Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Endpoint summary

Description of key information

The acute oral and inhalation toxicity studies indicate that the substance is of low toxicity if swallowed (rat LD0 > or = 2000 mg/kg bw) or inhaled (rat LC50 > or = 5250 mg/m3).

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:

23 April 2013 --- 13 June 2013

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

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 8 weeks old on the day of treatment
- Mean body weight at study initiation: 204 g (range: 182 g to 222 g)
- Fasting period before study: yes, during the night before treatment
- Housing: the animals were housed by three from the same group in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 14 May 2013 to 11 June 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Batch N° MKBH4894V

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 or 200 mg/mL
- Justification for choice of vehicle: as unsatisfactory solubility of the test item was obtained in drinking water treated by reverse osmosis, another vehicle was chosen from the following organic solvents (in order of preference): 0.5% methylcellulose aqueous solution and corn oil. A homogenous suspension was obtained in corn oil at the concentration of 200 mg/mL.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw.

DOSAGE PREPARATION (if unusual): the test item was administered as a homogenous suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
Fresh dose formulations were prepared on the day of each administration and kept at room temperature prior to administration.

CLASS METHOD (if applicable):
- Rationale for the selection of the starting dose: since no relevant toxicity data were available for the estimation of a lethal dose-level, the starting dose level was 300 mg/kg bw.

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

3 females per treatment step

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Clinical observations: frequently during the hours following treatment; then, at least once a day.
- Body weight: just before treatment, then on day of treatment (day 1) and on days 8 and 15.
- Necropsy of survivors performed: yes

Statistics:

None

Preliminary study:

Not applicable

Key result

Sex:

female

Dose descriptor:

LD0

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No unscheduled deaths occurred during the study.

Clinical signs:

other: No clinical signs were observed in any animals.

Gross pathology:

There were no findings considered to be related to the test item administration.
Dilatation of the uterus with translucent content was seen in one female given the test item at 2000 mg/kg (group 2). This is a common background finding in females rats related to the estrous cycle.
A white mass was found in the mammary gland from one female given the test item at 300 mg/kg. Although this macroscopic finding is unusual at this age, it was considered to be incidental in view of its isolated occurrence and the short duration of the observation period.

 Table 7.2.1/1: Mean body weight and body weight changes (g) in treated animals during the observation period compared to laboratory historical control data

Sex	Female
Group	Laboratory Historical control data	1	2	3
Dose-level (mg/kg)	0	300	2000	2000
Body Weight (g) ± SD
. Day 1	208 (± 11.7)	216 (± 9.8)	207 (± 8.2)	188 (± 10.1)
. Day 8	246 (± 12.7)	251 (± 12.5)	243 (± 16.3)	224 (± 10.7)
. Day 15	266 (± 14.0)	272 (± 12.4)	264 (± 9.6)	245 (± 9.1)
Body weight Change (g)± SD
. Days 1-8	+39 (± 5.1)	+35 (± 3.1)	+36 (± 10.0)	+36 (± 1.2)
. Days 8-15	+20 (± 6.3)	+21 (± 1.0)	+21 (± 6.9)	+21 (± 2.6)
. Days 1-15	+58 (± 5.8)	+56 (± 2.3)	+57 (± 3.5)	+57 (± 2.1)

SD: standard deviations.

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the oral LD0 of the test item was equal or higher than 2000 mg/kg bw in rats.

Executive summary:

The substance was tested for acute oral toxicity according to OECD 423 guideline and in compliance with Good Laboratory Practices.

The test item was administered once by gavage to 3 groups of 3 fasted female rats under a dosage-volume of 10 mL/kg. The test item was prepared in corn oil. Since no relevant toxicity data were available for the estimation of a lethal dose-level, the starting dose-level was 300 mg/kg bw for ethical reasons. After the first assay, the next higher dose-level of 2000 mg/kg bw was tested. Then, as no toxicity was observed at this higher dose-level, the results were confirmed in other 3 females at 2000 mg/kg bw.

Each animal was observed at least once a day for mortality and clinical signs for 15 days. Body weight was recorded before treatment then on day 1, 8 and 15. On completion of the observation period, the animals were sacrificed and then submitted for a macroscopic post-mortem examination.

No unscheduled deaths occurred during the study and no clinical signs were observed in any animals. Body weight gain was unaffected by the test item treatment, when compared to laboratory historical control data. The test item administration did not induce any macroscopic findings at necropsy.

