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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
4 weeks exposure
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2002 - February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
Adopted in 1981
Deviations:
no
Principles of method if other than guideline:
Study conducted according to OECD 412 with study conditions adjusted to fulfill both the TSCA § 798.2250 as well as the EU Guideline 92/69/EWG (1992).
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
446-620-9
EC Name:
-
Cas Number:
120983-72-4
Molecular formula:
Hill formula: C5H6Cl2O CAS formula: C5H6Cl20
IUPAC Name:
2-Chloro-1-(1-chlorocyclopropyl)ethanone
Test material form:
liquid
Details on test material:
purity: 92.3 %
Specific details on test material used for the study:
purity 93.2%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Hsd Cpb:WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2 - 3 months
- At the study start the variation of individual weights did not exceed ±10 per cent of the mean for each sex
- Housing: individually in Makrolon cages type II during the acclimation and study periods
- Diet: Altromin® 1324 diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2.0
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
Under dynamic conditions the test substance was evaporated from a glass flask (impinger) from which the substance was conveyed into the intake of the cylindrical inhalation chamber. The test substance was evaporated using a bubbler (glass flask, filling height: «5 cm, diameter: «1 cm, filled with approximately 10 g of test substance) that was maintained at 23 °C and 30 °C in groups 2-3 and 4-5, respectively. Specified flows of dry air were used for dilution of atmospheres whilst nitrogen was fed through the bubbler. The total air flow through the chamber was 30 L/min (for more details see Table 1, result section). The nitrogen bubbled through the impinger was metered using a precision TELAB piston pump.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of airborne test-substance were sampled and analytically determined. Briefly, the test substance was adsorbed on Florisil using the air flow rate described in the following section and was eluted from the Florisil matrix with toluene. Then, the analyte was quantified by gas chromatography.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Duration of exposure per day: 6 hours /day
Dosing regime: 5 days/week, for 4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0.115 mg/m³ air (analytical)
Dose / conc.:
0.411 mg/m³ air (analytical)
Dose / conc.:
1.5 mg/m³ air (analytical)
Dose / conc.:
5.63 mg/m³ air (analytical)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The 1-week pilot study (subacute inhalation rats, Pauluhn 2003) showed that the test substance induced a concentration dependent, irritant-related damage of the entire respiratory tract at 13.8 mg/m3 and above when exposed to sufficiently high concentrations of the vapor of this test substance. Taking all findings into account 1.9 mg/m3 constitutes an exposure level at which borderline effects of unclear toxicological significance appear to occur. Repeated exposures to 13.8 mg/m3 were causing damage of the entire respiratory tract with ensuing mortality whilst some borderline effects occurred in the same target organ at 1.9 mg/m3. Studies characterizing the irritant potency more precisely (e.g., RD5o-determination) not available so that this most critical endpoint was assessed on the basis of clinical observations. Based on respiratory tract irritation 5 mg/m3 is considered to be an adequate maximum concentration for repeated inhalation exposure studies, since higher concentrations would cause undue irritation. Accordingly, the following target concentrations were chosen: 0.15, 0.5, 1.5 and 5 mg/m3 air. The NO(A)EL was expected to be 0.5 mg/m3. Because of the more sensitive endpoints incorporated in the study design (histopathology) and the lack of an RD5o-study, the prediction of this irritant-based NO(A)EL was somewhat uncertain so that an additional low-level exposure group (0.15 mg/m3) was incorporated.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule:
twice on exposure days (before and after exposure), once on exposure-free days
- Cage side observations included but were not limited to: changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible, if applicable.

BODY WEIGHT: Yes.
- Time schedule for examinations:
twice per week during exposure and once per week during post-exposure period

FOOD CONSUMPTION:
- Food and water consumption determined twice per week.

The appearance and behavior of each rat was examined carefully at least twice on
exposure days (before and after exposure) and once a day during the exposure-free
days.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations:
once before start of the exposure period (study day -3), once towards the end of the exposure period (study day 22)

HAEMATOLOGY: Yes.
- Time schedule for collection of blood:
study days 28 - 30 and study days 56 - 57
- Anaesthetic used for blood collection: Yes. Pentobarbital narcosis.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood:
study days 28 - 30

URINALYSIS: Yes.
- Time schedule for collection of urine:
once on study days 24 and 25
- Metabolism cages used for collection of urine: Yes.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations:
once on study day 22

- Battery of functions tested: Visual placing response, grip strength (vertical, horizontal), tonus, cornea reflex, light reflex, pinna reflex, startle reflex (sound, touch), tail-pinch response, righting response (open field, drop method)

IMMUNOLOGY: No.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No.

