Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Endpoint summary

Description of key information

The acute toxicity of EGGE 2806-1 in rats is > 2000 mg/kg bw (LD50) after oral administration (Brockes, 2015) or > 3038 mg/m3 (LC50) after 4 hour inhalation exposure of a liquid aerosol (Kopf, 2015).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-Juni 2015

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The stability of test test item in the vehicle (0.05 and 200 mg/mL)for at least 7 days was confirmed by analysis


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The dosing preparations were prepared at room temperature and were well mixed prior administration. Before administration the dosing preparations were shortly heated up to 40°C in a cabinet.
- Final preparation of a solid: The test compound was heated to approximately 11 0°C for formulation and in orderto melt the amorphous mass so that an aliquot could be taken.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Homogeneity (visual inspection): The formulations 0.5 mglml and 200 mglml are clear solutions after preparation and therefore homogeneous.
The 200 mg/ml formulation showed a slight turbidity after cooling down at room temperature which disappeared after heating shortly in a cabinet at 40°C.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan GmbH, Horst, Netherlands
- Strain: HsdRCCHan:WIST
- Age at study initiation: approx. 8-12 weeks
- Weight at study initiation: 160-180 g
- Fasting period before study: 16-24 hours
- Housing: in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: Kolliphor HS 15 I Ethanol I Tap water (40/10/50)

Details on oral exposure:

- Application volume: 10 ml/kg bw

- Rationale for the selection of the starting dose:
As described in the flow charts of Annex 2, OECD guideline 423, the starting dose level should be that which is most likely to produce mortality in
some of the dosed animals. Therefore, the limit dose 2000 mg/kg bw was chosen as starting dose.

Doses:

2000 mg/kg

No. of animals per sex per dose:

2 x 3 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: at least once daily (clinical signs, mortality) or once weekly (weight gain)
- Necropsy of survivors performed: yes

Statistics:

none (limit test)

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

All 6 animals survived the treatment.

Clinical signs:

No clinical signs were observed in 6/6 animals.

Body weight:

Body weight development was not affected.

Gross pathology:

No gross pathological findings were observed.

Other findings:

none

Executive summary:

A single oral dose of 2000 mg/kg bw in Kolliphor HS 15/Ethanol/Tap water (40/10/50) was tolerated by female rats without mortalities, clinical signs, effects on weight gain and gross pathological findings. According to OECD guideline 423 the LD50 oral of the test item is > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

The study is GLP compliant and is of high quality (Klimisch score=1)

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2015 (inlife phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(2009)

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 39 (2009)

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: certified for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: certified by analysis

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland, AD Horst, The Netherlands
- Age at study initiation: approximately 2 months
- Weight at study initiation (mean): males 187.4-191.6 g, females 172.6-182.6; at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex.
- Housing: Singly in conventional Makrolon® Type IIIH cages with gnawing sticks.
- Diet and water: ad libitum
- Acclimation period: at least 5 days; during this period, rats were also acclimatized to the restraining tubes.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 60
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: liquid aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Lewatit© water

Mass median aerodynamic diameter (MMAD):

1.66 µm

Geometric standard deviation (GSD):

1.99

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Mode of exposure: Animals were exposed to the aerosolized test item in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals' size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided. This type of exposure principle is comparable with a directed-flow exposure design.The chambers used are commercially available (TSE, 61348 Bad Homburg) and the performance as well as their validation has been published.

- Description of apparatus: Dry conditioned air was used to generate the test item atmospheres The test atmosphere was conveyed through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimizes re-breathing of exhaled test atmosphere. Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. All air flows were monitored and adjusted continuously by means of calibrated and computer controlled mass-flow-controllers. A digitally controlled calibration flow meter was used to monitor the accuracy of mass-flow-controller. The ratio between supply and exhaust air was selected so that 85-90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Gas scrubbing devices were used for exhaust air clean-up. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H20. The exposure system was accommodated in an adequately ventilated enclosure. Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors. The sensors were located in the inhalation chamber.

- Source and rate of air: Inlet Air Flow (l/min): 15; Exhaust Air Flow (1imin): 12.8

- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a BEKO RA 55 compressed air dryer. Adequate control devices were employed to
control supply pressure.

- System of generating particulates/aerosols: A modified BGI 3-nozzle Collison nebulizer (Type CN-25 MRE, BGIInc., Waltham MA, USA) was used in order to attain a high and temporally stable concentration of exposure atmospheres. The representative dispersion pressure was approximately 420-450 kPa. The Collision Nebulizer was maintained at 25°C using a thermostat.