The acute oral LD0 of the test item was equal or higher than 2000 mg/kg bw.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Study complete and sufficient to fulfill the endpoint requirements.

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:

11 May 2015 ---- 23 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

yes

Remarks:

Identification methods in the protocol were tail mark and microchip; but only microchip method was used and actual animal identity was confirmed with a microchip reader. As all animals were uniquely identified, the integrity of the study was not affected.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 256 to 286 g (males) and 166 to 173 g (females)
- Fasting period before study:no
- Housing: The animals were housed three of one sex per cage
- Diet: Ad libitum, standard rodent diet (Harlan teklad 2014C Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.9 µm

Geometric standard deviation (GSD):

2.33

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, a single animal exposure section with 20 ports and a top section incorporating a central aerosol inlet.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 19 L/minute
- System of generating particulates/aerosols: Wright dust Feed(WDF) designed to produce and maintain test atmospheres containing dust by scraped from the surface of a compressed powder in a stream of dry air. The concentration of dust in the air was altered by changing the gear ratio of the mechanism and therefore the speed of rotation of the compressed powder towards the scraper blade.The WDF mechanism was attached directly to the top of the exposure chamber.
- Temperature, humidity, pressure in air chamber:
Chamber air temperature was measured using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. The mean chamber temperature value was 22.6°C.
- Humidity: Chamber relative humidity was measured using an electronic hygrometer probe inserted into the breathing zone of the animals via an unused exposure port. The mean chamber relative humidity was 18.7%
Pressure in air chamber was not reported.
- Air flow rate: Airflow were 19 L/minute.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 8-stage cascade impactor (Marple 298). A measured volume of air was drawn at a rate of 2 litres/minute from an unused exposure port on the exposure chamber through the cascade impactor and measured using a wet-type gas meter in line with a pump. Two samples were collected during the exposure. The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected 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 < 0.52, 0.93, 1.55, 3.5, 6.0 , 9.8, 14.8 and 21.3 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. The mean MMAD was 2.9 µm and the geometric standard deviation (GSD) was 2.33.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate of 20 L/minute. the airflow was filtered locally.

TEST ATMOSPHERE
- Brief description of analytical method used: A measured volume of air was drawn at a rate of 2 litres/minute from an unused exposure port on the exposure chamber through a glass microfibre filter, mounted in an open face filter holder and measured using a wet-type gas meter in line with a pump. Eight samples were collected during the exposure. The filters were weighed before and after sampling.. The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration. The mean achieved test atmosphere concentration was 5.25 +/- 0.541 mg/L

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target concentration: 5.0 mg/L
Mean achieved atmosphere concentration: 5.25 mg/L ; Standard deviation: 0.541

No. of animals per sex per dose:

3/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

OBSERVATIONS:
- Morbidity/Mortality: Animals were checked hourly during exposure, immediately following exposure and then at 1 h and 2 hrs post-exposure. During the 14-day observation period, the animals were observed twice daily for morbidity and/or mortality.

- Clinical Signs: All animals were observed for clinical signs at hourly intervals during exposure, as soon as practically possible following removal from restraint at the end of exposure, 1 h and 2-hrs after exposure and subsequently twice daily for 14 days.

- Bodyweight: Individual bodyweights were recorded prior to treatment, on the day of exposure (Day 1) prior to dosing and on Days 2, 4, 8 and 15 (or at death).

- Necropsy: All animals were subject to a gross necropsy which consisted of opening the cranial, thoracic and abdominal cavities.any macroscopic abnormalities in appareance of the organs were recorded.

Statistics:

None

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.25 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were 2 decedents during the study: Females 301 and 303 were sent to necropsy for welfare reasons approximately 3 or 1.75 hours respectively after completion of exposure. Clinical signs apparent for these animals immediately after exposure included unsteady gait, decreased activity, gasping, irregular or laboured breathing, wet rales, partially closed eyes and wet fur. These signs were generally present at the time of despatch to necropsy with piloerection, whole body pallor and hunched posture. Macroscopic examination revealed dark areas between 1 to 5 mm in diameter on all lung lobes.

Clinical signs:

other: Deep and/or laboured breathing was apparent in one female at 3 hours after start of exposure and in all animals at 3.5 hours after start of exposure. Immediately following exposure, clinical signs for the majority of animals included laboured deep breath

Body weight:

Body weight loss was observed in all males and the single remaining female on the day following the 4 hour exposure. The removal of food and water during exposure is considered to have contributed to the observed body weight loss and is therefore not solely attributed to treatment with the test substance.
All animals had recovered from the initial body weight loss by the next weighing occasion, and the group mean body weights from both sexes increased from Day 4 of the observation period onwards.

