(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Endpoint summary

Description of key information

Female rats: acute, oral LD50 >2000 mg/kg (GLP, OECD TG 425)

Female and male rats: acute, inhalation LC50 >2.53 mg/L (GLP, OECD 403)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Sex:

female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
Housing: Type II polypropylene/polycarbonate cages with deep wood sawdust (SAFE 3/4 S wooden chips, Arbocel crinklets natural nest building material)
Food: ssniff SM R/M autoclavable complete diet for rats and mice - breeding and maintenance, ad libitum
Water supply: tap water from municipal supply from 500 mL bottles ad libitum
Light: 12 hours daily, from 6 am to 6 pm
Temperature: 21.2 to 25.9 °C
Relative humidity: 30 - 70%
Ventilation: 15 - 20 air exchanges per hour
Number: 5 animals
Sex: female, nulliparous and non-pregnant
Age: young adult, 11-12 weeks old
Body weigth: 224 - 238 g
Acclimatisation before test: at least 25 days
Randomisation: selected by hand at time of delivery

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

The test substance was freshly formulated at concentrations of 17.5, 55 and 200 mg/mL in the vehicle, 0.5% carboxymethyl cellulose (batch BCBN1690V, Sigma-Aldrich) in distilled water (batch 8060219, Hungaro-Gal Kft). The dosed volume was between 2.2 and 2.4 mL (10 mL/kg bw).

Doses:

175, 550 and 2000 mg/kg bw

No. of animals per sex per dose:

175 mg/kg: one
550 mg/kg: one
2000 mg/kg: three

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: after 30 minutes, 1, 2, 3, 4 and 6 hours, then daily
- Frequency of weighing: on the day before treatment prior to removal of food, directly before dosing, on day 7 and on day 14 of the observation period
- Necropsy of survivors performed: yes
- Clinical signs including body weight: Individual observations were performed on the skin, fur, eyes, mucous membranes, somatomotor activity and behaviour pattern as well as respiratory, circulatory, autonomic and central nervous systems

Statistics:

The LD50 was calculated using the AOT425StatPgm program. This program was prepared for the US Environmental Protection Agency by Westat, May 2001 and updated by the US EPA June 2003. This program was constructed using the most appropriate method to estimate the LD50.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Not effects observed

Clinical signs:

other: Animals were symptom-free

Gross pathology:

No effects observed

Interpretation of results:

GHS criteria not met

Conclusions:

The substance was not acutely toxic to female rats following a single oral dose by gavage of 2000 mg/kg bw. The acute oral LD50 values were >2000 mg/kg.

Executive summary:

The acute oral toxicity of the substance to young adult female Wistar rats was studied under GLP to OECD TG 425. Healthy, nulliparous and non-pregnant animals were used in the study after an acclimatisation period of at least 25 days. The body weight of animals was between 224 and 238 g immediately before dosing. Animals were housed individually in polypropylene/polycarbonate cages with deep wood sawdust bedding, at a temperature ranging from 21.2 to 25.9 °C, a relative humidity of 35 to 70% and with 12 hours of light per day. The animals received standard rodent feed and water ad libitum. All animals were fasted overnight prior to treatment, but water was still available ad libitum overnight. Food was provided again three hours after the treatment. Animals were administered a single oral dose by gavage of the test substance dissolved in 0.5% aqueous carboxylmethyl cellulose. The administered volume was 10 mL/kg bw. The first animal was treated with an initial dose of 175 mg/kg bw, which was selected due to the lack of information on toxicity of the substance. Since no adverse effects were observed, a second animal was treated with a dose of 550 mg/kg bw, which also did not show any adverse effects. Therefore, three further animals were treated with 2000 mg/kg bw. All animals were observed for a period of 14 days after dosing. No mortality occurred during the study. No adverse clinical signs and no effects on body weight were observed. There were no findings during the gross observations at necropsy.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

Fully reliable GLP and guideline compliant study

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Remarks:

According to Regulation (EC) No 1907/2006, testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Type of information:

experimental study

Remarks:

This study was performed due to the relatively fine median particle size determined in the granulometry study and the anticipated increased likelihood of inhalation exposure of humans potentially occurring during the handling of the technical substance.

