N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Endpoint summary

Description of key information

Acute toxicity studies were conducted for N-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine by oral, dermal and inhalation route of exposure.

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:

12 November 2013 to 10 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

no

Species:

rat

Strain:

other: RccHan:(WIST)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 166-203 g
- Fasting period before study: Yes
- Housing: Individually in Type II polypropylene/polycarbonate cages
- Diet: Ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" ad libitum (except overnight prior to dosing and 3 hours after dosing)
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-22.2
- Humidity (%): 35-68
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 November 2013 To: 10 December 2013

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Doses:

2000, 550 mg/kg bw

No. of animals per sex per dose:

3 females per dose level

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 30 mins, 1, 2, 3, 4, 6 hours after dosing, then once daily for 14 days (observations); days -1 (prior to removal of food), 0 (prior to administration), 7 and 14 (body weight).
- Necropsy of survivors performed: Yes

Statistics:

Data was evaluated using the Acute Oral Toxicity (OECD Test Guidelines 425) Statistical Programme (AOT 425 Stat Pgm).

Sex:

female

Dose descriptor:

LD50

Effect level:

> 550 - < 2 000 mg/kg bw

Based on:

test mat.

95% CL:

550 - 2 000

Mortality:

3/3 at 2000 mg/kg bw, 0/3 at 550 mg/kg bw

Clinical signs:

other: Decreased activity (3/3), hunched back (2/3), prone position (3/3), incoordination (2/3), continuous tremors (3/3), piloerection (3/3), decreased respiratory rate (1/3) and cold to touch (1/3) at 2000 mg/kg bw. Hunched back (3/3), incoordination (3/3),

Gross pathology:

No treatment-related effects

Table 1: Mortality data

Dosing sequence / animal number	Dose (mg kg bw)	Outcome
4303	550	Survived
4304	2000	Died on day 2
4305	550	Survived
4306	2000	Died on day1
4307	550	Survived
4308	2000	Died on day 2

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information: H302: Harmful if swallowed

Conclusions:

Under the conditions of this study, the acute oral median lethal dose (LD50) of the test item, CA5204A, was found to be between 550 and 2000 mg/kg bw in female RccHan:(WIST) rats.

Executive summary:

An acute oral toxicity (up and down procedure) study was conducted with 6 animals (female RccHan:(WIST) rats). The starting dose was 550 mg/kg bw. Animals were treated with a single oral (gavage) dose of CA5204A at a dose level of 550 or 2000 mg/kg bw followed by a 14 day observation period. The animals were fasted overnight prior to treatment and food was returned approximately 3 hours after dosing. Surviving animals were observed individually after dosing at 30 minutes, 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter. Body weight was measured on Day -1 (prior to removal of food), Day 0 (prior to administration) and weekly thereafter. All surviving animals were examined macroscopically at the end of the observation period. Moreover, the animals found dead were examined macroscopically and body weight was recorded at necropsy. No mortality was observed at the dose level of 550 mg/kg bw. Treatment with CA5204A at the dose level of 550 mg/kg bw caused hunched back (3/3), incoordination (3/3), continuous tremors (2/3), piloerection (3/3) and hyperactivity (2/3). Surviving animals were symptom free from 8 days after the treatment. There were no treatment related body weight changes. Body weights were within the range commonly recorded for this strain and age. There was no evidence of the macroscopic observations in animals dosed at 550 mg/kg bw and terminated on Day 14. Mortality was observed in all three animals on Day 1 or 2 at 2000 mg/kg bw dose level. Treatment with CA5204A at the dose level of 2000 mg/kg bw caused decreased activity (3/3), hunched back (2/3), prone position (3/3), incoordination (2/3), continuous tremors (3/3), piloerection (3/3), decreased respiratory rate (1/3) and cold to touch (1/3).

