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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-06 to 2012-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
2009
Deviations:
yes
Remarks:
Because the present study was a concentration range finding study not all aspects of the guideline were implemented. Therefore, the study was based on OECD 413 guideline.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solidified melt
Details on test material:
White solidified melt
Batch number: A 4-1-14 of Wuxi
Purity: 98.75%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
- Source: Harlan Laboratories, Netherlands
- Strain: rats, Wistar (RccHan:WIST)
- Age: approx. 10 weeks when supplied
- Animals: 24 males
- Acclimatisation: 4 weeks
- Mean Body Weight at study initiation: male: 324 g
- Housing: 3 animals to a macrolon cage
- Diet: ad libitum, rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from SDS Special Diets Services, Whitham, England
- Water: tap water ad libitum, during exposure, the rats had no access to feed or water
- Temperature (°C): 22 +/- 2 °C,
- Humidity: about 45 - 65%
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Air exchange: 10 per hour

Administration / exposure

Route of administration:
other: vapor at low and mid conc., mixture of aerosol and vapor at high concentration
Type of inhalation exposure:
nose only
Vehicle:
other: clean air
Remarks on MMAD:
MMAD / GSD: The mean particle size (MMAD), determined by an aerodynamic particle sizer, was 0.675 µm (gsd of 1.27), 0.893 µm (gsd of 1.69) and 1.18 µm
(gsd of 1.97) for the low, mid and high concentration test atmospheres, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: see attachment, nose-only inhalation units consisting of a cylindrical polypropylene (group 1) or stainless steel (groups 2-4) column, surrounded by a transparent cylinder (see Figure 1). The column had a volume of 45 litres and consisted of a top assembly with the
entrance of the unit, a mixing chamber, a rodent tube section and at the bottom the base assembly with the exhaust port.
- Method of holding animals in test chamber: the animals were secured in plastic animal holders (Battelle), positioned radially through the outer
cylinder around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
- Source of air: compressed dryed air
- Method of conditioning air: the air pressure was regulated by a reducing valve and the air flow was measured with a mass view meter #
(MASSVIEW from Bronkhorst Hi Tec, Ruurlo, The Netherlands).
- System of generating particulates/aerosols: To generate the test atmospheres, the test material was first dissolved in demineralized water and then nebulized. The concentrations of test material in the aqueous solutions were about 0.08, 0.8 and 4 g/L for the low-, mid- and high-concentration, respectively. The solutions were prepared by serial dilution, in glass flasks. The highest concentration was placed in a heated ultrasonic bath
(the temperature did not exceed 60˚C) for about three hours and then stirred (magnetic stirrer) until the solution had a clear appearance. Fresh
solutions were prepared once per week and stored at ambient temperature.
The solution (amount controlled by a syringe pump) was nebulized using an air-driven atomizer (Schlick type 970/S, Coburg, Germany). The
atomizer was supplied with a stream of humidified air. The air pressure was regulated by a reducing valve and the air flow was measured with a
mass view meter (MASSVIEW from Bronkhorst Hi Tec, Ruurlo, The Netherlands). The solutions in the polypropylene syringes were filled up daily and
refreshed at the start of the second exposure week.
- Temperature, humidity in air chamber: 20.8b to 22.5 °C, humidity: 39.7 to 62.2 5%,
- Air flow rate: the total air flow through the units was at least 1 litre/min for each rat. The air entering the units was controlled at 22±2˚C and the
relative humidity was maintained between 30 and 70%, if possible.
- Air change rate: the total air flow, temperature and relative humidity were recorded hourly during exposure. The air flow was monitored by a mass
stream meter (MASSVIEW from Bronkhorst Hi Tec, Ruurlo, The Netherlands). Temperature and relative humidity were measured using an RH/T
device (Testo 635, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany).
- Method of particle size determination: Particle size distribution measurements were carried out using an APS (aerodynamic particle sizer, model
3321, TSI Inc. Shoreview, USA), at least once during preliminary generation of test atmospheres and once weekly after the start of the exposure
period for each concentration (low, mid and high). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd)
were calculated (APS user’s manual, 2010). Particle size measurement by means of a cascade impactor was not feasible for the low concentration
because the concentration was too low to obtain weighable amounts of material on the filters. Moreover, considering the vapour pressure of the
test material, evaporation of mass impacted on the stages before weighing of the filters could occur. Therefore, the cascade impactor was not used to measure particle size during the exposure period.
- Control group: exposure chamber for the control animals (group 1) was supplied with a stream of humified air only