Gross pathology:

The macroscopic examination of the two decedents revealed dark areas between 1 to 5 mm on all lung lobes. There were no macroscopic findings in the remaining animals.

Table 7.2.2/1: Signs associated with dosing -Individual observations on Day 1

Group /sex	Animal number	Day of death	Category	Observation	Day(s) observed
1 M					3hours after start of dosing	3.5hours after start of dosing	On return to home cage	1hour post dose	2hours Post dose	At the end of the working day
	0201	15	Abnormal gait	Unsteady			1	1	1	1
			Behavior	Decreased activity			1	1	1	1
				Salivation excessive			1
			Breathing	Labored, animal appears to be breathing deeply		1	1	1	1	1
				Rales,wet				1	1	1
	0202	15	Abnormal gait	Unsteady			1	1	1	1
			Behavior	Decreased activity			1	1	1	1
			Breathing	Labored, animal appears to be breathing deeply		1	1	1	1	1
				Rales,wet				1	1	1
	0203	15	Abnormal gait	Unsteady			1	1	1	1
			Behavior	Decreased activity			1	1	1	1
				Salivation excessive			1
			Breathing	Gasping			1	1	1
				Labored, animal appears to be breathing deeply		1		1	1	1
			Eyelids	Partially closed, bilateral			1	1	1	1

Only animals with observations are presented

Group /sex	Animal number	Day of death	Category	Observation	Day(s) observed
1F					3hours after start of dosing	3.5hours after start of dosing	On return to home cage	1hour post dose	2hours Post dose	At the end of the working day
	0301 WE	1	Abnormal gait	Unsteady			1	1	1
			Behavior	Decreased activity			1	1	1
				Salivation excessive			1
			Breathing	Gasping			1	1	1
				Irregular		1
				Labored	1	1	1	1	1
				Rales,Wet				1	1
			Coat	Piloerection					1
				Wet fur,Marked	1	1	1	1
			Posture	Hunched					1
	0302	15	Abnormal gait	Unsteady			1	1	1	1
			Behavior	Salivation excessive			1
			Breathing	Labored, animal appears to be breathing deeply		1	1	1	1	1
				Rales, Wet				1	1	1
			Coat	Piloerection					1	1
				Wet fur,Marked	1	1	1	1
			Eyelids	Partially closed, bilateral					1	1
			Posture	Hunched					1	1
	0303 WE	1	Abnormal gait	Unsteady			1	1
			Behavior	Decreased activity			1	1
			Breathing	Gasping			1	1
				Irregular			1	1
				Labored, animal appears to be breathing deeply		1
				Rales, Wet				1
			Coat	Wet fur,Marked			1	1
			Eyelids	Partially closed, bilateral			1	1

WE: euthanazied for welfare reasons

Only animals with observations are presented

Table 7.2.2/2: Signs associated with dosing -Individual observations on Days 2 to 15

Group /sex	Animal number	Day of death	Category	Observation	Day(s) observed
					Initial check	Additionnal observation	Additionnal observation	Additionnal observation	At the end of the working day
1F	0302	15	Breathing	Rales, Wet	2	2
			Coat	Piloerection	2	2	2	2	2
			Posture	Hunched	2-3	2	2	2	2-3

Only animals with observations are presented

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The LC50 of the test substance was in excess of 5.25 mg/L for male and female rats.
The substance is not classified according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
According to the Globally Harmonized Classification System (GHS; UNITED NATIONS), the substance is classified as Category 5.

Executive summary:

 

The substance was tested for acute inhalation toxicity according to the OECD 436 guideline and in compliance with Good Laboratory Practice.

A group of three male and three female Wistar rats was exposed, nose-only, to an atmosphere of the test item using a flow-through exposure system. The animals were exposed for four hours to a target concentration of 5 mg/L followed by a fourteen day observation period. Each animal was observed for mortality and clinical signs at hourly intervals during exposure, then one hour and two hours after exposure and at least twice a day during the 14 -day observation period. Bodyweights were recorded before treatment then on the day of exposure (day 1) and on days 2, 4,8 and 15. All surviving animals were necropsied at the end of the observation period.

