4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 - 29 March 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EG Guideline 92/69/EWG. Journal of the European Community - Legal Specifications L 2383 A, 35.

Version / remarks:

29 December 1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency. Pesticide assessment guidelines, subdivision F, hazard evaluation: Human and domestic animals (Revised) § 81-3 Acute Inhalation Toxicity Study. NTIS Report PB86-108958, Washington, DC

Version / remarks:

1984

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency. Hazard evaluation division: Standard evaluation procedure, inhalation toxicity testing, NTIS Report PB89-100366, Washington, DC.

Version / remarks:

1988

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

EC Number:

605-140-1

Cas Number:

158237-07-1

Molecular formula:

C16H20ClN5O2

IUPAC Name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd Win: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
- Weight at study initiation: 170 - 205 g (males), 169 - 187 g (females)
- 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: dust

Type of inhalation exposure:

nose only

Vehicle:

other: test substance was aerosolized as dust without a carrier or vehicle

Mass median aerodynamic diameter (MMAD):

ca. 2.6 µm

Geometric standard deviation (GSD):

ca. 2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure tubes (TSE, Bad Homburg, Germany)
- Exposure chamber volume: 3.8 L (dimensions of the inhalation chamber: inner diameter =14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm)

- Source and rate of air: compressed air supplied by Boge compressors
- Method of conditioning air: automatically by a VIA compressed air dryer
- System of generating particulates/aerosols: In order to obtain the high concentration of respirable aerosol an EXACTOMAT 4200 (TSE, 61348 Bad Homburg, Germany) and for the lower concentration a wright-dust-feeder (BGI Inc., Waltham, MA, USA) was used.
- Method of particle size determination: gravimetric analyses (cascade impactors)
- Treatment of exhaust air: the exhaust air was purified via cotton-wool/activated charcoal and HEPA filters. These filters were disposed of.
- Temperature, humidity, pressure in air chamber: 22°C, humidity: control group: 15%, 535 mg/m³: 8% and 5085 mg/m³: 4%

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis with cellulose-acetate filter
- Samples taken from breathing zone: yes

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

535 mg/m³
5085 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 and 28 days
- Frequency of observations and weighing: clinical signs: several times on the day of exposure and at least once daily thereafter; body weights: measured prior to exposure and on Day 3 and 7 and weekly thereafter
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathology

Statistics:

Statistically significant differences (vs control) are indicated by asterisks (*for p < 0.05, **for p < 0.01).

Necropsy: evaluation of respiratory tract findings in surviving rats(pairwise Fisher test after the RxC chi-squared test) The Fisher test was only performed if differences occurred between groups in the RxC chi-squared test or if a frequency value of < 5 was calculated (according to with Gad and Weil, 1982). For calculation of the unilateral p value a symmetrical distribution was assumed (p unilateral = (p bilateral)/2).

Body weights: Means and single standard deviations of body weights are calculated. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group (one-way ANOVA (vide infra))

Physical measurements: means + single standard deviation

Physiological data: rectal temperature measurements (ANOVA (vide infra))

Calculation of the LC50: according to the method of A.P. Rosiello et al (1977) as modified by Pauluhn (1983) (based on the maximum-likelihood method of C.I. Bliss (1938)). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a x²-homogeneity test is not performed. The interpolated concentration at 50% lethality in this case was designated the approximate LC50.

Randomization: computerized list of random numbers (animals)

Analysis of variance (ANOVA): groups are compared at a confidence level of (1-oc) = 95% (p = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 groups were compared. If the F-test shows that the intra-group variability> inter-group variability, this is shown as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) (Games and Howell modification of the Tukey-Kramer significance test)

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 085 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occured until the end of the observation period.

Clinical signs:

other: All rats tolerated the treatment without any clinical sign.

Body weight:

Decreased body weights were determined in both treatment groups. However, these changes were minor and transient in nature and are therefore considered to be toxicologically insignificant.

Gross pathology:

No findings related to treatment were observed.

Other findings:

Rectal temperature:
Statistical comparison between groups exposed to the test substance with those in the control group did indicate concentration-dependent effect on body temperature in all groups exposed to the test compound. The changes observed, however, are considered to be toxicologically insignificant.

Any other information on results incl. tables

Aerosol generation:

The aerosol generated was of adequate respirability in the lower exposure group (i.e. MMAD ca. 2.6 |um, GSD ca. 2, relative mass < 3 \im ca. 60%). It appeared not to be feasible to generate concentrations approximating 5000 mg/m³ air using a cyclone to seperate off larger particles. This, in fact, resulted in a decreased respirability of particles in the highest esposure group. Despite the lower respirability rats experienced a hypothermia in the higher exposure groups.

Table 2: Summary of acute inhalation

Group/Sex	Gravimetric Concentration (mg/m³)	Toxicological Result	Onset and duration of signs	Rectal temperature (°C)	Onset of Mortality
1/m	0	0/0/5	-	38.2	-
2/m	535	0/0/5	-	37.3**	-
2/m	5085	0/0/5	-	35.8**	-
1/f	0	0/0/5	-	38.6	-
2/f	535	0/0/5	-	37.0**	-
3/f	5085	0/0/5	-	35.4**	-

m = males, f = females, — not applicable

Values given in the 'Toxicological results' column are:

1st = number of dead animals.

2nd = number of animals with signs after cessation of exposure.

3rd = number of animals exposed.

 

Table 3: Mean Bodyweights with standard deviations [g]

Dose [mg/m^3]	Sex	exposure day	3 days after exposure	1 week after exposure	2 weeks after exposure
control	m	197.2 ± 5.1	202 ± 4.6	221.0 ± 6.3	254.8 ± 9,5
535	m	170.8 ± 1.3	177 ± 4.4	204.4 ± 5.9	239.4 ± 9.5
5085	m	192.6 ± 6.2	200.6 ± 8.4	224.2 ± 10.0	255.0 ± 10.6
control	f	185.2 ± 1.5	184.0 ± 2.5	187.2 ± 2.5	198.2 ± 5.6
535	f	171.6 ± 2.9	167.0 ± 7.6	174.8 ± 7.5	185 ± 8.0
5085	f	178.2 ± 4.4	174.6 ± 6.3	176.8 ± 6.9	188.4 ± 7.3

m male, f female

 

Table 4: Gross pathology findings

Group	Dose [mg/m^3]	Animals with effects	Time of death	Sacrificed after	Pathology findings
1 m	control	0/5	-	28 days	no observable effects
2 m	535	1/5	-	14 days	lung: foci slightly dark red
3 m	5085	0/5	-	14 days	no observable effects
1 f	control	0/5	-	28 days	no observable effects
2 f	535	0/5	-	14 days	no observable effects
3 f	5085	0/5	-	14 days	no observable effects

m male, f female

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

In this acute inhalation toxicity study in rats a LC50 value of > 5085 mg/m³ air was determined.