- Method of particle size determination: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor.

- Treatment of exhaust air: The exhaust air was purified via filter systems.

- Temperature, humidity, pressure in air chamber:
Air pressure (kPa): 450 (control); 420 (test item); Rel. Humidity (mean, %): 94.5 (control); 83.4 (test item); Temperature (mean, "C): 21.4 (control); 21.9 (test item)

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Gottingen, Germany; postsampling conditioning period of 30 min (at 70°C); 10 min equilibration in an exsiccator; digital balance). The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford,Massachusetts, USA). Samples were taken continuously from the vicinity of the breathing zone.

- Samples taken from breathing zone: yes

VEHICLE

- Concentration of test material in vehicle: 50% (w/v)
- Justification of choice of vehicle: To reduce viscosity and increase the maximum attainable concentration.


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Mass < 3 µm (%): 80.5
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.66/1.99

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric analysis

Duration of exposure:

4 h

Concentrations:

Target concentration: 5000 mg/m³
Analytical concentration: 3038 mg/m³ (technically maximal attainable concentration)

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Bodyweights were recorded prior to exposure (day 0) and on days 1, 3, and 7, and 14. The appearance and behavior of each rat were examined carefully at least two times on the day of exposure and at least once daily thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257). Rectal temperatures were measured shortly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats.

Statistics:

one-way ANOVA (vide infra) (body weight, rectal temperature)

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 038 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: technically maximal attainable concentration

Mortality:

Mortality did not occur at 3038 mg/m³.

Clinical signs:

other: Neither animals exposed to the vehicle nor animals exposed to the test item revealed any clinical symptoms.

Body weight:

Comparisons between the control and the exposure groups did not reveal significant differences in the incremental gain of body weights after exposure.

Gross pathology:

A few gray areas in the lungs of 2/5 female rats and few dark-red foci in the lungs of 2/5 male rats were found in the test item exposed animals.

Other findings:

- Rectal temperatur: Statistical comparisons between the control and the exposure groups did not reveal any significant changes in body temperature at 3038 mg/m³ compared to control animals.
- Reflex measurements: No differences between animals exposed to 3038 mg/m³ test item compared to the control group were found.

Executive summary:

A study on the acute inhalation toxicity was conducted according to OECD TG 403 and OECD GD 39. Male and female rats were nose-only exposed to the liquid aerosol of test item at the maximal technical attainable concentration of 3038 mg/m³. Lewatit© water was used as vehicle to reduce viscosity and increase the maximum attainable concentration. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.66 µm, GSD 1.99). Rats exposed to 3038 mg/m³ did neither reveal clinical symptoms, reduction in incremental body weight, changes in reflexes nor reduction in body temperature after the exposure. Four out of ten rats showed gross pathological findings in the lungs on day 14 (e.g. few gray areas, few dark-red foci). The LC50 (4 h) was thus concluded to be > 3038 mg/m³.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

3 038 mg/m³

Quality of whole database:

The study is GLP compliant and is of high quality (Klimisch score=1)

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A single oral dose of 2000 mg/kg bw in Kolliphor HS 15/Ethanol/Tap water (40/10/50) was tolerated by female rats without mortalities, clinical signs, effects on weight gain and gross pathological findings

(Brockes, 2015). According to OECD guideline 423 the LD50 oral of the test item is > 2000 mg/kg bw.

A study on the acute inhalation toxicity was conducted according to OECD TG 403 and OECD GD 39 (Kopf, 2015). Male and female rats were nose-only exposed to the liquid aerosol of test item at the maximal technical attainable concentrationof 3038 mg/m³. Lewatit© water was used as vehicle to reduce viscosity and increase the maximum attainable concentration. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.66 µm, GSD 1.99). Rats exposed to 3038 mg/m³ did neither reveal clinical symptoms, reduction in incremental body weight, changes in reflexes nor reduction in body temperature after the exposure. Four out of ten rats showed gross pathological findings in the lungs on day 14 (e.g. few gray areas, few dark-red foci). The LC50 (4 h) was thus concluded to be > 3038 mg/m³.

 

Justification for classification or non-classification

Acute toxicity: oral

Based on the LD50 > 2000 mg/kg bw a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.

Acute toxicity: inhalation

Based on the LC50 (4h) > 3038 mg/m3 air (no mortality at the maximum technically attainable concentration) a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.