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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
Study period:
30 September 2009 - 06 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: performed according to OECD test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification Diformal
Chemical name Bis-(2-chlorethyl)-formal; Bis-(2-chloroethoxy)methane
Structure
Molecular formula C5H10Cl2O2
Molecular weight 173.0377
CAS Number 111-91-1
Description Clear yellowish liquid (determined at NOTOX)
Batch 54600-235530
Purity 90.4 %
Test substance storage At room temperature in the dark under nitrogen
Stability under storage conditions Stable
Expiry date 31 December 2011

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species Rat: Crl:WI(Han) (outbred, SPF-Quality)
Recognised by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals 12; 3 males and 3 females (females were nulliparous and non-pregnant) per exposure level. Two exposure levels were used.
Age and body weight Young adult animals were selected (approximately 11 weeks old).
Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean.
Identification Earmark
Health inspection A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0C (actual range: 18.4 – 21.8C), a relative humidity of 40-70% (actual range: 31 - 76%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
During exposure the temperature and relative humidity were between 19.2 to 21.3oC (mean 20.6 ± 0.6 oC) and 10 to 14% (mean 11 ± 1%) respectively.

Accommodation
Group housing of 3 animals per sex per cage in labelled Macrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
After exposure on Day 1, the animals exposed at 0.55 mg/L were housed in stainless steel wire mesh cages to prevent suffocation in case of bad health condition and in order to recover. At the end of Day 1 the animals were housed as described above.

Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. Animals were housed with maximally 5 animals per cage per sex as described above.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance.

Water
Free access to tap water except during exposure to the test substance.

Results of analysis for each batch of diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Animals were exposed to the test substance via the inhalatory route. For this purpose the animals were placed in restraining tubes, connected to the exposure chamber.
The design of the exposure chamber was based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 2.0 L/min, which ensures an adequate oxygen supply to the test animals. The main inlet of the test atmosphere was located at the top section and the main exhaust outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal.
The placement of the individual animals in the inhalation chamber is shown in figure 2. All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained a slight negative pressure.

Since during method development of the atmosphere generation of 0.55 mg/L the test atmosphere contained droplets, the FTIR readings were compared with the results of samples measured gravimetrically. Gravimetrically a mean of 0.41 mg/L (n=4) was calculated for the aerosol.
By placing a filter (PTFE, 0.2 um) in the FTIR sample line the amount of gaseous phase was measured and a mean of 0.14 mg/L (n=8) was calculated. Since the FTIR readings at development showed a concentration of 0.57 mg/L it was concluded that the both phases were collected and measured correctly by the FTIR and that no gravimetrical measurements were needed for the main study.

Method development for the 0.055 mg/L showed gravimetrically an aerosol concentration of approximately 0.008 mg/L (n=2) at FTIR concentrations of approximately 0.056 mg/L, indicating an aerosol content of approximately 14%.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Infrared spectrophotometer
Duration of exposure:
4 h
Concentrations:
0.55 and 0.055 mg/L
No. of animals per sex per dose:
3 males / 3 females
Control animals:
no
Details on study design:
Target concentrations were based on the cut off values specified in the UN and EC classification guidelines for dusts and mists (0.05, 0.5, 1 or 5 mg/L.) increased with 10% in order to avoid the actual mean concentrations to fall below the cut off values due to experimental variation.

Based on available toxicity data of the test substance and since it is the intention to minimize the number of animals to be used, 0.55 mg/L was selected as starting concentration. Three males and three females were exposed and were found dead on Days 1 (Day of exposure) or day 2. In consultation with the sponsor, a second group of three males and three females were exposed at 0.055 mg/L.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 0.05 - <= 0.5 mg/L air
Exp. duration:
4 h
Mortality:
All animals exposed to 0.55 mg/L were found dead between Days 1 and 2 post-exposure. No mortality occurred at 0.055mg/L.
Clinical signs:
other: During exposure, no clinical signs were noted. After exposure, lethargy, hunched posture, ventral-lateral recumbence, slow breathing and/or piloerection were noted among the animals exposed at 0.55 mg/L. No clinical signs were noted in the animals expose
Body weight:
Overall body weight gain in surviving males and females were within the range expected for rats of this strain and age used in this type of study
Gross pathology:
No treatment abnormalities were found at macroscopic post mortem examination of the animals.

