(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate

Endpoint summary

Description of key information

The oral toxicity was determined in an OECD 423 guideline study using Sprague Dawley rats (MB Research, 2016). The oral LD50 determined for the test item is greater than 2000 mg/kg b.w. The oral LD50 calculated for Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate was 918 mg/kg b.w. 

 

The inhalation toxicity was determined in an OECD 436 guideline study using Wistar rats (strain Crl:WI (Han), Charles River Deutschland). Based on the experimentally results, the LC50 value for Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate is considered higher than 1979.9 mg/m3, which is the maximum technically achievable aerosol concentration resulting in a MMAD in the respirable range from 1-4 µm .

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

Batch# 1776194

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were received from Charles River, Raleigh NC, on 15 Oct 2015 and 05 Nov 2015. Following an acclimation period of at least five days, three male and three healthy, non-pregnant and nulliparous female Sprague Dawley rats were assigned to treatment groups without conscious bias.
The animals were born on 19 Aug 2015 and 09 Sep 2015. The pretest body weight range was
250 - 258 grams for males and 200 - 207 grams for females. The weight variation of the animals used did not exceed ±20% of the mean body weight of the previously dosed animals within a sex.
The animals were identified by cage notation and indelible body marks, and housed in suspended wire cages; five per sex per cage prior to dosing and three per sex per cage following dosing. Absorbent paper bedding was placed beneath the cages and changed at least three times per week. Fresh PMI Rat Chow (Diet #5012) was freely available except for 16-20 hours prior to dosing. Water was available ad libitum. The animal room, reserved exclusively for rats on acute tests, was temperature controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test article was used as received and the dose was based on the sample weight as calculated from the specific gravity. A single dose was administered orally by syringe and dosing needle to three female rats and three male rats.

Doses:

Dose level of 2000 mg/kg was administered to three female rats and three male rats.

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

In Vivo – Animals were observed at 15 minutes, 1, 2 and 4 hours postdose and once daily for 14 days for toxicity and pharmacological effects and twice daily for mortality. Body weights were recorded immediately pretest, weekly, at death and at termination in the survivors.
Post Mortem – All animals were humanely sacrificed using CO2 and were examined for gross pathology following study termination.
Analysis of Data

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

918 mg/kg bw

Based on:

act. ingr.

Remarks on result:

other: LD50 was calculated for the registered substance based on its content in the tested product (45.9%).

Mortality:

Two female rats survived following a single 2000 mg/kg oral dose. One animal was found dead on Day 1.All three male rats survived following a single 2000 mg/kg oral dose.

Clinical signs:

other: Female Prior to death, abnormal physical signs including piloerection, lethargy, and dyspnea were observed. Among the survivors, prostration, lethargy, sagging eyelids, and piloerection were observed. Male Abnormal physical signs including piloerection, a

Gross pathology:

Female
The gross necropsy, of the animal that died, revealed red staining of the nose/mouth area and abnormalities of the gastrointestinal tract. No observable abnormalities were observed among the survivors.
Male
The gross necropsy revealed emaciated appearance, bloated abdomen, chromodacryorrhea, chromorhinorrhea, and abnormalities of the gastrointestinal tract.

Conclusions:

The oral LD50 determined for the test item by OECD 423 method is greater than 2000 mg/kg b.w. The oral LD50 calculated for Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate was 918 mg/kg b.w..

Executive summary:

The oral LD50 determined for the test item by OECD 423 method is greater than 2000 mg/kg b.w. The oral LD50 calculated for Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate was 918 mg/kg b.w. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

918 mg/kg bw

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:

