ to brain weight ratios revealed minor, but statistically significant deviations from the corresponding controls. Since no correlation with results of clinical laboratory investigations and pathological examinations could be established these deviations are considered to be of coincidental origin without any toxicological significance. There were no treatment-related macroscopic alterations detected at necropsy. The microscopic examination revealed no morphological evidence of toxicity produced by the treatment with

DMPPA_701-402-5 . Based on the results obtained in this study, the "no-adverse-effect-level" of DMPPA_701-402-5 is 1000 mg/kg body weight for males and females.

 

- Repeated dose toxicity, inhalation:

Currently no study to assess the repeated dose inhalation toxicity potential of DMPPA_701-402-5 is available. However, the vapour pressure for the substance was found to be low (extrapolated <3 x 10E-02 Pa at 25 °C). Hence the substance is considered to have low volatility. Synthesis and formulation of this chemical is performed in a closed process; the final product consists of clear-viscous liquid with a low vapour pressure. All related products are not foreseen for spray application. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Further, the chemical is found to have high water solubility of 8000 g/L, hence in the case of mists or vapours are inhaled and entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. No systemic toxicity was observed when DMPPA_701-402-5 was administered up to 1000 mg/kg bw/day via gavage in a 28-day repeated dose toxicity study. Taking above arguments into consideration, low toxicity potential is expected on repeated exposure of DMPPA_701-402-5

via inhalation route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for DMPPA_701-402-5 is considered to be scientifically not necessary.

- Repeated dose toxicity, dermal:

Currently no study to assess the repeated dose dermal toxicity of DMPPA_701-402-5 is available. It has water solubility of 8000 g/L and n-octanol/water partition coefficient (log P) of -1.86, indicating it being too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance will be low. No systemic toxicity was observed when DMMPA_701-402-5 was administered upto 1000 mg/kg bw/day via gavage in a 28-day repeated dose toxicity study. Similarly, absence of systemic toxicity in skin irritation as well as sensitization studies, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Further experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon dermal exposure and not after systemic application. Taking into consideration the above arguments, low toxicity potential is expected on repeated exposure of DMPPA_701-402-5

via dermal route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for DMPPA_701-402-5 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the available data, DMPPA_701-402-5 is not hazardous when exposed repeatedly via oral route. Therefore, no classification according to Regulation (EC) No. 1272/2008 is deemed to be required.