The mean achieved atmosphere concentration was 5.25 mg/L and the mean mass median aerodynamic diameter was 2.9 µm.

There were two decedents during the study: 2 Female were sent to necropsy for welfare reasons approximately 3 or 1.75 hours respectively after completion of exposure on Day 1. Clinical signs apparent for these animals immediately after exposure included unsteady gait, decreased activity, gasping, irregular or laboured breathing, wet rales, partially closed eyes and wet fur. These signs were generally present at the time of despatch to necropsy with piloerection, whole body pallor and hunched posture also evident at this point. Macroscopic examination revealed dark areas between 1 to 5 mm in diameter on all lung lobes. Deep and/or laboured breathing was apparent in one female at 3 hours after start of exposure and in all animals at 3.5 hours after start of exposure.

Clinical signs for the majority of animals immediately following exposure included laboured deep breathing or gasping, unsteady gait and decreased activity; partially closed eyes, excessive salivation and wet fur. The majority of these signs were still evident at the 1 and 2 hour post-exposure and end of day checks with the exception of excessive salivation, wet fur or gasping that were not present at the 1 hour post-exposure, 2 hour post-exposure or end of day time points respectively. In addition, for a proportion of animals, wet rales was apparent from 1 hour post-exposure with hunched posture and piloerection evident from 2 hours post-exposure; these signs persisted for the surviving female up to Day 2 (piloerection and wet rales) or Day 3 (hunched posture). All males were considered normal from Day 2 and the surviving female from Day 4.

There were no adverse bodyweight effects observed. The macroscopic examination revealed dark areas between 1 to 5 mm in diameter on all lung lobes of the 2 decedents. There were no macroscopic findings on the remaining animals.

The 4 -hour inhalation LC50 was found to be greater than 5.25 mg/L (ie 5250 mg/m3).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

5 250 mg/m³ air

Quality of whole database:

Study complete and sufficient to fulfill the endpoint requirements.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route:

In an acute oral toxicity study performed according to OECD 423 guideline and in compliance with Good Laboratory Practices, groups of three females rats received by gavage a single dose of 2000 or 300 mg/kg bw of the test substance. The starting dose-level was 300 mg/kg bw for ethical reasons. After the first assay, the next higher dose-level of 2000 mg/kg bw was tested. Then, as no toxicity was observed at this higher dose-level, the results were confirmed in three other females. Animals were then observed for 14 days following exposure.

No deaths occurred during the study and no clinical signs were observed in any animals. Body weight gain was unaffected by the test item treatment, when compared to laboratory historical control data. The test item administration did not induce any macroscopic findings at necropsy.

The acute oral LD0 of the test item was found equal or higher than 2000 mg/kg bw (Papineau, 2013a).

Inhalation:

The substance was tested for acute inhalation toxicity according to the OECD 436 guideline and in compliance with Good Laboratory Practice.

A group of three male and three female rats was exposed for four hours to a target concentration of 5 mg/L followed by a fourteen day observation period.

The mean achieved atmosphere concentration was 5.25 mg/L and the mean mass median aerodynamic diameter was 2.9 µm.

There were two decedents during the study: 2 Female were sent to necropsy for welfare reasons approximately 3 or 1.75 hours respectively after completion of exposure. Macroscopic examination revealed dark areas on all lung lobes.

Deep and/or laboured breathing was apparent in one female at 3 hours after start of exposure and in all animals at 3.5 hours after start of exposure.

Immediately following exposure, clinical signs for the majority of animals included laboured deep breathing or gasping, unsteady gait and decreased activity; partially closed eyes, excessive salivation and wet fur. In addition, for a proportion of animals, wet rales was apparent from 1 hour post-exposure with hunched posture and piloerection evident from 2 hours post-exposure; these signs persisted for the surviving female up to Day 2 (piloerection and wet rales) or Day 3 (hunched posture). All males were considered normal from Day 2 and the surviving female from Day 4.

There were no adverse bodyweight effects observed. There were no macroscopic findings on the surviving animals.

The 4 -hour inhalation LC50 was found to be greater than 5.25 mg/L (ie 5250 mg/m3) (Beebe, 2015a).

Justification for classification or non-classification

No mortalities were noted in rats after treatment by oral route with a single dose of 2000 mg/kg body weight and the 4 -hour inhalation LC50 was found to be greater than 5.25 mg/L (ie 5250 mg/m3).

On the basis of these results and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to acute oral and inhalation toxicities.