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Description of key information

Envigo Research Limited, 2017: An acute oral toxicity in the Wistar strain rat has been performed, according the 420 OECD guideline in order to assess the LD50 of the test item.

Following a sighting test at dose levels of 50, 300 and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a suspension in arachis oil BP, at a dose level of 300 mg/kg. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. At 2000 mg/kg, one animal was killed because of evident sign of systemic toxicity, which was confirmed by necropsy. At 50 and 300 mg/kg dose levels, no mortality, no sign of systemic toxicity (also in necropsy) were observed. Therefore, the LD50 ot the test item in the female Wistar strain rat is estimated to be in the range of 300 -2000 mg/kg.

 

Lent et al 2012: Mortality occurred in all male rats at doses of 300.0 mg/kg and greater, occurring 3 ± 1.7 hours after administration of the test substance. Mortality occurred in female rats dosed at 300.0 mg/kg and greater with the exception of the rat dosed at 450.0 mg/kg which survived the 14-day observation period. Mortality occurred in female rats 4 ± 1.7 hours after administration of the test substance.

Clinical signs including lethargy, rapid respiration/laboured breathing, prostrate posture, and salivation were noted in male rats at dose level of 88.9 mg/kg and greater and in female rats at dose level of 133.3 mg/kg and greater. Female rats additionally exhibited chromodacryorrhea. Clinical signs were apparent approximately fifteen to thirty minutes after dosing and persisted throughout the first day of observation in surviving animals.

 

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:
26 May - 17 December, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
not applicable
Remarks:
Due to the short term nature of the study, no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affec the integrity or validity of the study.
Qualifier:
according to
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
not applicable
Remarks:
Due to the short term nature of the study, no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affec the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state/Apparence : pale yellow powder
- Source and lot/batch No.of test material: 55503625
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was prepared as a suspension in arachis oil BP.
The arachid oil BP was used because the test item dod not dissolve/suspend in distilled water.
Species:
rat
Strain:
Wistar
Remarks:
RccHan : WIST
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Females nulliparous and non-pregnant
- Age at study initiation: 8-12 weeks of age
- Fasting period before study: an overnight before dosing and for approximately 3 to 4 hours after dosing
- Housing: animal were foused in groups of up to four in suspended solid-floor polypropylene cages surnished with woodflakes
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 5 days
- Body Weight: the body weight variation did not exceed +/- 20% of the mean body weight at the stard of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/ 12

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
Using available information on the toxicity ot the test item, 50 mg/kg was chosen as the starting dose
A single animal was treated with 50mg/kg.
In the absence of mortality at this dose level, an additional animal was treated with 300 mg/kg.
In the absence of mortality at this dose level, an additional animal was treated with 2000 mg/kg.
Due to mortality at the dose level of 2000 mg/kg, an additional group of 4 animals was treated at 300 mg/kg.
Doses:
50, 300, 2000 mg/kg
No. of animals per sex per dose:
1 animal for 50 mg/kg and 2000 mg/kg
5 animal for 300 mg/kg
Control animals:
no
Details on study design:
- Duration of clinical observation period following administration: 30 min, 1, 2 and 4 hours after dosing and the daily for up to 14 days.
- Frequency of weighing: day 0 and on day 7 and 14 or at death.
- Frequanecy of morbidity and mortality : twice daily, early and late during normal working day, and once daily at weekends and public holidays.
- Euthanasia : animals were killed by cervical dislocation
- Necropsy of survivors performed: yes for all animals

Evaluation of data included identification of the number of animals that died during the study (or that were killed for humane reasons), and determination of the nature, severity, onset and duration of the toxic effects. If possible, the signs of evident toxicity were described.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
At 50 mg/kg: no mortalilty
At 300 mg/kg: no mortality
At 2000 mg/kg: the animal was killed for humane reasons, 2 hours after dosing, due to the occurence of clinical signs of toxicity that approached the severity
limit set forth in th UK Home Office Project License.
Clinical signs:
At 50 mg/kg: Hunched posture and ataxia were noted during the day of dosing. The animal appeared normal 1 day after dosing.

At 300 mg/kg: No signs of systemic toxicity during the observation period.

At 2000 mg/kg: Signs of systemic toxicity noted were loss of righting reflex, decreased respiratory rate, labored respiration, pallor of the extremities, pilo-
ereection, prostration ans hypothermia.
Body weight:
At 50 mg/kg and 300 mg/kg: The animals showed expected gains in body weight over the observation period.

At 2000 mg/kg: Not applicable
Gross pathology:
At 50 mg/kg and 300 mg/kg: No abnormalities were noted at necropsy.