TEST ATMOSPHERE
- Brief description of analytical method used: the concentration of the test material in impinger liquid was measured by a High Performance Liquid
Chromatography – Mass Spectrometry (HPLC-MS) method.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On each exposure day, the actual concentration of the test material in the test atmosphere was determined by chemical analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled amounts of test atmosphere at 0.465 Ln/min through glass impingers filled with methanol. The sampling time was 120 minutes for the low concentration and 40 minutes for the higher
concentrations. The concentration of the test material in impinger liquid was measured by a High Performance Liquid Chromatography – Mass
Spectrometry (HPLC-MS) method.
Duration of treatment / exposure:
5 days per week for 2 weeks
Frequency of treatment:
5 days/week x 6 hours/day
Doses / concentrations
Remarks:
Doses / Concentrations:
0.14, 0.62, 5.7 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
6 animals per dose group = 24 males
Control animals:
yes, concurrent vehicle
Details on study design:
Administration route of inhaltion was chosen because humans may be exposed to the test material by inhalation.
Male rats were selected since sex differences were not expected and the use of a single sex with more animals per group increases statistical power.
Positive control:
not nesessary

Examinations

Observations and examinations performed and frequency:
Animal observations:
- Clinical signs: Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity.
All animals were observed before and after the exposure, on days without exposure once a day. During exposure observation was limited due to
the animals’ stay in restraining tubes and attention was directed towards any breathing abnormalities and restlessness. All abnormalities, signs of
ill health, and reactions to treatment were recorded.
- Body weights: The body weight of each animal was recorded 1 day before the start of exposure and prior to exposure on the first day (day 0).
Subsequently, animals were weighed on day 1, 3, 7, 10 and 14
- Food consumption: measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was
measured over two successive 7-day periods, starting on day 0

Sacrifice and pathology:
Pathology
- Necropsy with gross pathological examination: All animals on Day 15.
- Organ weight determination: All animals, fresh weights of heart, adrenals, testes, lungs with trachea and larynx, kidneys, livers and spleens,
at necropsy.
- Tissue preservation: Weighted organs of all animals were preserved
- Histopathological examination: The nasopharyngeal tissues of all animals of the control and high concentration groups were examined
histopathologically (by light microscopy). The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994, see attachment,
with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT). As treatment-related changes were observed in animals of the high concentration group, examination of the nasopharyngeal tissues (levels III, IV and V) was extended to the animals of the
intermediate concentration groups.
Other examinations:
no other examinations
Statistics:
- Body weight data: ‘Ancova & Dunnett’s Test’
- Organ weights: ‘Generalised Anova/Ancova Test’
- Food consumption results (experimental unit is the cage): Dunnett’s multiple comparison test.
- Incidences of histopathological changes: Fisher’s exact probability test.
Arithmetic means and standard deviations (abbreviation SD) are given in the tables of continuous and semi-continuous data. Tests are performed as
two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01. Because numerous variables are subjected to statisticalanalysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final
interpretation of results is based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
details see below
Mortality:
no mortality observed
Description (incidence):
details see below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
details see below
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
details see below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
details see below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
details see below
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
details see below
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
details see below
Details on results:
- Clinical signs: All animals survived until scheduled sacrifice. The observations made about halfway through each exposure day showed no
abnormalities. The observations before and after exposure revealed no treatment-related clinical signs either.
- Body weight: Body weight was not affected by treatment as shown by the absence of (statistically) significant differences between animals exposed to the test material and controls.
- Food consumption: Food consumption was comparable in all groups.
- Organ weights: Absolute and relative organ weights showed no (statistically) significant differences between animals exposed to the test material
and controls.

- Pathology:
Macroscopic examination: Macroscopic observations at scheduled sacrifice were unremarkable. The few findings represent normal background
pathology in rats of this strain and age and occurred only incidentally.

Microscopic examination: Exposure-related effects essentially concentrated in the three middle sections of the nasal passages,
i.e. levels III, IV and V.

The most prominent effect was a mixed (granulocytes as well as monocytes/lymphocytes) inflammatory cell infiltration in the nasal mucosa of
animals of the mid and high concentration groups. The animals of the high concentration group were more affected than those of the mid
concentration group, both in incidence and in the number of inflammatory cell foci (‘multifocal’ was restricted to the high concentration animals).