Under the conditions of this study, the acute oral median lethal dose (LD50) of the test item, CA5204A, was found to be between 550 and 2000 mg/kg bw in female RccHan:(WIST) rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 049 mg/kg bw

Quality of whole database:

1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Remarks:

The study on acute inhalation was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2015 to 01 october 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Test type:

fixed concentration procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at treatment: Young adult rats, 9 weeks old at sighting, 9 weeks old in the Main Study.
- Weight at study initiation: 228 to 404 g (males: 379 g, 404 g; females: 228 g, 235g) at sighting, 234 g to 373 g (males: 360-373 g; females: 234-247 g) at the Main Study
- Fasting period before study: No
- Housing: Animals were grouped by sex (up to 5 animals per cage) in Type III solid floor cages with stainless steel mesh lids. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
- Diet (e.g. ad libitum): ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-26.0°C
- Humidity (%): 40-62%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Geometric standard deviation (GSD):

>= 1 - <= 3

Remark on MMAD/GSD:

Measurements of aerodynamic particle size were performed from the animal’s breathing zone using a cascade impactor

Details on inhalation exposure:

The test item formulation was aerosolised using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. The rate of formulation use was controlled by a syringe pump. Compressed air was supplied by means of an oil-free compressor passed through a suitable filter system prior to introduction to the nebuliser.

The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprised of 2 concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.7 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere as it is about twice the respiratory minute volume of a rat.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones (Pauluhn, 1994).

Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99) (Silver, 1946), a main group of 10 rats (5 male and 5 female) was exposed to the mean achieved concentration for a period 4 hours.

Prior to atmosphere generation, the non-volatile component of the test material was determined by adding a small, known amount of the material to glass fibre filters (Type GF/C, Whatman, Germany). The filters were then dried, at atmospheric pressure, in a desiccator at room temperature for at least 24 hours and weighed again. The difference in the two weights was taken as the volatile content of the test material and the non-volatile component was calculated as a percentage. The mean non-volatile content of the batch used for the animals' exposure was found to be 96.75% (n = 9) with a standard deviation 0.26%.
The test atmosphere was sampled at regular intervals during the exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume of test atmosphere through weighed GF10 glass fibre filters (Type GF10, Whatman, Germany). 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.
After sampling, the filters were dried (under the same conditions as those previously described) and weighed again 24 hours later. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was the chamber concentration in terms of non-volatile component.

Based on the results of the preliminary work, these figures were adjusted to obtain a true figure for the test material concentration in the test atmospheres.
The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that went through the chamber during the same period.
The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

4.93 mg/L for the sighting study and 5.12 mg/L for the main study

No. of animals per sex per dose:

4 rats (2 male and 2 female) for the sighting study
10 rats (5 male and 5 female) for the main study

Control animals:

no

Details on study design:

All animals were observed individually at hourly intervals during exposure, as soon as possible following removal from restrains at the end of exposure, 1 hour after exposure and twice daily for 14 days thereafter. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Individual bodyweights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14 or at death.
All animals were subjected to macroscopic examination. All animals were exsanguinated under pentobarbital anaesthesia (Euthanimal 40% injection). After examination of the external appearance, the thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any gross macroscopic changes were recorded. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

Preliminary study:

In sighting, slight laboured respiration, noisy respiration (slight to moderate), slow superficial respiration, activity decreased (slight to moderate), incoordination and tremors were observed. The animals were symptom free by Day 7. Slight bodyweight loss was noted in the day of exposure. From Day 3, normal body weight gain was noted in both sexes.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.12 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

One female animal died in the main study on Day 3.

Clinical signs:

other: Wet fur, ruffled fur and/or red brown fur staining were recorded in animals on the day of exposure and 5 days after exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not

Body weight:

Body weight losswas recorded in the day of exposure in both sexes. In the nine surviving animals normal body weight gain was observed from Day 3.

Gross pathology:

A single four hours nose-only exposure of CA5204A to Crl (WI) strain rats led to the death of one female dosed exposed to the concentration of 5.12 mg/L during Main study. Dark/red discoloration of the collapsed lungs was considered to be associated with the administration of the test item.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Under the experimental conditions of the study, one death occurred in a group of 10 rats exposed to 5.12 mg/L of the substance for 4 hours. The acute inhalation median lethal concentration in CRL:WI Wistar strain rats is therefore considered to be greater than 5.12 mg/L.

Executive summary:

The study was performed to assess the acute inhalation toxicity of the test item following a 4 hour exposure at the mean achieved concentration of 5.12 mg/L to 5 male and 5 female rats. A single sighting exposure was performed prior to the main study with 2 males and 2 females at the mean achieved concentration of 4.93 mg/L.

