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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
Acute Toxic Class Method, 07 September, 2009.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate of GLP Compliance No G-044 from 17.07.2021, valid until 17.07.2023, Slovak National Accreditation Service
Test type:
acute toxic class method

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2-bis(hydroxymethyl)butanoic acid
EC Number:
424-090-1
EC Name:
2,2-bis(hydroxymethyl)butanoic acid
Cas Number:
10097-02-6
Molecular formula:
C6H12O4
IUPAC Name:
2,2-bis(hydroxymethyl)butanoic acid
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar, outbreed
Sex:
male/female
Details on test animals or test system and environmental conditions:
6 males and 6 females

Source: The Lab Animals Breeding Center, Branch of the Institute of Bioorganic Chemistry RAS (www.spf-animals.ru) Nauki 6, Pushchino, Moscow region, Russia

Characteristic:
Nulliparous and non-pregnant animals with defined microbiological health status. The animal health monitoring is performed by breeder under FELASA-guidelines quarterly in AnLab, s.r.o. (Czech Republic). Health monitoring report from the breeder is attached to the study materials.

Date of batch receiving and age:
26.08.2021, 18 males of 5 weeks old, and 18 females of 7 weeks old

Age at day of exposure: Males: 7-8 weeks old; Females: 9-10 weeks old

Body weight at day of exposure:
Males: 222 ± 5 g, N = 6
Females: 199 ± 2 g, N = 6

Water: Tap water filtered with MilliRO system was provided ad libitum in standard water bottles.

Diet: Animals were fed Laboratory Rodent Diet ad libitum

Environmental Conditions
- actual mean temperature ranged from 21 °C to 24 °C
- mean relative humidity ranged from 30 % to 70 %.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 4 µm
Remark on MMAD/GSD:
Group 1: 2.84 ± 3.06 μm (sample 1) and 3.03 ± 2.73 μm (sample 2)
Group 2: 3.57 ± 2.89 μm (sample 1) and 3.74 ± 2.98 μm (sample 2)
Details on inhalation exposure:
For the inhalation, rats (3/sex/level) were placed in exposure tubes connected via the ports to the inhalation chamber of the TSE Inhalation System.
Food and water were withdrawn from rats during exposure.

Each aerosol exposure consisted of a 30-minute T95 equilibration period, a 240-minute exposure period and a 30-minute de-equilibration period equal to the T95 equilibration period. T95 equilibration period was established in the preliminary sighting tests and constantly monitored by online data recording.
After each aerosol exposure, animals were removed from the exposure tubes and returned to the home cage with ad libitum feed and water.
In Step 1, the animals selected to Group No.1 (3 males and 3 females) were exposed at the 1.1 mg/L expected level.
In Step 2, exposure was performed with a maximum feasible concentration that has reached the actual level 4 mg/L and provided the respirable particle size (MMAD 1-4 μm).
The period of four days between the exposures at each concentration level has been allowed for the observation of delayed toxicity
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Remarks on duration:
Inhalation (Head-Nose only). The animals were adapted to the exposure tubes for 5 days with gradually increased time to a total duration of 4 hours by the fifth day.
Post-exposure period: 14 days
Concentrations:
Concentration of test material in vehicle:

Nominal concentration = 4.5 mg/L (Group 1) and 9.6 mg/L (Group 2). Achieved mean concentration was 1.11 mg/L or 24.7 % of output (Group 1) and 3.67 mg/L or 38.2 % of output (Group 2).
No. of animals per sex per dose:
3 males and 3 females (Group 1),
3 males and 3 females (Group 2).
Control animals:
other: A concurrent negative control group is not necessary according to OECD 436.
Statistics:
Body weight means and standard deviations were calculated separately for males and females.

Calculation of the mean particle size (mass median aerodynamic diameter, MMAD) with geometric standard deviation (GSD), and percentage of particles <4.0 μm were done using PSD (Particle Size Distribution for Windows) software, TSE System.