The incidence of mononuclear cell infiltrates (monocytes/lymphocytes) was very low in the controls (only one control animal at level IV). The
incidences in the exposed groups were high, particularly in the low and high concentration groups. Due to the lack of a concentration-effect
relationship these high incidences could not be attributed to the exposure to the test material with certainty.

A few focal epithelial changes appeared related to the exposure, namely goblet cell hyperplasia (levels IV and V) and focal metaplasia of the
respiratory epithelium (level IV) in the mid and high concentration groups. Goblet cell hyperplasia was also seen at level III in a single animal of the
low and mid concentration groups. The goblet cell hyperplasia at level III was not attributed to the exposure because it occurred only incidentally
and was not seen in the high concentration group.

In summary, exposure-related effects were seen in the middle part of the nasal passages, at the mid and high concentrations. The effects consisted of a distinct mixed inflammatory cell infiltration in the nasal mucosa and, much less prominent, epithelial changes.

Effect levels

Dose descriptor:
NOAEC
Effect level:
0.14 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:
Based on microscopic changes observed in the nasal passages of animals of the mid and high concentration groups and the absence of adverse
effects in the low concentration group, the No-Observed-Adverse-Effect-Concentration (NOAEC) of decamethylenediamine was placed at the low
concentration, namely 0.14 mg/m3 (actual concentration measured by chemical analysis).
Executive summary:

The objective of this study was to provide data on the sub-acute (14-day) toxicity in rats of decamethylenediamine after exposure by inhalation. The results of this 14-day study may serve as a basis for selection of the concentration levels for a future sub-chronic (90-day) inhalation study with this substance.

 

The study included four groups of 6 male Wistar rats each. The animals were exposed nose-onlyfor 6 hours/day, 5 days/week over a 14-day period (resulting in 10 exposure days) to three different concentrations of the test material or to clean air for the control group. The highest and the middle target concentrations were 5 and 0.5 mg/m3, respectively. The intended lowest target concentration was 0.05 mg/m3. However, as the actual concentrations obtained on exposure days 1-3 came out about two-fold higher than 0.05 mg/m3, it was decided to continue the exposure of the lowest concentration group at the generation settings used on days 1-3. Observations and measurements to detect adverse effects included daily clinical observations, body weight, food consumption, organ weights, macroscopic examination at sacrifice on the day after the last exposure and microscopic examination of the nasopharyngeal tissues.

 

The mean actual concentrations (± standard deviation) of decamethylenediamine in the test atmospheres were 0.14 (± 0.03), 0.62 (± 0.10) and 5.7 (± 0.7) mg/m3 for the low, mid and high concentration level, respectively. Although the analytical method (HPLC-MS) was not optimal, these values should be regarded as a reasonable indication of the achieved exposure levels. The generation efficiency calculated on the basis of these values was 36%, 52% and 73% for the low, mid and high concentration, respectively. The mid and high concentration test atmospheres were also sampled for analysis by gravimetry. The mean concentrations (± standard deviation) were 1.13 (± 0.03) and 7.7 (± 0.3) mg/m3at the mid and high level, respectively. These gravimetric concentrations approached the nominal concentrations, resulting in a generation efficiency >90%, which is unlikely for an aerosol. Possibly, the gravimetric concentrations were increased due to a reaction of the test material with CO2.

 

The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 0.675 µm (with a geometric standard deviation (gsd) of 1.27), 0.893 µm (gsd of 1.69) and 1.18 µm (gsd of 1.97) for the low, mid and high concentration test atmospheres, respectively.

 

All animals survived until scheduled sacrifice. There were no treatment-related clinical signs.

 

Body weight or food consumption were not affected by the test material.

 

Organ weight data and macroscopic examination at scheduled sacrifice showed no treatment-related changes.

 

Microscopic examination of the nasopharyngeal tissues revealed exposure-related effects in the middle part of the nasal passages (levels III, IV and V) in the mid and high concentration groups. The most prominent effect consisted of a distinct mixed inflammatory cell infiltration in the nasal mucosa. The animals of the high concentration group were more affected than those of the mid concentration group. The other effects were much less prominent and consisted of epithelial changes at levels IV and V (goblet cell hyperplasia and focal metaplasia of the respiratory epithelium).

 

 

Conclusion

Based on microscopic changes observed in the nasal passages of animals of the mid and high concentration groups and the absence of adverse effects in the low concentration group, the No-Observed-Adverse-Effect-Concentration (NOAEC) of decamethylenediamine was placed at the low concentration, namely 0.14 mg/m3(actual concentration measured by chemical analysis).