Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2017 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
yes
Remarks:
Exposure chamber humidity was not kept at 30-70 % in the second exposure. One animal was not weighed at death.
Qualifier:
according to
Guideline:
EU Method B.52 (Acute Inhalation Toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
Exposure chamber humidity was not kept at 30-70 % in the second exposure. One animal was not weighed at death.
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in the dark
- Stability under test conditions: Confirmed by GC-MS analysis.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material) : The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A

Test animals

Species:
rat
Strain:
other: RccHan:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 200-300 g
- Fasting period before study: No
- Housing: The animals were housed in groups of up to three by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet (e.g. ad libitum): Ad libitum; 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70 (apart from the second exposure 14 %)
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 21 August 2017 To: 22 January 2018

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.11 - <= 2.22 µm
Geometric standard deviation (GSD):
>= 2.27 - <= 2.38
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply. Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: 40 L/hour. Compressed air.
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature, humidity, pressure in air chamber: Temperature: 19-20 ºC; Humidity: 14-41 %; Air Pressure: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test item was measured by determined by gas chromatography (GC) using an external standard technique. The test atmosphere was sampled after theoretical chamber equilibration and then at approximately 30 minute intervals during the exposure period. Samples were then submitted for analysis.
- Samples taken from breathing zone: Yes

VEHICLE
- Composition of vehicle (if applicable): N/A
- Concentration of test material in vehicle (if applicable): N/A
- Justification of choice of vehicle: N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference. The mean amount for each stage was used to determine the cumulative amount below each cut off point size. In this way, the proportion (%) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 µm was calculated.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The resulting values were converted to probits and plotted against Log10 cut point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (percentage) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Mean achieved concentrations:

5.67 mg/L and 1.02 mg/L
No. of animals per sex per dose:
3 males and 3 females per test concentration
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:

Clinical observations- All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any deaths or evidence of overt toxicity was recorded at each observation.
Body weight - Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14 or at death.
Necropsy- At the end of the 14 observation period surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals including those that died or were humanely killed during the study were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, organ weights, necropsy
Statistics:
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects. Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - <= 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
2.5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: result is based on the cut-off value as determined in Annex 3 of the OECD TG 436
Mortality:
5.67 mg/L group: 3/3 males and 3/3 females
1.02 mg/L group: 0/3 males and 0/3 femlaes
Clinical signs:
5.67 mg/L group: During exposure all animals exhibited decreased respiratory rate. One animal died after 102 minutes of exposure, two animals died after 120 minutes of exposure. Labored respiration and gasping respiration were also exhibited by two of the surviving animals after 120 minutes of exposure. One animal died after 160 minutes of exposure and a further animal died after 170 minutes of exposure. Ataxia, hunched posture and areas of red/brown staining around the snout were also exhibited by the surviving animal after 180 minutes of exposure. This animal was humanely killed due to the occurrence of clinical signs of toxicity that were considered likely to exceed the severity limit set forth in the UK Home Office Project License after 230 minutes of exposure.

1.02 mg/L group: During exposure, on removal from the chamber and one hour post-exposure, all animals exhibited decreased respiratory rate. On removal from the chamber all animals exhibited ataxia, which was severe in one animal, and there were occasional instances of gasping respiration, labored respiration, noisy respiration, areas of red/brown staining around the snout with isolated instances of clonic convulsions, lethargy and areas of red/brown staining around the eyes. Improvement in the condition of the animals was noted one hour post-exposure.

One day after exposure, all animals exhibited hunched posture and one female also exhibited noisy respiration, pilo-erection and areas of red/brown staining around the eyes.

Animals recovered to appear normal from Days 2 to 5 post-exposure. One male that recovered to appear normal on Day 2 post-exposure subsequently exhibited sneezing on Day 3 post exposure. Sneezing was also exhibited by one female from Days 2 to 4 post-exposure.
Body weight:
5.67 mg/L group: None of the animals survived until the end of the day of exposure and as such no significant body weight data was collected.

1.02 mg/L group: With the exception of one female, all animals showed body weight loss on Day 1 post exposure. Animals showed expected gains in body weight during the remainder of the recovery period with the exception of two males that showed body weight loss or no gain in body weight from Days 1 to 3 post-exposure, one female that showed no gain in body weight from Days 3 to 7 post-exposure and one female that showed no gain in body weight from Days 7 to 14 post-exposure.
Gross pathology:
5.67 mg/L group: The following macroscopic abnormalities were detected at necropsy of animals that died or were humanely killed during exposure:
Lungs – hemorrhagic, pale and/or abnormally red.

1.02 mg/L group: No macroscopic abnormalities were detected at necropsy of one animal that survived until the end of the recovery period.

