Registration Dossier

Administrative data

Description of key information

Acute Oral Toxicity; OECD 423; Dreher, D. (2017); LD50: >300 - 2000 mg/kg. Based on the cut-off value the LD50 used in the CSA is 500 mg/kg bw.

Acute Inhalation Toxicity; OECD 436; Bradshaw, J. (2018); LC50: >1 - 5 mg/L. Based on the cut-off value the LD50 used in the CSA is 2.5 mg/L.

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:
03 March - 30 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline valiity criteria were met.
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
European Community (EC). Council Regulation (EC) No 440/2008 (31 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17 December 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
Qualifier:
according to
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147, Japanese Ministry of Agriculture, Forestry and Fisheries of Japan
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
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 C, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: The test article was dispersed in corn oil for the 50 mg/kg dose level. Corn oil was chosen as it was capable of forming a homogeneous solution as described in Covance GLP Study 8359421.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was used as supplied for dose levels of 300 and 2000 mg/kg. For the 50 mg/kg/day group, the test item was formulated in corn oil.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as supplied, or in a corn oil formulation (50 mg/kg/day group only)

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

OTHER SPECIFICS: N/A
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 7-10 weeks
- Weight at study initiation: The weight variation did not exceed ±20% of the mean weight.
- Fasting period before study: Night before dosing
- Housing: The animals were housed in groups of up to five during the acclimatisation period in suspended, solid floor cages with wire lids. From the day prior to dosing (Day-1), the rats were housed in groups of three in similar cages. Bedding was provided on a weekly basis to each cage by use of clean European soft wood bedding (Datesand Ltd., Manchester, UK). Each batch of bedding was analysed for specific constituents and contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.
- Diet (e.g. ad libitum): Throughout the study the animals had access to 5LF2 EU Rodent Diet 14%, which was freely available to the animals at all times, except for a period of fasting from the evening of the day prior to dosing (Day-1) until approximately 3 hours after dosing. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. No contaminants were present in diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage mounted water bottles. The water was periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: 6-15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 14 March 2017 To: 25 May 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
50 mg/kg/day group only
Details on oral exposure:
VEHICLE
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg bw
- Justification for choice of vehicle: Was found to form a homogenous formulation in Covance GLP Study 8359421. Corn oil is also an guideline recommended vehicle.
- Lot/batch no. (if required): Not reported
- Purity: Not reported

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg

DOSAGE PREPARATION (if unusual): N/A

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: N/A
Doses:
2000, 300 and 50 mg/kg
No. of animals per sex per dose:
6 females (2 x groups of 3)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Treated rats were observed closely for clinical signs of reaction to treatment. Clinical signs were recorded immediately post-dose, at approximately 15 and 30 minutes post- dose, hourly between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once daily from the fifth to last day of the observation period.
Individual body weights were recorded on Day-1 (day before dosing) and on Days 1, 4, 8 and 15. Decedent carcass weights were also recorded prior to necropsy
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, free-hand histopathology (sectioning) of liver and kidneys, examination of representative sections of mucosal surfaces of the stomach, small and large intestines
Statistics:
No
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
500 mg/kg bw
Based on:
test mat.
Remarks on result:
other: result is based on the cut-off value as determined in Annex 2 of the OECD TG 423
Mortality:
50 mg/kg = 0/6 mortality
300 mg/kg = 0/6 mortality
2000 mg/kg = 3/6 mortality
Clinical signs:
Decreased activity and hunched posture were noted 3 and 4 hours after dosing, in one animal treated at 2000 mg/kg. Hypothermia, ptosis, piloerection, laboured breathing and prone posture were noted in the animal that was killed in extremis. No clinical signs were seen prior to death in the two animals that were found dead. No clinical signs were seen in animals treated at 50 or 300 mg/kg.
Body weight:
All surviving rats achieved body weight gains during the first and second weeks of the study.
Gross pathology:
No abnormalities were noted at necropsy of animals that died or were killed in extremis during the study or at necropsy of those that were killed at the end of the study.

Table 2      Number of animals dead (and with evident toxicity)

 

Dose

(mg/kg bw)

Mortality

(# dead / total)

Time range of deaths

(hours)

Number with evident toxicity

(# / total)

Male

Female

Combined

Male

Female

Combined

2000

-

3 / 6

3/ 6

24 -48

-

2 / 6

2 / 6

 300

-

0 / 6

0 / 6

n/a

-

0 / 6

0 / 6

 50

-

0 / 6

0 / 6

n/a

-

0 / 6

0 / 6

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based of the experimental conditions of this study, the test article was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

OECD 423 (2017) - This study was conducted to assess the acute toxicity of the test article (Reaction Mass of N,N,N',N-tetrabutylmethylenediamine and dibutylamine), following a single oral administration to small groups of rats. The study design provides information for hazard assessment and classification and enables a chemical to be assigned to toxicity classes but severely restricts animal usage.

 

A group of three female fasted rats was given the test article as a single dose by oral gavage at a dose level of 2000 mg/kg. Based on the results from this dose level, further groups of three female fasted rats were treated at dose levels of 50 mg/kg,


300 mg/kg and 2000 mg/kg. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.