Adequacy of study:

key study

Study period:

Start of experiment: 8.10.2021, End of experiment: 11.11.2021

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

GLP compliance:

yes (incl. QA statement)

Test type:

fixed concentration procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age in sighting study: 11 to 12 weeks
Age in main study: 8 weeks
Weight in sighting study: 491-423 g (males), 267-272 g (females)
Weight in main study: 295-313 g (males), 206-224 g (females)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
Housing: in groups of 2 or 3 animals per cage by sex
Acclimatisation period: 43 days for sighting study, 7 days for main study
Diet: ssniff SM R/M 'Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance' ad libitum
Water: tap water ad libitum
Temperature: 20.0 to 25.6 °C (outside of expected range of 22 ± 3 °C, no impact on study results expected)
Humidity: 28 to 77% (outside of expected range of 30-70%, , no impact on study results expected)
Air changes: 15-20 per hour
Photoperiod: 12 hours light to 12 hours darkness

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.52 µm

Geometric standard deviation (GSD):

2.38

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The substance was aerolised using Wright's Dust Feed System (TSE Systems GmbH, Bad Homburg, Germany; Serial Numbers: 040804-60) located at the top of the exposure chamber. Animals were exposed nose-only to an atmosphere of the test substance using a TSE Rodent Exposure system (TSE Systems GmbH, Bad Homburg, Germany). It comprises of two concentric anodised aluminium chambers and a computer control system with pressure detectors and mass flow controllers. Fresh aerosols were constantly generated and supplied to the distribution chamber from where it was distributed to the exposure ports. Animals were held in polycarbonate restraint tubes which allowed only the animal's nostrils to enter the exposure port. After passing through the animal's breathing zone, used aerosols entered the outer cylinder from where it was exhausted through a suitable filter system. The flow of air through each port was at least 0.5 L/minute.

TEST ATMOSPHERE
The test atmosphere was sampled at regular intervals during the exposure period. Samples were taken from an unoccupied exposure port to represent the animal's breathing zone. A suitable known volume of test atmosphere was pulled through weighed glass fibre filters (Type GF10, Whatman GmbH, GE Healtcare UK Limited, Lot Numbers: A29518736). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the achieved test atmosphere concentration.

VEHICLE
No vehicle was used and the test substance was used as received.

PARTICLE SIZE DISTRIBUTION
The particle size distribution of the test atmosphere was determined three times during the exposure period using a 7-stage impactor (TSE Systems GmbH, Bad Homburg, Germany). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, 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 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 μm was calculated. From these data, using software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.

OTHER CONDITIONS
The measured air flow into the equipment (inner plenum) was 15.5 to 30.8 L/minute. The air flow out of the equipment was 30.4 to 30.9 L/minute. The temperature in the chamber was 22.8 to 23.9 °C, and the relative humidity was 20.4 to 22.9%. The chamber volume of the inner plenum was 3.85 L.

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

Sighting study: 2.58 mg/L
Main study: 2.53 mg/L

No. of animals per sex per dose:

Sighting study: 2 per sex
Main study: 5 per sex

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed for clinical signs hourly during the exposure, then twice on the day of exposure following removal from the chamber. Clinical signs were checked daily during the observation period. Individual body weights were recorded prior to the exposure and on days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes (macroscopic examination of external appearance and of tissues and organs after opening of thoracic and abdominal cavities)
- Clinical signs including body weight: yes

Statistics:

No statistics were performed.

Preliminary study:

The mean achieved concentration in the sighting study was 2.58 mg/L. The MMAD was 3.76 µm (GSD of 2.39). The respirable fraction (below 4 µm) was 52.8%. One male animal in preliminary study was euthanised on day 5 due to animal welfare reasons. This animal showed laboured, noisy respiration, distended abdomen, gasping respiration, piloerection, fur staining and red-brown staining around the eyes and the nose. The surviving male animal had similar symptoms, which disappeared on day 4 of the observation period. The female animals also had laboured, noisy respiration, sneezing, fur staining, red-brown staining on the head, wet fur and hunched back. These symptoms disappeared on day 8 of the observation period.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.53 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

One male animal was euthanised on day 8 of the experiment due to animal welfare reasons. One male animal was found dead on day 10.

Clinical signs:

other: laboured, gasping, noisy respiration, wet fur, fur staining, red-brown staining on nose and around eyes, piloerection, sneezing

Body weight:

In the sighting study, the surviving male animal had slight body weight loss on Days 0-3. The body weight gain was normal on Days 3-14. In the preterminal euthanised male animal slight to moderate body weight loss was observed on Days 0-3. In one female animal, slight to moderate body weight loss was observed on Days 0-3 and in the other female animal, slight body weight loss was observed on Days 0-1. The body weight gain was normal on Days 3-14 both animals.
In the main study, In one (1/3) surviving male animal, moderate body weight loss was noted on Days 0-1 and in of the other two (2/3) surviving animals, slight to moderate body weight losses were observed on Days 1-3. The body weight gain was normal on Days 3-14 in all surviving male animals. In the preterminal euthanised and found dead male animals, slight to moderate body weight losses were observed on Days 0-7. In the female animals, slight to moderate body weight losses were observed on Day 0-1 and in two out of five animals slight body weight loss was observed on Days 1-3. The body weight gain was normal on Days 3-14 in all female animals.