LUNG BURDEN: No.

OTHER:
RECTAL TEMPERATURE
- Time schedule for examinations: study day 0, 4, and 23

METHEMOBLOBIN-DETERMINATION
- Time schedule for examinations: study days 14 - 17
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All rats were subjected to gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.
HISTOPATHOLOGY: Yes
Statistics:
For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical tests are used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variate in question can be considered approximately normally distributed with equal variances across treatments, the Dunnett test is used, if heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA are carried out, i. e. the Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures (Miller (1981)). Within such a family, the experimentwise error is controlled. If not otherwise noted, all pair-wise tests are two-sided comparisons.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Concentrations up to
and including 1.5 mg/m3 were tolerated by all rats without clinical effects. At 5.63
mg/m3 some rats experienced mild and transient signs of respiratory tract irritation
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no consistent, i.e., concentration-dependent effect on body weights up to and including the 1.5 mg/m3 group. At 5 mg/m3 the rats experienced a decrease in body weights especially towards the end of the exposure period. During the exposure free postexposure period male rats recovered rapidly whereas in females the incremental loss of body weights remained.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no toxicologically consistent effect on food consumption up to and including 1.5 mg/m3. At 5 mg/m3 a significantly decreased food intake was observed. The diminished food intake became normal or was overcompensated during the early postexposure period and differences between the control and 5 mg/m3 groups resolved entirely within the postexposure period. Overall, the cumulative food intake at this exposure level was decreased.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no toxicologically consistent effect on water consumption amongst the groups.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There was no clear concentration dependent change in any of the hematological parameters. Therefore, the isolated statistical significance's are considered to be of no pathodiagnostic relevance due to a lack of consistent changes throughout the groups. Also the decreased number of reticulocytes (males, 5 mg/m3 group) and increased hepatoquick (females, 5 mg/m3 group) did not show any conclusive pattern of changes indicative of a specific toxic response.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no clear concentration dependent change in any of the clinical parameters.
Therefore, the isolated statistical significance's are considered to be of no
pathodiagnostic relevance due to a lack of consistent changes throughout the
groups. The mild increase in serum ALAT activity (5 mg/m3, females) appears to
have some association with the increased hematocrit observed at this level
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Collectively, there was a trend of a decrease in liver, spleen and thymus weights in rats of the high-level exposure group. In female rats of this group somewhat increased adrenal weights were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After the end of the 4-week exposure period, inflammatory as well as concentration dependent epithelial changes (metaplasia, necrosis) were observed in the upper respiratory tract (nasal cavity, pharynx, larynx) of rats exposed at 1.5 and 5 mg/m3. In none of the groups evidence of test substance-related findings were detected in the trachea or lung. At the end of the recovery period, epithelial metaplasia, necrosis and inflammatory findings were still detectable in the nasal cavity, however, the incidence and the grading were lower as compared to the end of the exposure period. In the pharynx, some minimal inflammatory infiltrates whereas in the larynx of rats of these groups, epithelial changes or metaplasia could not be detected anymore. In the trachea and lung and in all other organs/tissues examined, the histopathological findings were equally distributed between groups and are therefore considered to be spontaneously occurring.
In summary, the histopathological examinations showed a clear anterior-posterior gradient of intensity of changes. Based on the most sensitive endpoint, i.e., irritant related changes occurring the upper airways, the no-observed-effect level was 0.5 mg/m3.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Rectal Temperatures:
In comparison to the concurrent control group, there was some mild decrease body (rectal) temperature in the 5 mg/m3 group during the last exposure week. In the males of this group this effect gained (marginal) statistical significance.
Details on results:
Mortality did not occur in any of the exposure groups. Concentrations up to and including 1.5 mg/m3 were tolerated by all rats without clinical effects. At 5.63 mg/m3 some rats experienced mild and transient signs of respiratory tract irritation (serous nasal discharge, stridor, tachypnea, irregular breathing pattern, limp, ungroomed hair-coat, piloerection, mild hypothermia). In the rats of this group significantly decreased body weights and a decreased food consumption were observed. There was no evidence of hematological effects considered to be patho-diagnostically relevant. Clinical-pathology did not reveal any evidence of concentration-dependent effects considered to be causally related to the exposure to JAU 6476 - Chloromethylketon. There were no statistically significant or conclusive changes in absolute or relative organ weights up to and including 1.5 mg/m3. However, there was a trend of significantly decreased liver, spleen and thymus weights in rats of the high-level exposure group. In female rats of this group somewhat increased adrenal weights were observed. At the end of the 4-week exposure period, inflammatory as well as concentration dependent epithelial changes (metaplasia, necrosis) were observed in the upper respiratory tract (nasal cavity, pharynx, larynx) of rats exposed at 1.5 and 5.63 mg/m3. In none of the groups evidence of test substance-related findings were detected in the trachea or lung. Following the 6-week recovery period, epithelial metaplasia, necrosis and inflammatory findings were still detectable in the nasal cavity, however, the intensity of changes decreased appreciably as compared to the findings observed at the end of the exposure period. There was no effect on extrapulmonary organs.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
0.411 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1.5 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
pharynx
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 2: Summary of 4-weeks subacute inhalation toxicity (6 h/day on 5 days per week)

































































