Test item should be allocated to one of four hazard categories based on acute toxicity estimate (ATE) according to the numeric cut-off criteria of the GHS for dusts and mists [2]:
Category 1: ATE < 0.05 mg/L
Category 2: 0.05 < ATE < 0.5 mg/L
Category 3: 0.5 < ATE < 1.0 mg/L
Category 4: 1.0 < ATE < 5.0 mg/L
The method of ATE – LC50 was not applied due to lack of mortality.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Based on effects on the maximal feasible concentration 3.67 ± 0.51 mg/L that was lower than the cut-off value 5 mg/L, the acute inhalation LC50 in rats is considered to be more than 5 mg/L.
Mortality:
There were no mortality in the Group 1 and Group 2 male and female rats exposed to a 4-hour inhalation to the test item 2,2-bis(hydroxymethyl)butanoic acid at concentrations 1.11 ± 0.25 mg/L and 3.67 ± 0.51 mg/L air.
Clinical signs:
other: In Group 1, the most animals showed ocular/nasal discharge on day 1 (after end of exposure until 3 hours). In Group 2, the ocular/nasal discharge was recorded in one female until day 2, one female had post-inhalation noisy breathing, and the pallor appear
Body weight:
The reduction in body weight gain was observed in two of three females in Group 1 and two of three females in Group 2. The mean total body weight gain was slightly lower in males inhaled with high concentration than in males treated with low concentration. However, the final mean body weight gain was positive in Groups 1 and 2.
Gross pathology:
During scheduled necropsy on day 15, no gross findings in respiratory organs (nasopharynx, trachea, lungs) and other organs were found.

Applicant's summary and conclusion

Interpretation of results:
other: not classified as dangerous for inhalation acute toxicity according to Regulation (EC) No 1272/2008 (CLP Regulation)
Conclusions:
The inhalatory LC50 (4 hours) for the test item 2,2-bis(hydroxymethyl)butanoic acid in male and female rats is considered to be > 5 mg/L.
Executive summary:

The acute inhalation toxicity study with 2,2-bis(hydroxymethyl)butanoic acid was carried out in accordance to the OECD Guideline 436: Acute inhalation toxicity – Acute toxic class method (2009) in GLP conditions.


The study was performed with 3 males and 3 females of rats (Rattus norvegicus, strain Wistar) for each tested group. Concentration of test material in air (vehicle) was: nominal concentration = 4.5 mg/L (Group 1) and 9.6 mg/L (Group 2) , achieved mean concentration (measured in breathing zone using gravimetric and analytical techniques was 1.11 mg/L or 24.7 % of output (Group 1) and 3.67 mg/L or 38.2 % of output (Group 2).
Type of inhalation exposure was Head-Nose only and the animals were adapted to the exposure tubes for 5 days with gradually increased time to a total duration of 4 hours by the fifth day.
All the rats were observed for mortality and clinical signs during exposure and daily during the subsequent 14-day post-exposure period
The maximal cut-off value of exposure concentration 5 mg/L was failed to reach achieving a respirable particle size (MMAD = 1-4 μm). For Group 2, exposure was performed with a feasible concentration of 3.67 ± 0.51 mg/L with a maximal analytical value of 4.6 mg/L for one sample.
There were no animal’s morbidity and mortality on 1.11 ± 0.25 mg/L and 3.67 ± 0.51 mg/L exposure. Clinical signs in some animals treated with lower and higher concentrations were observed 24-48 hours after exposure and considered to be caused by the irritative effect of the test item. The total weight gain in all animals was positive, except for one female in group 1, which showed a fall in body weight on the last day of the study for an unclear reason. No gross findings were revealed during necropsy.
Based on effects on the maximal feasible concentration 3.67 ± 0.51 mg/L that was lower than the cut-off value 5 mg/L, the acute inhalation LC50 in rats is considered to be more than 5 mg/L.