Gross pathology:

In the sighting study, the preterminal euthanised male animal had diffuse, pale discoloration of all lobes of lungs and multifocal dark red discoloration of all lobes of lungs. Moreover, dilatation with gas of cecum, colon, duodenum, oesophagus, ileum, jejunum and rectum were observed, as well as yellow focal discoloration and focal thickness of the wall and nonglandular region of the stomach. Small spleen and small thymus were also recorded. In the surviving male and female animals, no macroscopic observations were seen on necropsy Day 14.
In the main study, the preterminal euthanised male animal had diffuse, pale discoloration of all lobes of lungs and soft, diffuse, of the right cranial, caudal and middle lobes were observed. Moreover, dilatation with gas of cecum, colon, duodenum, ileum, jejunum and stomach was recorded, as well as small bilateral coagulation gland, epididymis and seminal vesicles. Small spleen, prostate and thymus were observed. The focal, glandular region of stomach was thin. In the found dead animal dark red discoloration of all lobes of collapsed lungs were observed. Moreover, dilatation with gas of cecum, colon, duodenum, ileum, jejunum, rectum and stomach were recorded. Small bilateral testis, coagulation gland, epididymis and seminal vesicles, small spleen and small prostate were observed. In the surviving animals, no macroscopic observations were seen on necropsy Day 14.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, two male rats in a group of 10 rats died when exposed to 2.53 mg/L of the test substance over a period of four hours. The acute inhalation median lethal concentration is considered to be >2.53 mg/L.

Executive summary:

The acute inhalation toxicity of the substance was studied under GLP to OECD TG 403 (fixed dose) as a limit test using the highest achievable concentration. The test substance administered as supplied. A sighting study was performed with 2 male and 2 female rats to estimate the inhalation toxicity of the test substance, identify sex differences in susceptibility and assist in selecting exposure concentrations for the main study. Based on the sighting study, a main test was performed with 10 (5 males and 5 females) Crl:WI Wistar rats as a limit test. The animals were exposed to aerosols generated with a suitable dust feed system for 4 hours using a suitable nose-only exposure system, followed by a 14-day observation period. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. The achieved mean concentration in the main study was 2.53 mg/L, and the MMAD was 3.52 µm (GSD of 2.38). In the main study, one male was killed on day 8 of the experiment due to animal welfare reason, and a second male was found dead on day 10. All other test animals survived until the end of the 14-day observation period. Animals showed laboured, gasping and noisy respiration, sneezing, wet fur, fur staining on the head, around the nose and the eyes, piloerection or distended abdomen. The surviving males were smptom free from day 7, and the females from day 9 of the observation period. Slight to moderate body weight loss was noticed in treated males until day 3 of the observation period. Body weight gain was normal on days 3 to 14 in surviving males. Slight to moderate body weight loss was also observed in females in the main study until day 3. The body weight gain was normal on days 3 to 14 in the female rats. In the preterminal euthanised male animal, diffuse, pale discoloration of all lobes of lungs and soft, diffuse, of the right cranial, caudal and middle lobes were observed. Moreover, dilatation with gas of cecum, colon, duodenum, ileum, jejunum and stomach was recorded, as well as small bilateral coagulation gland, epididymis and seminal vesicles. Small spleen, prostate and thymus were observed. The focal, glandular region of stomach was thin. In the found dead animal dark red discoloration of all lobes of collapsed lungs were observed. Moreover, dilatation with gas of cecum, colon, duodenum, ileum, jejunum, rectum and stomach were recorded. Small bilateral testis, coagulation gland, epididymis and seminal vesicles, small spleen and small prostate were observed. In the surviving animals, no macroscopic observations were seen on necropsy Day 14. Based on the results and the number of two dead animals out of ten rats treated at a mean concentration of 2.53 mg/L for a period of 4 hours by inhalation exposure nose-only, the LC50 value was considered to be >2.53 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

> 2.53 mg/L air

Physical form:

inhalation: aerosol

Quality of whole database:

Fully reliable GLP and guideline compliant study

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information the substance is not classified for acute toxicity in accordance with Regulation (EC) No. 1272/2008.