The following macroscopic abnormalities were detected at necropsy of the remaining animals that survived until the end of the recovery period:
Lungs – pale, dark patches, abnormally red, abnormally dark.

Due to the observations noted during the study and at necropsy it is considered that the deaths noted during the study may have been mainly attributable to local toxicity.

Any other information on results incl. tables

Table 1       Mortality Data

Mean achieved atmosphere concentration

(mg/L)

Deaths

Male

Female

Total

5.67

3/3*

3/3

6/6

1.02

0/3

0/3

0/6

* 1 male killed in extremis due to evert signs of clinical toxicity

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
None of the animals died in a group of six rats exposed to a mean achieved atmosphere concentration of 1.02 mg/L. However, all animals died in a group of six rats exposed to a mean achieved atmosphere concentration of 5.67 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item, in the RccHanTM : WIST strain rat, should be considered to be > 1.0 – 5.0 mg/L (Globally Harmonized Classification System – Category 4). This equates to an LD50 based on the cut-off value of 2.5 mg/L.
Executive summary:

OECD 436 (2018) - In an acute inhalation toxicity study, groups of young adult RccHan:WIST strain rats (6 male and 6 female) were exposed by inhalation route to Reaction Mass of N,N,N',N-tetrabutylmethylenediamine and dibutylamine for 4 hours to nose only at mean measured concentrations of 1.02 and 5.67  mg/L.  Animals then were observed for 14 days after which time they were sacrificed and underwent necropsy.

 

Mortality

 

In the 5.67 mg/L group, all 6 rats were found dead within 102-170 minutes exposure. There was no mortality observed in the 1.02 mg/L group.

 

Clinical signs

 

5.67 mg/L group: During exposure all animals exhibited decreased respiratory rate. One animal died after 102 minutes of exposure, two animals died after 120 minutes of exposure. Labored respiration and gasping respiration were also exhibited by two of the surviving animals after 120 minutes of exposure. One animal died after 160 minutes of exposure and a further animal died after 170 minutes of exposure. Ataxia, hunched posture and areas of red/brown staining around the snout were also exhibited by the surviving animal after 180 minutes of exposure. This animal was humanely killed due to the occurrence of clinical signs of toxicity that were considered likely to exceed the severity limit set forth in the UK Home Office Project License after 230 minutes of exposure.

 

1.02 mg/L group: During exposure, on removal from the chamber and one hour post-exposure, all animals exhibited decreased respiratory rate. On removal from the chamber all animals exhibited ataxia, which was severe in one animal, and there were occasional instances of gasping respiration, labored respiration, noisy respiration, areas of red/brown staining around the snout with isolated instances of clonic convulsions, lethargy and areas of red/brown staining around the eyes. Improvement in the condition of the animals was noted one hour post-exposure.

One day after exposure, all animals exhibited hunched posture and one female also exhibited noisy respiration, pilo-erection and areas of red/brown staining around the eyes.

Animals recovered to appear normal from Days 2 to 5 post-exposure. One male that recovered to appear normal on Day 2 post-exposure subsequently exhibited sneezing on Day 3 post‑exposure. Sneezing was also exhibited by one female from Days 2 to 4 post-exposure.

 

Body Weight

 

5.67 mg/L group: None of the animals survived until the end of the day of exposure and as such no significant body weight data was collected.

1.02 mg/L group: With the exception of one female, all animals showed body weight loss on Day 1 post‑exposure. Animals showed expected gains in body weight during the remainder of the recovery period with the exception of two males that showed body weight loss or no gain in body weight from Days 1 to 3 post-exposure, one female that showed no gain in body weight from Days 3 to 7 post-exposure and one female that showed no gain in body weight from Days 7 to 14 post-exposure.

 

Necropsy

 

5.67 mg/L group: The following macroscopic abnormalities were detected at necropsy of animals that died or were humanely killed during exposure:

Lungs – hemorrhagic, pale and/or abnormally red.

 

1.02 mg/L group: No macroscopic abnormalities were detected at necropsy of one animal that survived until the end of the recovery period.

The following macroscopic abnormalities were detected at necropsy of the remaining animals that survived until the end of the recovery period:

Lungs – pale, dark patches, abnormally red, abnormally dark.

 

Due to the observations noted during the study and at necropsy it is considered that the deaths noted during the study may have been mainly attributable to local toxicity.

None of the animals died in a group of six rats exposed to a mean achieved atmosphere concentration of 1.02 mg/L.  However, all animals died in a group of six rats exposed to a mean achieved atmosphere concentration of 5.67 mg/L.  It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item, in the RccHanTM : WIST strain rat, should be considered to be > 1.0 – 5.0 mg/L (Globally Harmonized Classification System – Category 4).  This equates to an LD50 based on the cut-off value of 2.5 mg/L.