The study started April 25, 2022, final report not yet available

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on Sept. 7, 2009

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar rats [strain Crl:WI (Han)]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6-7 weeks of age at delivery
- At start of study initiation: for set 1 the mean bodyweight of males was 281.53 g (S.D. 24.26), and for females 171.80 g (S.D. 10.09), for set 2 the mean bodyweight of the males was 263.57 g (S.D. 43.93), for the females 197.27 g (S.D. 15.34)
- Housing: Makrolon® (polycarbonate) cages type IV, three rats of the same sex per cage, and were maintained under conventional laboratory conditions. Cages and absorbing softwood bedding material (Lignocel BK8-15) were changed twice a week or more often, if necessary.
- Diet (e.g. ad libitum): Ssniff V1534, purchased from Ssniff-Spezialdiäten (Soest, Germany)
- Water (e.g. ad libitum): tap water
- Acclimation period: approximately two weeks
Upon arrival all animals were thoroughly examined by a veterinarian or his designate. This examination included the whole body, especially the skin and hair coat, eyes, ears, nose, orifices and the respiratory tract. This clinical examination assured that only animals with a satisfactory general health status were included into the study. Additional examination was performed to clarify a suspected impaired health status, if necessary. Prior to the start of the treatment period, the following procedures were completed for all rats with satisfactory general health status:
1. The animals were allowed to adjust and become acclimatized to the Fraunhofer ITEM environment for approximately two weeks.
2. Clinical observations were made at least once a day. Body weight will be measured during the acclimatization period (once; randomization weighing).
Rats were accepted for the study upon completion of item 1 and 2 and absence of disease as demonstrated by general physical condition during the acclimatization period.
-Identification: To each animal in the study a unique four-digit identification number was assigned. These four digits are necessary for data storage in the computer system. The number assigned is DSNN where DS denotes the two-digit (D=dose group: 1; S=sex, i.e. males=1; females=2) dose group and sex number, and NN is the two-digit (01-07) consecutive animal number. All data collected from an animal will be filed under that number.
Three rats of the same group and sex within one cage were labeled individually (tail) by coloured marker pen. In addition, a metal plate on the cage indicating the study no., the dose group no. and the consecutive animal numbers was used for identifying the animals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° + 2 °C
- Humidity (%): 55% + 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: April 27, 2022 To: May 18, 2022

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

water

Mass median aerodynamic diameter (MMAD):

> 2.86 - < 3 µm

Geometric standard deviation (GSD):

> 2 - < 2.2

Remark on MMAD/GSD:

Set 1: mean MMAD = 2,86 µm; GSD 2,00
Set 2: mean MMAD = 3,00 µm; GSD 2,20

Details on inhalation exposure:

AEROSOL GENERATION
-EXPOSURE SYSTEM:
The working solution was prepared one day before the day of exposure by dissolving the test item in purified water.
Preparation of working solution:
Dose (mg/m³): 1000
Purified water amount (mL): 36
Test item PAMMS amount (g): 5,35

The test item was aerosolized into a dilution cylinder as aqueous solutions using an operated syringe pump and a spraying nozzle (developed by Fraunhofer ITEM) with pressurized air. Permanent monitoring of the aerosol concentration was conducted by aerosol photometers. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. This signal was used to control the feed rate of the sprayer in order to keep the aerosol concentration in the inhalation unit constant. Actual test item concentrations were measured in the breathing zone of the animals.

The animal's snout protrudes in the anterior end of the tube, which is connected to the exposure cylinder by means of a push fit. The aerosol enters the nasal region of the animal through a small tube. This ensured that a continuous air flow was passing through the animal's breathing zone. In this system, the aerosol was supplied to each rat individually, and exhaled air was immediately removed. Therefore, oxygen supply was always sufficient and measurement of the oxygen concentration was not necessary. The exposure unit is capable to house up to 16 animals on one level (in this study 3/3 m/f for each dose group/set). The airflow to each rat was approximately 1 l/min which is calculated to be laminar.

Filter sample of the aerosol were taken to control the aerosol concentration. These sample were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats were inhaling the aerosol. The mass median aerodynamic diameter (MMAD) was determined during the exposure period by a cascade impactor.

MONITORING AND CONTROLLING THE EXPOSURE ATMOSPHERES
Air flow , temperature and relative humidity were measured continuously and recorded by 10-minute means (Conc. 1) and 5-minute means (Conc. 2). The limits were set at 22° C + 2° C for temperature and 55 % + 15 % for relative humidity. Animal room lighting was on a 12 hour light/dark cycle controlled by an automatic timing device.
For adjustment of the photometer the aerosol concentration was determined gravimetrically using filter samples (4 times per 4-hour inhalation, in one hour intervals). Additionally, the MMAD was determined twice during the exposure period for test item exposure unit by a cascade impactor.

Set1:
Actual aerosol concentrations (mg/m3): 996.4; SD: 149,7, N=4
Mean MMAD (µm): 2,86; SD: 2,00; N=2

Set2:
Actual aerosol concentrations (mg/m3): 1979,7; SD: 206,1, N=4
Mean MMAD (µm): 3,00; SD: 2,20; N=2

It is noticeable that the MMAD of the 2nd impactor collection is smaller in each case. Possibly, a reduction of the nozzle gap due to deposits of the test item is the cause for this.
This is also supported by the fact that the flow through the nozzle has become smaller in the course of the exposure.

