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Toxicological information

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:
supporting study
Study period:
2012-04-24 to 2012-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
In the study plan it was indicated (erroneously) that the study plan was drafted according to the OECD 413 guideline. Because the present study was a concentration range finding study not all aspects of the guideline were implemented.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Appearance : slightly yellowish liquid
Purity1 : >99%
Batch number : VP BL-PTSA-Kat.1106

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
- Source: Harlan, netherlands
- Strain: rats, Wistar (RccHan:WIST)
- Age: approx. 8 weeks on first exposure day
- Animals: 24 males
- Acclimatisation: 12 days
- Mean Body Weight at study initiation: male: 260g
- 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
- 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:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: humdified compressed air
Remarks on MMAD:
MMAD / GSD: The average mass median aerodynamic diameter (MMAD) for the low, mid and high concentration groups was 1.29, 1.49 and 2.45 µm, with
corresponding average geometric standard deviations (gsd) of 2.11, 2.40 and 2.13, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: see attachment, nose-only inhalation units (a modification of the chamber manufactured by ADG Developments Ltd.,
Codicote, Hitchin, Herts, SG4 8UB, United Kingdom;
- Method of holding animals in test chamber: animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder
around the central column
- Source of air: humdified compressed air, 22 +/- 2 °C, humidity between 30 and 70 %
- Air flow: at least 1 litre/min for each rat,
- System of generating particulates/aerosols: An amount of test material, controlled by a syringe pump (groups 2 and 3) or a peristaltic pump
(group 4), was nebulized using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) which was wrapped up in electric heating tape and
aluminium foil (the test material was too viscous for atomizing without heating) . The atomizer was supplied with a stream of humidified air. The
pressure was regulated by a reducing valve and the flow was measured with a mass stream meter (MASSVIEW from Bronkhorst Hi Tec, Ruurlo, The
Netherlands). The top section of the exposure unit was cooled by one or two plastic pots containing dry ice.
- Temperature, humidity in air chamber: 20.0 to 24.3 °C, humidity: 29.0 to 46.3 5%,
- Air flow 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).
- Method of particle size determination: using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA),
- Control group: The exposure chamber for the control animals (group 1) was supplied with a stream of humified air only

TEST ATMOSPHERE
- Analyses of dust particleize: once weekly and at least once during preliminary generation of the test atmosphere for each exposure condition
- Graimetric analyses: The actual concentration of the test material in the test atmosphere was determined at least three times per day for each
exposure condition by gravimetric analysis

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative test atmosphere samples were obtained from the animals’ breathing zone during the study by passing mass flow controlled amounts of test atmosphere (230, 23 and 4.6 Ln , corresponding to about 250, 25 and 5 L, at the low, mid and high concentration, respectively) at 4.6 Ln/min through fiber glass filters. Filters were weighed before and after sampling. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.
Duration of treatment / exposure:
5 days per week for 2 weeks
Frequency of treatment:
5 days/week x 6 hours/day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air (nominal)
Remarks:
Control
Dose / conc.:
20 mg/L air (nominal)
Remarks:
Low dose
Dose / conc.:
200 mg/L air (nominal)
Remarks:
Mid dose
Dose / conc.:
2 000 mg/L air (nominal)
Remarks:
High dose
Dose / conc.:
31 mg/L air (analytical)
Remarks:
Low dose
Dose / conc.:
202 mg/L air (analytical)
Remarks:
Mid dose
Dose / conc.:
2 032 mg/L air (analytical)
Remarks:
High dose
No. of animals per sex per dose:
6 males per dose, 4 doses (incl. control)
24 males
Control animals:
yes, concurrent vehicle
Details on study design:
The concentration levels were chosen in consultation with the sponsor.
Male rats were chosen since the results of an acute study with this test material (TNO Triskelion study 9901/12) did not show sex differences and a selection of a single sex with more animals per group increases the statistical power
Positive control:
not nesessary