 

The test article was used undiluted for the 300 and 2000 mg/kg dose levels and was dispersed in corn oil for the 50 mg/kg dose level. Surviving animals were killed on Day 15. All animals underwent a full necropsy.

 

Two animals treated at 2000 mg/kg were found dead on Day 2. One other animal was humanely killed due to the severity of the clinical signs on this day.

 

There were no deaths at dose levels of 50 or 300 mg/kg.

 

Decreased activity and hunched posture were noted 3 and 4 hours after dosing, in one animal treated at 2000 mg/kg. Hypothermia, ptosis, piloerection, laboured breathing and prone posture were noted in the animal that was killed in extremis. No clinical signs were seen prior to death in the two animals that were found dead.

No clinical signs were seen in animals treated at 50 or 300 mg/kg.

 

All surviving rats achieved body weight gains during the first and second weeks of the study.

No abnormalities were noted at necropsy of animals that died or were killed in extremis during the study or at necropsy of those that were killed at the end of the study.

Based of the experimental conditions of this study, the test article was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Based on the observed effects the LD50 using the cut-off value was estimated to be 500 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
500 mg/kg bw
Quality of whole database:
The study is reliable and meets the data requirements according to the Registrants tonnage band submission (up to 100 T/y), according to the REACH Regulation (EC) No 1907/2006 Annex VIII, Section 8.5.

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:
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.
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
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
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
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
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
2.5 mg/m³
Quality of whole database:
The study is reliable and meets the data requirements according to the Registrants tonnage band submission (up to 100 T/y), according to the REACH Regulation (EC) No 1907/2006 Annex VIII, Section 8.5.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In line with the REACH regulation (EC no. 1907/2006) Annex VIII Section 8.5.3, Column 2, a study on the acute toxicity via the dermal route has not been conducted as inhalation is the most likely route of concern based on the substances physical chemical properties. Dibutylamine has harmonised classification and labelling, due, to the classification of dibutylamine and its percentage within the substance the substance will be classified as Acute Tox. Dermal Cat. 3. In line with the most recent self classification by the Lead Registrant.
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 423 (2017) - This study was conducted to assess the acute toxicity of the test article (Reaction Mass of N,N,N',N-tetrabutylmethylenediamine and dibutylamine), following a single oral administration to small groups of rats. The study design provides information for hazard assessment and classification and enables a chemical to be assigned to toxicity classes but severely restricts animal usage.

 

A group of three female fasted rats was given the test article as a single dose by oral gavage at a dose level of 2000 mg/kg. Based on the results from this dose level, further groups of three female fasted rats were treated at dose levels of 50 mg/kg,


300 mg/kg and 2000 mg/kg. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.

 

The test article was used undiluted for the 300 and 2000 mg/kg dose levels and was dispersed in corn oil for the 50 mg/kg dose level. Surviving animals were killed on Day 15. All animals underwent a full necropsy.

 

Two animals treated at 2000 mg/kg were found dead on Day 2. One other animal was humanely killed due to the severity of the clinical signs on this day.

 

There were no deaths at dose levels of 50 or 300 mg/kg.

 

Decreased activity and hunched posture were noted 3 and 4 hours after dosing, in one animal treated at 2000 mg/kg. Hypothermia, ptosis, piloerection, laboured breathing and prone posture were noted in the animal that was killed in extremis. No clinical signs were seen prior to death in the two animals that were found dead.

No clinical signs were seen in animals treated at 50 or 300 mg/kg.

 

All surviving rats achieved body weight gains during the first and second weeks of the study.

No abnormalities were noted at necropsy of animals that died or were killed in extremis during the study or at necropsy of those that were killed at the end of the study.

Based of the experimental conditions of this study, the test article was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Based on the observed effects the LD50 using the cut-off value was estimated to be 500 mg/kg bw.

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

The following observations considered to be associated with the restraint procedure were detected: Wet fur was noted both during and for a short period after exposure.  Hunched posture and pilo-erection were noted on removal and for a short period on removal from the chamber.

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:  All Group 2 animals survived until the end of the recovery period.  No macroscopic abnormalities were detected at necropsy for one animal that survived until the end of the recovery period.

The following macroscopic abnormalities were detected at necropsy of the remaining Group 2 animals:

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 are 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.

Acute dermal toxicity:

In line with the Lead Registrants most recent self classification dibutylamine is classified as acute dermal toxicity (Category 3) (sourced May 2018: https://echa.europa.eu/substance-information/-/substanceinfo/100.003.565).

In accordance with Regulation (EC) No. 1272/2008 (CLP), as the substance is above the cut-off value (>1 %) within the mixture, the classification must be accounted for in the substance. Therefore, Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine must be classified as acute dermal toxicity (Category 3).

Justification for classification or non-classification

Reaction Mass of N,N,N',N-tetrabutylmethylenediamine and dibutylamine meets the criteria for classification for acute oral toxicity (category 4) and acute inhalation toxicity (category 4) according to Regulation (EC) No 1272/2008 (CLP).

Based on the classification of dibutylamine, the substance is classified as acute dermal toxicity (Category 3) in accordance with Regulation (EC) No. 1272/2008 (CLP), as the substance is above the cut-off value (>1 %) within the mixture.