Any other information on results incl. tables

The mean actual concentrations were 0.55 ± 0.07 mg/L and 0.058 ± 0.011 mg/L. The nominal concentrations were 1.895 and 0.99 mg/L and the generation efficiencies (ratio of actual and nominal concentration) were 29 and 6% respectively.

 

During the exposure to 0.55 mg/L, the Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. The MMAD was 3.5mm and 4.1mm respectively and the gsd was 1.6 and 1.5 respectively. The MMAD of the second measurement was just abovethe recommended range of 1 – 4µm. Since one MMAD value was within the range and the other close to the upper limit and since the gsd were appropriate (i.e. between 1.5 and 3), it can be assumed that deposition of particles in the lower respiratory tract had occurred.For the 0.055 mg/L, an attempt was made to determine the MMAD and gsd but due to the very small amount of aerosol present (approximately 0.008 mg/L) the measurement did not show a normal distribution and therefore no MMAD and gsd were calculated.

Applicant's summary and conclusion

Interpretation of results:
Category 2 based on GHS criteria
Remarks:
Migrated information
Conclusions:
The inhalatory LC50, 4h value of Diformal in Wistar rats was established to be within the range of
0.05 – 0.5 mg/L.
Executive summary:

Assessment of acute inhalation toxicity withDIFORMALin the rat (acute toxic class method)

 

 

The study was carried out based on the guideline described in Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.436, "Acute Inhalation Toxicity-Acute Toxic Class Method", September 2009.

 

 

DIFORMALwas aerosolized at concentrations of 0.55 and 0.055 mg/L. Two groups of three male and three female Wistar rats each were exposed by inhalation to one of these concentrations for a single period of 4 hours. Animals were subjected to daily observations and weekly determination of body weight.Macroscopic examination was performed on the day of death or at terminal sacrifice (day 15).

 

 

The mean actual concentrations were 0.55 ± 0.07 mg/L and 0.058 ± 0.011 mg/L. The nominal concentrations were 1.895 and 0.99 mg/L and the generation efficiencies (ratio of actual and nominal concentration) were 29 and 6% respectively.

 

During the exposure to 0.55 mg/L, the Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. The MMAD was 3.5mm and 4.1mm respectively and the gsd was 1.6 and 1.5 respectively. The MMAD of the second measurement was just abovethe recommended range of 1 – 4µm. Since one MMAD value was within the range and the other close to the upper limit and since the gsd were appropriate (i.e. between 1.5 and 3), it can be assumed that deposition of particles in the lower respiratory tract had occurred.For the 0.055 mg/L, an attempt was made to determine the MMAD and gsd but due to the very small amount of aerosol present (approximately 0.008 mg/L) the measurement did not show a normal distribution and therefore no MMAD and gsd were calculated.

 

All animals exposed to 0.55 mg/L were found dead between Days 1 and 2 post-exposure. No mortality occurred at 0.055mg/L.

 

During exposure, no clinical signs were noted. After exposure, lethargy, hunched posture, ventral-lateral recumbence, slow breathing and/or piloerection were noted among the animals exposed to 0.55 mg/L. No clinical signs were noted in the animals exposed to 0.055 mg/L.

 

Overall body weight gain in surviving males and females were within the range expected for rats of this strain and age used in this type of study

 

No treatment abnormalities were found at macroscopic post mortem examination of the animals.

 

 

The inhalatory LC50, 4hvalue of Diformal in Wistar rats was established to be within the range of

0.05 – 0.5 mg/L.