Examinations

Observations and examinations performed and frequency:
- Animal observations: All animals, before and after the exposure, on days without exposure once a day. 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 the periods day 0-1, 1-7 and 7-14.
Sacrifice and pathology:
- Necropsy with gross pathological examination: All animals on Day 15.
- Organ weight determination: All animals, fresh weights of heart, adrenals, testes, kidneys, lungs with trachea and larynx, livers and spleens at
necropsy.
- Tissue preservation: The organs were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde
(10% solution of formalin). They will be discarded after completion of the final report.
Other examinations:
no other examinations
Statistics:
- Body weight data: ‘Ancova & Dunnett’s Test’
- Pretreatment and terminal body weights, weight changes and organ weight data: ‘Generalised Anova/Ancova Test’
- Food consumption results (experimental unit is the cage): Dunnett’s multiple comparison 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:
effects observed, treatment-related
Description (incidence and severity):
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 clinical signs in animals of the high concentration group only, namely dyspnoea (4 animals) and blepharospasm (2 animals) after the first exposure, and grunting (1 animal) prior to exposure on days 8-9. These clinical signs were considered to be related to treatment.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
n the second week of the study, growth was reduced, concentration-dependently, in all groups exposed to the test material. This effect was most marked on the days that the animals were exposed. Between days 7 and 10 (3 successive exposure days) most animals of the mid and high concentration groups lost weight and the animals of the low concentration group gained less weight than controls. In the period day 10-14 (no exposure on days 11 and 12), all exposed animals gained weight but statistically significantly less than controls.
The weight change results in the first exposure week were confounded by wide interanimal variation which probably resulted from the switch from pelleted to powdered feed on the day before initiation of treatment. Consequently, it is not clear whether or not growth was affected by treatment in the first exposure week. Compared to the control group, the mean terminal body weights in the low, mid and high concentration group were decreased by 2%, 6% and 4%, respectively. The differences in mean body weight did not reach the level of statistical significance at any time point.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
On the first exposure day, animals of the high concentration group consumed less food than controls. In the period day 1-7, food consumption of treated animals and controls was comparable. Thereafter, the treated animals tended to eat somewhat less than controls but the differences showed no concentration-related response and, except in the mid concentration group, they were not statistically significant.
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The weight of the lungs (absolute and relative) and kidneys (relative only were statistically significantly increased in the high concentration group. The weights of the other organs were not affected. In the absence of a similar change in the high concentration group, the statistically significantly lower liver weight (absolute and relative) observed in the mid concentration group was considered to be unrelated to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic observations at scheduled sacrifice showed incompletely collapsed lungs in two animals and patches on the lungs in one animal of the high concentration group. These findings were possibly related to treatment.
The other findings were unremarkable. They represent normal background pathology in rats of this strain and age and occurred only incidentally.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEC
Effect level:
202 mg/m³ air
Based on:
test mat.
Sex:
male

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study no adverse effects occurred in the low and mid concentration groups. Therefore, the mid concentration of
202 mg/m3 (actual concentration measured by gravimetry) was a No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic effects of the test item.
Furthermore, this 14 day repeated dose inhalation study reveal no indications for local effects up to concentrations of 2032 mg/m3.
Executive summary:

The objective of this study was to provide data on the sub-acute (15-day) toxicity in rats of the test item after exposure by inhalation. The results of this 15-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 inhalation toxicity of the test item was studied in a sub-acute (15-day) inhalation toxicity study in Wistar rats. Four groups of 6 male rats were exposed nose-only to target concentrations of 0 (control) 20, 200 or 2000 mg/m3 of the test material for 6 hours/day, 5 days/week over a 15-day period, with a total of 10 exposure days. Observations and measurements to detect adverse effects included daily clinical observations, body weight, food consumption, organ weights and macroscopic examination at sacrifice on the day after the last exposure.

 

The mean actual concentrations (± standard deviation) of the test item (solvent free), based on gravimetric analysis, were 31 (± 16), 202 (± 10) and 2032 (± 139) mg/m3 for the low, mid and high concentration level, respectively. The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 1.29 µm (with a geometric standard derviation (gsd) of 2.11), 1.49 µm (gsd of 2.40) and 2.45 µm (gsd of 2.13) for the low, mid and high concentration test atmospheres, respectively.

 

All animals survived until scheduled sacrifice. Treatment-related clinical signs were seen in the high concentration group only and consisted of dyspnoea (4 animals) and blepharospasm (2 animals) after the first exposure, and grunting (1 animal) prior to exposure on days 8-9.

 

In the second week of the study, growth was reduced, concentration-dependently, in all groups exposed to the test material. This effect was most marked on the days that the animals were exposed (between days 7 and 10, most animals of the mid and high concentration groups lost weight). Growth data of the first week were inconclusive due to wide inter animal variation which probably resulted from the switch from pelleted to powdered feed on the day before initiation of treatment. Despite the effect on growth, mean body weights showed no statistically significant intergroup differences. Therefore, the effect on growth was considered to be of little toxicological significance.

 

Food consumption was decreased on the first exposure day in the high concentration group and, to a limited degree, in the second week in all exposed groups.

 

Absolute and relative lung weight and relative kidney weight were statistically significantly increased in the high concentration group.

 

Macroscopic examination at scheduled sacrifice showed incompletely collapsed lungs (two animals) and patches on the lung (one animal) in the high concentration group.These findings were possibly related to treatment.

 

Conclusion

Under the conditions of this study no adverse effects occurred in the low and mid concentration groups. Therefore, the mid concentration of 202 mg/m3 (actual concentration measured by gravimetry) was a No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic effects of the test item. Furthermore, this 14 day repeated dose inhalation study reveal no indications for local effects up to concentrations of 2032 mg/m3.