(6S,7S)-7-Amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

yes

Remarks:

The optimum MMAD achieved was 5.75 μm instead of 1-4 μm stated in the guidelines, due to the nature of the test item.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

The optimum MMAD achieved was 5.75 μm instead of 1-4 μm stated in the guidelines, due to the nature of the test item.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Deviations:

yes

Remarks:

The optimum MMAD achieved was 5.75 μm instead of 1-4 μm stated in the guidelines, due to the nature of the test item.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

435-680-1

EC Name:

-

Cas Number:

24701-69-7

Molecular formula:

C9H12N2O4S (Hill formula) C9H12N2O4S (CAS formula)

IUPAC Name:

(6R,7R)-7-amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Test item: 7-AMCA
Chemical name: 5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-amino-3-(methoxymethyl)-8-oxo, (6R,7R)-
Batch No.: B345118
CAS No.: 24701-69-7
Other components: water
Appearance: Light brown, weakly odorous powder
Manufacturing date: 01 February 2016
Retest date: 31 January 2017
Storage: Refrigerator (2-8ºC), protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL (WI) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Rat, CRL (WI) BR of Wistar origin
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the test: Good conventional
Number of animals: 6 animals in the main study (3 male and 3 female)
Sex: Male and female. Female were nulliparous, non-pregnant
Age of animals: Young adult rats, 8-12 weeks old (at start of the study)
Planned body weight range at starting: The weight variation was exceed ± 20% of the mean weight for either sex
Acclimatization time: At least 5 days

Animal health: Only healthy animals were used. The breeder certified the healthy status.
Hygienic level during the test: Good conventional
Housing: Group caging (up to 3 animals, by sex, per cage)
Cage type: Polypropylene/polycarbonate (type III) with stainless steel mesh lids.
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %. Actually: 14.5 - 34.7 %.
Ventilation: Above 10 air exchanges/hour by central air-condition system.
Housing/Enrichment: Rats are group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

Feed: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water: Tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). The quality of the bedding material is guaranteed by the supplier. The bedding is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

3.81 µm

Geometric standard deviation (GSD):

2.67

Remark on MMAD/GSD:

Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor type 02-150 (IN-TOX Products, N.M., USA). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. During the exposure period samples were collected three times with one sample taken during the first hour of exposure and one during the last hour. Samples were collected from vacant animal exposure ports (animals breathing zone) and were determined gravimetrically. The log-probit plots of the resulting data was used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage < 4 μm (considered to be inhalable in the rat).

Details on inhalation exposure:

The animals were exposed to an atmosphere of the test item for a single, continuous four-hour period, generated according to the system and flow rates determined during the technical trials. The four-hour exposure period was not start until theoretical chamber concentration equilibration calculated according to Silver S. D. (1946) has been reached.
Atmosphere generation
The test item was aerosolised using a Wright Dust Feeder II. (CH Technologies (USA), Inc., 263 Center Ave, Westwood, NJ 07675, USA; Serial Number: 052) located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.

Animal Exposure Conditions
The animals were held in polycarbonate restraining tubes and exposed “nose-only” under dynamic air flow conditions, using an anodised aluminium Flow Past Exposure Chamber. (CR Equipment SA, Switzerland). Validation of the same type of system was published by J. Pauluhn. (1994).
The exposure unit is placed in closed hood in order to avoid cross-contamination and contamination of the laboratory environment.
The inhalation chamber used is modular and for this study was assembled from 4 identical vertical modules, with 8 animal ports in each module. The animals were distributed on one module (levels 1, counted from the top) of the exposure chamber.
Each module of the chamber consists of two concentric cylinders with inner diameter of 25 mm and 80 mm, respectively. The height of the modules is 110 mm. In this system the air carrying the test substance enters primary from above in the central cylinders and is distributed in each module through 8 radial tubes (diameter: 5 mm, length: 40 mm) to the animal ports, i.e. to the openings (diameter: 30 mm) on the wall of the outer cylinder where the animal restraining tubes can be attached. From the animal ports the air turns back into the outer cylinder towards the vacuum pump. In the particular set-up the radial tubes as well as the animal ports in the lowest module were closed by plastic stoppers. In such a way the air containing particles of the test item could flow out from the central plenum only through the 16 openings of the two upper levels. Here 6 animal ports were occupied by the animals, 1 by the Temperature-Humidity Sensor, 1 port was used for sampling on aerosol filters and the Cascade Impactor. All eight animal ports on the 2nd level (but not the radial tube) were closed by a plastic stopper.
The total volume of the exposure chamber was 2.24 L. The air flow rate of 19.3 L/min (1.2 L/min to each animal port) excluded re-breathing of the animals and ensured the continuous presence of fresh aerosol and of natural oxygen concentration in the breathing zones.
The air was supplied by an oil-free air compressor and was filtered in a two-stage filter set. The temperature of the air was regulated by a heat exchanger. The air was not humidified in order to avoid modification of particle size distribution.