N



Target



 



Onset and



 



Group



Concentration



Toxicological



Duration of



Onset



/sex



(mg/m3)



Result



Signs



of Mortality



1 /m



0



0/0/20



-



-



2/m



0.15



0/0/10



-



-



3 /m



0.5



0/0/10



-



-



4 /m



1.5



0/0/10



-



-



5/m



5



0/13/20



9d - 26d



-



1/f



0



0/0/20



-



-



2 / f



0.15



0/0/10



-



-



3 / f



0.5



0/0/10



-



-



4/f



1.5



0/0/10



-



-



5/f



5



0/13/20



4d - 26d



-



m = males, f = females, - not applicable, day 0: first exposure day


Values given in the Toxicological results' column are:


1 st = number of dead animals.


2nd = number of animals with signs


3rd = number of animals exposed.

Applicant's summary and conclusion

Conclusions:
Taking all findings into account 0.411 mg/m3 (0.000411 mg/l) constitutes the no-adverse-observed effect-level (NOAEL).
Based on the repeated inhalation study results classification according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, STOT-RE catagory 1 (28d study rat, inhalation,  vapour) is warranted.
Executive summary:

Mortality did not occur in any of the exposure groups. Concentrations up to and including 1.5 mg/m3 were tolerated by all rats without clinical effects. At 5.63 mg/m3 some rats experienced mild and transient signs of respiratory tract irritation (serous nasal discharge, stridor, tachypnea, irregular breathing pattern, limp, ungroomed hair-coat, piloerection, mild hypothermia). In the rats of this group significantly decreased body weights and a decreased food consumption were observed. There was no evidence of hematological effects considered to be patho-diagnostically relevant. Clinical-pathology did not reveal any evidence of concentration- dependent effects considered to be causally related to the exposure to the test substance. There were no statistically significant or conclusive changes in absolute or relative organ weights up to and including 1.5 mg/m3. However, there was a trend of significantly decreased liver, spleen and thymus weights in rats of the high-level exposure group. In female rats of this group somewhat increased adrenal weights were observed. Collectively, the changes in body weights and food consumption are considered to be the major cause of the liver weight changes observed at 5.63 mg/m3, despite the somewhat increased hepatoquick and ALAT values observed in the female rats of this group. At the end of the 4-week exposure period, inflammatory as well as concentration dependent epithelial changes (metaplasia, necrosis) were observed in the upper respiratory tract (nasal cavity, pharynx, larynx) of rats exposed at 1.5 and 5.63 mg/m3. In none of the groups evidence of test substance-related findings were detected in the trachea or lung. Following the 6-week recovery period, epithelial metaplasia, necrosis and inflammatory findings were still detectable in the nasal cavity, however, the intensity of changes decreased appreciably as compared to the findings observed at the end of the exposure period. There was no effect on extrapulmonary organs. In summary, this study demonstrates that the outcome of study is governed by irritant-related effects at the portal-of-entry. The histopathological examinations showed a marked anterior-posterior gradient of intensity of changes. Based on the most sensitive endpoint, i.e., irritant-related changes occurring the upper, extrathoracic airways, the NO(A)EL is 0.411 mg/m3. At 1.5 mg/m3 histopathological whilst at 5.63 mg/m3 additional clinical evidence of upper respiratory tract irritation existed (mainly nasal discharge). The mildly affected organ weight (decreased liver, spleen and thymus weights) and increased adrenal weights at 5.63 mg/m3 are of unclear toxicological significance and may be associated to irritant-related stress (upper respiratory tract irritation). Taking all findings into account 0.411 mg/m3 constitutes the no-adverse-observed effect-level (NOAEL).

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