At 2000 mg/kg: Abnormalities noted at necropsy were patchy pallor of the liver, dark kidneys and clear liquid present in the stomach.
Other findings:
NA
Interpretation of results:
Category 4 based on GHS criteria
Executive summary:

An acute oral toxicity in the Wistar strain rat (A. Sanders, 2017, Envigo) has been performed, according the 420 OECD guideline in order to assess the LD50 of the test item.

Following a sighthing test at dose levels of 50, 300 and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a suspension in arachis oil BP, at a dose level of 300 mg/kg. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

At 2000 mg/kg, animal was killed because of evident sign of systemic toxicity, confirmed by necropsy.

At 50 and 300 mg/kg, no mortality, no sign of systemic toxicity (also in necropsy) were observed.

Therefore the LD50 ot the test item in the female Wistar strain rat is estimated to be in the range of 300 -2000 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
300 mg/kg bw
Quality of whole database:
All studies Klimisch 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:
Study completed February 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
Slight differences in temperature and humidity observed due to the heat required to generate DNAN aerosol. Due to the relatively short duration of the rats to
these slight temperature, humidity and oxygen disgressions, the environmental conditions with the exposure chamber were considered to be acceptable and did not affect the validity of the study
Deviations:
not applicable
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
The MMAD was higher than required ranges (1-4 um). It was due to the method of generation, which was a direct result of the physical properties of DNAN. It
is heated and generated as a condensation aerosol, which actually represented a realistic exposure scenario for individuals involved with this material in a
munition plant. Condensation aerosols make it difficult to control the particule size of the test atmosphere due to various factors (sublimation, condensate nuclei...). This deviation is considered to be acceptable for assessing the inhalation toxicity of DNAN.
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Test type:
fixed concentration procedure
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BAE Systems, Ordnance Systems, 4509 West Stone Kingsport, TN 37660 ; Batch number = 10DNAN9-9 ; lot number = BAE10>H281-008
- Expiration date of the lot/batch: N/A
- Purity test date: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Practically insoluble in water
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachussets
- Females nulliparous and non-pregnant
- Age at study initiation: 7 weeks old
- Housing: singly in solid bottom polycarbonate boxes, suspended on a cage rack
- Diet : ad libitum, exept during the 4-hour exposure period
- Water :ad libitum , exept during the 4-hour exposure period
- Acclimation period: 5-day acclimatization

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71 +/-0.6
- Humidity (%): 47 +/- 2.1

- Photoperiod (hrs dark / hrs light): a 12/ 12

Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 10 µm
Geometric standard deviation (GSD):
>= 1.3 - <= 1.8
Remark on MMAD/GSD:
2 particles size samples collected from the 2 exposures
GSD for first sample : 1.8-1.8
GSD for second sample : 1.3-1.4
The desired MMAD for inhalation toxicology studies typically ranges from 1-4. However For this study it was higher than desired.
The reason for the increased particule size was due to the method of generation , which was a direct result of the physical properties of the test material which is a wax-like solid that could not be dispersed in the air as a dry powder.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a glass cylinder
- Exposure chamber volume: 36 liters
- Method of holding animals in test chamber: Only the heads of rats were positioned within the exposure chamber and their bodies were positioned outside the exposure chamber. Rats were positioned in the exposure cylinder such that their noses were at the conical end of the cylinder. in order to secure the rat in this position, a plastic disk with a hole in the center was inserted over the tail of each rat and positioned within the cylinder close to the base of the rat's tail so as to prevent the rat frombacking out of the rearof the cylinder. Each exposure cylinder was inserted into one of the holes in the faceplate of the exposure chamber such that the head of each rat extended into the exposure chamber (nose-only).


- Source and rate of air:10L/min
- Method of conditioning air: air was introduced via a port on the top of the exposure chamber to provide adequate test atmospheremixing in the chamber and to maintain oxygen levels above 19%
- System of generating particulates/aerosols: Chamber atmospheres of DNAN aerosol were generated dynamically in air within the exposure chamber. Test atmospheres were generated by heating powdered DNAN in a fabricated 3-neck glass generation vessel to approximately 270°C with a 600mL beaker heating mantle.
- Method of particle size determination: Measurement of the Mass Median Aerodynamic Diameter (MMAD)
- Treatment of exhaust air: Test atmospheres were exhausted through air impinger containing acetone to scrub the DNAN using a Radeco model AVS-28A vacuum pump prior to discharge into a fume hood.
- Temperature, humidity, pressure in air chamber: Chamber temperature for the exposures ranged from 21 to 25°C and were slightly outside of the targeted range of 22+/-2°C during certain periods of exposure. Chamber relative humidity for the exposures ranged from 15 to 33% and was slightly lower than the targeted range of 30 to 70% due to the heat required to generate DNAN aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: Numerous tube samples were collected in the line with the gravimetric sampling train and analyzed by gas chromatography to determine if vapor component was present. The gravimetric analysis was the most accurate method to characterize the condensation aerosol present in the chamber during the animal exposures.
- Samples taken from breathing zone: Atmospheric concentration of the DNAN condensation aerosol was determined during all inhalation exposures. A total of 10 to 17 samples were collecteded from the exposure chamber during each of the exposures.







Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Remarks on duration:
Due to problems with the generation system, the first LC50 exposure was terminated approximately 2 hours early. Data not reported.
Concentrations:
0.73 +/- 0.10 mg/L and 2.4 +/- 0.25 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
For the first LC50 inhalation exposure : rats were weighed on test days 1, 2, 3, 6, 8, 13 and 15. for the second LC50 inhalation exposure, at test days 1, 2, 3, 7, 10 and 15
Rats were observed approximately every hour during each oth the inhalation exposures and each day during the 14-day post-exposure
- Necropsy of survivors performed: yes
- Other examinations performed: clinicals signs of toxicity and gross pathological examination after euthanasia by carbon dioxide asphyxiation.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
>= 2.4 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality during the 4-hour exposure and subsequent 14-day recovery period.
Clinical signs:
other: During the exposure, most rats generally began to form a yellow crustaround their noses and on their whiskers within the first hour of exposure. The yellow crust remained throughoutthe exposure but did not appearto effect respiration. After removal from e
Body weight:
2 of 5 male rat exhibited a 1-2% weight loss and 4 of 5 female exhibited a 1-5% weight losson post-exposure day 1 but all animals began to gain weight at a normal rate by post-exposure day 2.
Gross pathology:
All animals exposed during the 2 LC50 exposures had minimal to mild yellow staining on various parts of the head, chest, and/or forearms at the time of necropsy.
One male rat exposed during the first LC50 exposure also had a mildly enlarged spleen.
One male rat exposed during the second LC50 exposure had mottled lungs with froth in the lower trachea and a second male rat had a slightly enlarged spleen.
Interpretation of results:
Category 5 based on GHS criteria
Executive summary:

According to the Health Effects Test Guidelines OPPTS 870.1300, an acute inhalation toxicity study has been performed, using groups of 10 rats (5 male and 5 female), exposed nose-only to a 2.4 mg/L aerosol atmosphere of DNAN during 4 hours. The condensation aerosol atmosphere of DNAN has been obtained by heating powdered DNAN. By GC analysis, no significant vapor component was present in the chamber. 2.4 mg/L is the highest-achievable concentration of DNAN aerosol.

No test compound-related mortalities occured in rats exposed during the study and no adverse toxic signs, body wheight changes, or gross necropsy findings were observed in exposed rats after exposure and after a 14 -day observation period.

Therefore, the LC50 will be reported as > 2.4 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An acute inhalation toxicity study (Crouse and O'Neill, 2015) was performed according to US EPA guidelines (OPPTS 870.1300, 1998) even though, for REACH, an acute inhalation test could be waived due to measured vapour pressure of DNAN ie 1x10 ^-6 kPa at 25°C (Chapter R.7a: Endpoint specific guidance, Version 6.0 - July 2017 p367). In order to generate a test atmosphere the test substance DNAN, which is a waxy solid, was first ground to a fine consistency then heated to ca. 270°C and the resultant DNAN condensation aerosol atmosphere was used to perform the test. The mean atmospheric concentration of the DNAN condensation aerosol in the exposure chamber during the LC50 tests were determined to be 2.4 ± 0.25 mg/L. The MMAD of the LC50 exposure atmosphere ranged from 5.6 to 5.8 μm, with 0 percent of the particles less than 1 μm, 13-17 percent of the particles less than 4 μm, and 94 percent of the particles less than 10 μm. The particle sizes generated during this study were the result of attempts to deliver the most reasonable MMAD based on physical constraints of the test material.

 

The larger particles contained in a test atmosphere are typically retained in the mucous-lined head region and tracheobronchial airways where they may be absorbed or transported out of the respiratory airways via ciliary action. The particles transported out of the airways may then either be expelled or swallowed to the gastrointestinal tract, essentially leading to an additional oral dose. The smaller, soluble particles reaching the alveolar (deep lung) regions may then be absorbed into the blood. Therefore, for DNAN, there is the potential for the respiratory system to be exposed to a large proportion of the exposure atmosphere and blood concentration measurements taken during another phase of this study demonstrate this.

 

As, despite all efforts, the maximal exposure atmosphere that could be generated was 2.4 mg/L and as there were no compound-related mortalities and no adverse toxic signs, body weight changes, or gross necropsy findings observed in exposed rats, DNAN does not cause any adverse effects when tested at the highest achievable concentration.

Justification for classification or non-classification


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