A mean aerosol concentration of 996.4 mg/m3 PAMMS was achieved in set 1. An actual mean aerosol concentration of 1979.9 mg/m³ of the test item PAMMS in set 2 represents the maximum technically feasible concentration.
The respirability of the aerosol (OECD guideline 436 recommendations allow the following ranges: MMAD = 1-4 µm; GSD = 1.5 - 3) was achieved.

EXPOSURE OF RATS
The rats were placed around the exposure cylinder in tapered acrylic glass tubes with adjustable backstops. The exposure tubes are arranged around a cylinder capable to take up 16 tubes per platform. The rat nose is located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder.
The animals were exposed to the test item once for 4 hours. An equilibration of the chamber concentration occurs immediately as the cylinder capacity is limited. Following the exposure, 3 rats per group and sex were kept in Makrolon® cages under observation for additional 14 days; thereafter, these animals were sacrificed.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

The animals were exposed once for 4 hours.

Concentrations:

Set1:
Actual aerosol concentrations (mg/m3): 996.4; SD: 149,7, N=4
Mean MMAD (µm): 2,86; GSD: 2,00; N=2

Set2:
Actual aerosol concentrations (mg/m3): 1979,7; SD: 206,1, N=4
Mean MMAD (µm): 3,00; GSD: 2,20; N=2

No. of animals per sex per dose:

3; 1 female for the pretest, 1 female as a sentinel

Control animals:

no

Details on study design:

STUDY DESIGN AND DOSING SCHEME:
Exposure to the test item was conducted in animal room T1.040. The rats were exposed to the exposure atmosphere in a direct flow nose-only inhalation exposure system. The inhalation route of exposure is appropriate since one potential route of human exposure to the test item will be by inhalation. For 2 weeks before exposure, rats were trained to the exposure tubes avoiding undue stress on the animals. Animal restraining tubes are constructed in such a way that hyperthermic effects on rats cannot occur. In this system the aerosol is supplied to each animal individually, and exhaled air is drawn off immediately. The rats are placed around the exposure cylinder in tapered acrylic glass tubes with adjustable backstops. Historical measurements have confirmed, that there are no differences in concentrations among the different outlets.
To allow for adequate exposure of all relevant regions of the respiratory tract, it is intended to generate an aerosol with a mass median aerodynamic diameter (MMAD) in the range of 1-4 µm and a geometric standard deviation (GSD) in the range of 1.5 to 3.0.
A pretest without animals was conducted prior to the animal pre- and main test to ensure that the chamber conditions can be achieved.

PRETEST (as part of aerosol generation):
Due to animal welfare reasons, one female rat was exposed to a test item concentration of 1000 mg/m3 to check whether the concentration is tolerated as a starting concentration. This concentration was well tolerated, so the main test could be carried out at 1000 mg/m3, whereby the risk of severe exposure of the animals in the main test was considerably minimized. The pretest was performed in a female rat, as they tend to be more sensitive.

MAIN TEST:
The criterion for a positive main test was the determination of an LC50 value from up to three dose groups.
Depending on the results of the pretest and the recommendations of the OECD TG 436 the first group was exposed once for 4 hours to a test item concentration of 1 mg/L (equivalent to 1000 mg/m3). The study design is shown in Table 3.
Depending on the results of the first concentration (6 animals, 3M/3F), one further concentration according to OECD TG 436, ANNEX 3c was tested in one further dose group. The tested dose depended on the results of dose group 1 and was 2 mg/L. The testing of a third dose was not necessary.

CLINICAL OBSERVATION:
On the day of treatment, all animals were observed frequently for clinical symptoms (prior to, during and after exposure). In the following 2 weeks, all animals were clinically observed in their cages once a day. Once a week, they were inspected outside their home cages and carefully examined for abnormalities concerning their general condition, especially for the following parameters:
- general condition, fur, grooming activity
- visible mucous membranes
- behaviour and locomotor activity (lethargy, coma, convulsions, diarrhea, salivation)
- central nervous symptoms
- breathing pattern
- reflexes (at least 1, 24, 48 h after treatment in compliance with ITEM SOP 010625)
- rectal temperature once after treatment (after 1 hour in compliance with ITEM SOP 010624)

BODY WEIGHT DATA:
Individual body weight was recorded to the nearest 0.1 g once during the acclimatization period, on the day of exposure prior to exposure (day 0), and at least on days 1, 3, 7, 10 and 14 before necropsy, and at the time of death or euthanasia for all animals.