The main study group, 10 (5 male and 5 female) CRL:WI Wistar strain rats, was exposed to the mean achieved concentration of 5.12 mg/L of test item. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14 day observation period. The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and bodyweights were recorded throughout the study and at the end of the scheduled period the animals were euthanised and subjected to a gross examination post mortem.

In sighting, slight laboured respiration, noisy respiration (slight to moderate), slow superficial respiration, activity decreased (slight to moderate), incoordination and tremors were observed. The animals were symptom free by Day 7.

In the Main Study, noisy respiration (slight to moderate), slow superficial respiration, activity decreased (slight to moderate) and tremors were recorded. The animals were symptom free from Day 6.

In the sighting, slight bodyweight loss was noted in the day of exposure. From Day 3, normal body weight gain was noted in both sexes. In the Main Study, body weight loss was recorded

in the day of exposure in both sexes. In the nine surviving animals normal body weight gain was observed from Day 3.

A single four hours nose-only exposure of test item to Crl (WI) strain rats led to the death of one female dosed exposed to the concentration of 5.12 mg/L during Main study. Dark/red discoloration of the collapsed lungs was considered to be associated with the administration of the test item.

In surviving animals subjected to the necropsy on Day 14, no macroscopic changes at concentration level of 4.93 mg/L or 5.12 mg/L were seen during Sighting exposure or Main study, respectively.

The acute inhalation median lethal concentration of CA5204A, in CRL:WI Wistar strain rats is considered to be greater than 5.12 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 120 mg/m³ air

Quality of whole database:

1

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Remarks:

The study on acute dermal toxicity was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2013 to 29 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: RccHan:(WIST)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: 220-257 g
- Fasting period before study: No
- Housing: Individually in Type II polypropylene/polycarbonate cages
- Diet: Ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7-24.3
- Humidity (%): 31-54
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 November 2013 To: 29 November 2013

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: Back (shaved 24 hours prior to application).
- % coverage: Approximately 10% total body surface.
- Type of wrap if used: Gauze pads kept in contact with the skin with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Residual test item was removed, using body temperature water.
- Time after start of exposure: 24 hours.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.54 mL/kg bw (x1300 mg/mL = 2000 mg/kg bw).

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

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: 1, 5 hours after application and once daily thereafter (observations); Day 0 (before the experiment) and on Days 7 and 14 (body weights).
- Necropsy of survivors performed: Yes.

Statistics:

Not applicable.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality

Clinical signs:

other: Decreased activity (3/10), hunched back (8/10), incoordination (9/10), irritability (3/10) and continuous tremors (9/10). All animals were symptom free from 7 days after the treatment. No signs of dermal irritation.

Gross pathology:

No treatment-related findings.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The median lethal dose of the test substance after a single dermal administration (dermal LD50) was greater than 2000 mg/kg bw in male and female RccHan:(WIST) rats.

Executive summary:

Five male and five female RccHan:(WIST) rats were treated with a single, 24 hour, semi occlusive dermal application of the test substance at a dose of 2000 mg/kg bw. Clinical observations along with a check of viability and mortality were assessed in all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. All animals were killed and subjected to a gross macroscopic examination at the end of the 14-day observation period (Day 14).

There was no mortality. Decreased activity (3/10), hunched back (8/10), incoordination (9/10), irritability (3/10) and continuous tremors (9/10) were seen but all animals were symptom free from 7 days after the treatment. No treatment related skin irritation was observed in any animal throughout the study. Slight body weight loss was noted in one male rat and four female rats between Days 0 and 7. Bodyweights of these animals had recovered by the end of the observation period. There was no evidence of the any observations at a dose level of 2000 mg/kg bw at necropsy.

The median lethal dose of the test substance after a single dermal administration (dermal LD50) was greater than 2000 mg/kg bw in male and female RccHan:(WIST) rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

1

Justification for classification or non-classification

Based on the above stated assessment of the acute oral toxicity, N-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine does have to be classified as Acute Toxicity Oral Category 4 (H302 harmful if swallowed) according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council. The substance does not need to be classified for acute dermal toxicity and acute inhalation toxicity according to according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.