Actual Test Atmosphere Concentrations
The actual concentration of generated atmosphere was measured at regular intervals during exposures by pulling a suitable, known volume of test atmosphere from the exposure chamber, through GF10 glass fibre filters (GE Healthcare Life Science, Whatman GmbH, Germany). Since it was proved that the concentration is stable within ±10% during the technical trials then at least 8 samples were taken during each exposure. Sampling was performed shortly after chamber equilibration and then uniformly distributed, at regular intervals. Samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item.
The amount of material collected on glass fibre filters was determined gravimetrically. The filter holder was opened after sampling and filter was weighed right before exposure. Immediately after sampling the filters were weighed again.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetrically.

Duration of exposure:

4 h

Remarks on duration:

The four-hour exposure period was not started until the theoretical chamber concentration equilibration, calculated according to Silver S. D. (1946), has been reached.

Concentrations:

Target concentration: 5 mg/L.

No. of animals per sex per dose:

3m + 3f.

Control animals:

no

Details on study design:

A group of six animals (three males and three females) was exposed for a four hour period to the target concentration of 5 mg/L. As exposure to the mean achieved concentration of 5.12 mg/L results in no mortalities then no further exposures were undertaken.

Morbidity/Mortality were checked twice daily.
Clinical observations were performed after one, two and three hours exposure whilst the animals are still restrained. Following exposure clinical observations were performed at least twice on the day of exposure and then at least once daily for fourteen days.
Cage-side observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Body weights were recorded on the day of exposure day 0 (prior to exposure) and days 1, 3, 7 and 14.
Necropsy/Pathology: A gross necropsy was performed on all animals euthanised by exsanguination following intra-peritoneal injection of pentobarbital solution at the end of the observation period.
A complete examination of the abdominal and thoracic cavities was made and special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Histopathology: During necropsy no organs were found with macroscopic abnormalities therefore no organ has been preserved.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.12 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Standard deviation: 0.15 mg/L.

Mortality:

No animals died during the study.

Clinical signs:

other: Only dyspnoea (3/3♂, 3/3♀) and reddish staining around snout (3/3♂, 3/3♀) were recorded in the animals during and/or shortly after the exposure. All animal recovered and no significant clinical signs were recorded from the day following exposure until the

Body weight:

Normal bodyweight gain was noted during the two weeks observation period.

Gross pathology:

No test item-related macroscopic alteration was detected.

Other findings:

Test Atmosphere Concentration:
Target Concentration: 5 mg/L
Mean achieved Concentration: 5.12 mg/L
Standard Deviation: 0.15
Nominal Concentration: 8.04 mg/L

Particle Size Analysis:
Mean achieved concentration (mg/L): 5.10 mg/L
Mean Mass Median Aerodynamic Diameter (MMAD): 3.81 μm
Geometric Standard Deviation (GSD): 2.67
Inhalable Fraction (% < 4μm): 50.0 %

Test Chamber Environmental and Equilibration Data:
Mean Air Flow Application (Inner Plenum) (L/min): 30.2
Air Flow Out (Outer Cylinder) (L/min): 39.9
Temperature (°C): 24.6
Relative Humidity (%): 19.9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute inhalation LC50 of the test substance in Wistar rats is >5.12 mg/L.

Executive summary:

An acute inhalation toxicity study according to the OECD guideline 436 was performed. The only study group, consisting of 6 Wistar rats (3 males and 3 females), was exposed to the target concentration of 5 mg test substance/L as an aerosol. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14-day observation period. Aerosol concentration was 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.

Results:

Gravimetric results of the test atmosphere analysis:

The mean achieved concentration in the study was 5.12 mg/L. The mass median aerodynamic diameter (MMAD) was 3.81 μm with a geometric standard deviation (GSD) 2.67. The quality of the test atmosphere complied with criteria documented in the guideline.

Mortality: No animals died during the study.

Clinical observations: Only dyspnoea (3/3 m, 3/3 f) and red stating around snout (3/3 m, 3/3 f) were recorded in the animals during and/or shortly after the exposure. All animal recovered and no significant clinical signs were recorded from the day following exposure until the end of the observation period.

Bodyweight: Normal bodyweight gain was noted during the two weeks observation period.

Necropsy: No test item-related macroscopic alterations were detected.

The acute inhalation LC50 of the test substance in Wistar rats is >5.12 mg/L.