GROSS NECROPSY:
During the study period, all animals at terminal sacrifice were killed humanely after lethal anesthesia with Narcoren® followed by exsanguination.
Animals were necropsied immediately and subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.

Statistics:

Data Collection and Documentation: All data to be collected were recorded either directly (on-line) using electronic data processing system or manually using data sheets. Recording and analysis of animal data like body weight, clinical observations, and mortality was performed using the PROVANTIS system (version 8.4.3.1) or manually using data sheets.

Statistics: Mean values and standard deviations were calculated.

Preliminary study:

A pre-test during the experimental set-up (under non-GLP conditions) using one extra rat (#1207) was conducted on April 27, 2022. The rat was exposed to an aerosol concentration of approx. approx. 1 mg/L. This concentration was well tolerated.
Based on this result, a single exposure to a test concentration of approx. 1 mg/L for 4 hours was justified as start concentration in the acute toxicity test.

Aerosol concentrations – pre test (animal #1207)
Target aerosol concentration (mg/m3) # of measurement Approx. 1000
Actual aerosol concentrations (mg/m3) 1 618.6
2 1013.0
3 947.4
4 1081.4
Mean: 915.1
SD: 177.6
N: 4

Clinical observations – pre test(animal #1207):
Animal Time after start of exposure ClinObs
1207 1 hr 20 min slight flank breathing
3 hr 15 min slight shallow breathing

Upon necropsy, no macroscopical findings were observed in animal #1207.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 979.7 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality was observed.

Clinical signs:

irregular respiration

Remarks:

Clinical observations did show signs of mild respiratory distress in some animals of set 2, starting approx. 1 hr and 20 min after start of exposure. This could be caused by exposure to the test item.

Body weight:

Body weight data were in the normal range of rats of the given age.

Gross pathology:

All animals were sacrificed at scheduled dates. Upon necropsy, some macroscopical findings were observed. The lung lobe of one animal showed isolated dark red districts up to 1 mm in diameter (animal 1101), another animal (animal 1103) showed a greyish discoloration of the liver margin. It is unlikely that these findings are related to the exposure of the test item.

Other findings:

Rectal temperatures
Rectal temperatures were measured in all rats approx. 1 hour after end of exposure. They were in the range of historical control animals.

Reflexes after end of exposure
All animals were without significant changes in reflexes after end of exposure, so reflexes were not measured after 24 and 48 hours.

Conclusions:

No acute toxicity.

Executive summary:

In an acute nose-only inhalation study 3 male and 3 female rats were exposed for 4 hours to an aerosol concentration of approx. 1 mg/L (set 1) and 2 mg/L PAMMS (set 2). No premature deaths occurred during the study period. Upon cessation of exposure some rats exposed to the test item showed mild signs of respiratory distress in form of flank breathing. However, all animals survived and recovered fully within 24-48 hours. All rats showed a good general health status during the remaining post-exposure observation period up to 14 days. Body temperature means measured rectally 1 hour after exposure were in the range of historical controls. Reflex analysis revealed no treatment-related effects in all rats 1 hour after end of exposure. Two rats did show gross pathological findings upon necropsy, but it is unlikely that the findings are related to the exposure of the test item.

An LC50 was not determined in this study. Based on the experimentally results, the LC50 value is considered higher than 1979.9 mg/m3, which is the maximum technically achievable aerosol concentration resulting in an MMAD in the respirable range from 1-4 µm . 

From the results observed in this acute 4-hour nose-only inhalation study the following classification was derived for the test item PAMMS: Unclassified (according to Regulation (EC) No. 1272/2008; EU, 2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 1 979.9 mg/m³ air

Physical form:

inhalation: aerosol

Justification for classification or non-classification

The oral LD50 determined for the test item by OECD 423 method  is greater than 2000 mg/kg b.w. The oral LD50 calculated for Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate was 918 mg/kg b.w. resulting in GHS Cat. 4 classification for oral toxicity according to the criteria of EC Regulation 1272/2008.

 

The inhalation toxicity value LC50 determined for the test item Propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate by OECD 436 method  is higher than 1979.9 mg/m3, which is the maximum technically achievable aerosol concentration resulting in a MMAD in the respirable range from 1-4 µm. Based on the results observed in this acute 4-hour nose-only inhalation study, the test item needs no classification according to CLP Regulation (EC) No. 1272/2008; EU, 2008.