Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Endpoint summary

Description of key information

Acute toxicity:
Oral (OECD 423), rat: LD50 ≥ 5000 mg/kg bw (LD50 cut-off value)
Dermal (OECD 402), rabbit: LD50 > 2000 mg/kg bw (limit test)
Inhalation (OECD 403), rat: LD50 > 3513 mg/m³ (limit test)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb - 15 Mar 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted 17 Dec 2001

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Wistar HsdCpb:Wu

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan/Winkelmann, Borden, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 10 -14 weeks.
- Body weight at study initiation: 161 g - 177 g (167 ± 6.4 g)
- Fasting period before dosing: Approx. 16 - 24 h before administration of the test compound, and approx. 2 - 4 h after administration.
- Diet: Standard diet "Provimi Kliba 3883.0.15 Maus/Ratte Haltung. Nutritive composition and the contaminant content checked and analyzed routinely, ad libitum.
- Water: Tap water was of drinking water quality, ad libitum.
- Acclimitazation period: At least 5 days.
- Housing: Group caged conventionally in polycarbonate cages on low dust wood granulate bedding.

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 2 °C
Air humidity: 55 ± 5%
Ventilation: Approx. 10 air changes per hour.
Light/ Dark cycle: 12/12 hours

Route of administration:

oral: gavage

Vehicle:

other: tap water with the aid of 2% Cremophor EL

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

CLASS METHOD
- Rationale for the selection of the starting dose: In accordance with procedure described in the flow charts of Annex 2, OECD guideline 423, three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight, for this study 2000 mg/kg bw. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.:
• no further testing is needed,
• dosing of three additional animals, with the same dose,
• dosing of three additional animals at the next higher or the next lower dose level.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

6 (3 + 3) female

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical signs and mortality rates were determined several times on the day of administration and subsequently at least once daily. Weight gain of the animals was checked weekly.
- Necropsy of survivors performed: Yes, gross examination of organs and tissues at necropsy.
- Clinical signs including body weight: Yes, nature, duration and intensity of clinical signs and body weight measurements.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

>= 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: LD50 cut-off according to OECD 423

Mortality:

There were no deaths during the study period

Clinical signs:

other: No clinical signs of toxicity were observed during the 14-day observation period

Gross pathology:

Necropsies performed at the end of the study revealed no specific or treatment-related findings

Interpretation of results:

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

Conclusions:

The study was performed in accordance to OECD TG 423 under GLP conditions and is considered reliable. The LD50 was determined to be ≥ 5000 mg/kg bw (LD50 cut-off of value).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

>= 5 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Apr - 28 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted 7 Sep 2009

Deviations:

yes

Remarks:

Animals weren't acclimatised to the test apparatus prior to testing. Animals weren't group housed during the non-exposure periods.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Wistar Hsd Cpb:WU (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen (Germany).
- Age at study initiation: Approx. 2 months.
- Weight at study initiation: 180 - 199 g for males and 164 - 179 g for females.
- Housing: Individually in conventional Makrolon® cages.
- Diet: Standard fixed formula diet (KLIBA 3883 = NAFAG 9441 pellets, PROVIMI KLIBA SA, Switzerland), ad libitum.
- Water: Drinking quality municipality tap-water, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12; artificial light from 6.00 a.m to 6.00 p.m.

IN-LIFE DATES: From: 2004-04-14 to: 2004-04-28

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.24 µm

Geometric standard deviation (GSD):

1.82

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: See figure 1 for a diagram of the exposure chamber (attached background material). Animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle. Under dynamic conditions the test substance was aerosolized into the inlet of the cylindrical inhalation chamber. The cyclone was used to prevent larger particles from entering the inhalation chamber. The cyclone was designed so that particles larger than 10 µm are retained in the cyclone.
- Exposure chamber volume: The aluminium inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume - about 3.8 L).
- Method of holding animals in test chamber: Restrained in the exposure tubes.
- Source and rate of air (airflow): Compressed air was supplied by Boge compressors. At each exposure port a minimal air flow rate of 0.75 l/min was provided.
- Method of conditioning air: Air was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: The test substance was aerosolized using a Wright-Dust-Feeder {BGI Inc., Waltham, MA, USA).
- Method of particle size determination: The particle-size distribution was analysed using an ANDERSEN cascade impactor (Hauke, Gmunden, Austria).
- Treatment of exhaust air: Purified via cotton-wool/HEPA filters.
- Temperature and humidity air chamber: 21 °C. Relative humidity < 7%. (Temperature and humidity measurements were made using a computerized system (Hydra, Fluke-Philips)).

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: The test-substance concentration was determined by gravimetrical analysis (filter: glass fibre filters, Sartorius, Gottingen, Germany; digital balance). The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected on an hourly basis. All analytical concentrations reported refer to mg of test substance/m3 air. Nominal concentrations were not calculated since this would have required a derangement of the dust generating system and in doing so this may had caused an increase in the temporal (day-to-day) variability of test concentrations.
- Samples taken from breathing zone: Yes (chamber samples were taken in the vicinity of the breathing zone).
- Time needed for equilibrium of exposure concentration before animal exposure: 5 minutes

- Particle size distribution:

Measurement I:

Respirability (percent < 1.0 µm):
Mass related: 2.3% (measured)
Number related: 38.7% (extrapolated)

Respirability (percent < 3.0 µm):
Mass related: 43.7% (measured)
Number related: 94.5% (extrapolated)

Respirability (percent < 5.0 µm):
Mass related: 76.3% (measured)
Number related: 99.3% (extrapolated)

Measurement II:

Respirability (percent < 1.0 µm):
Mass related: 2.3% (measured)
Number related: 38.7% (extrapolated)

Respirability (percent < 3.0 µm):
Mass related: 43.7% (measured)
Number related: 94.5% (extrapolated)

Respirability (percent < 5.0 µm):
Mass related: 76.3% (measured)
Number related: 99.3% (extrapolated)

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric analysis

Duration of exposure:

4 h

Concentrations:

5000 mg/m³ (nominal concentration), 3513 mg/m³ (analytical concentration, maximum achievable concentration)

No. of animals per sex per dose:

5

Control animals:

yes

Remarks:

Controls were exposed to an atmosphere using essentially similar exposure conditions as were used for the test substance (15 L air/min; conditioned dry air; duration of exposure = 1 x 4h; 5 males and 5 females per group).

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights were measured before exposure, on Days 3 and 7, and weekly thereafter. Individual weights are also recorded at death, if applicable. The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs). Following exposure, observations were made and recorded systematically; individual records were maintained for each animal. Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucus membranes, respiratory, circulatory, autonomic and central nervous system, and 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. Since these signs can only be assessed adequately from freely moving animals, no specific assessment was performed during exposure while animals were restrained. Each rat was first observed in its home cage and then individually examined. The following reflexes were tested, based on recommendations made by Irwin (1968): visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinna! reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back). rectal temperatures were measured shortly after cessation of exposure (approximately within 30 minutes after the end of exposure) using a digital thermometer with a rectal probe for rats.
- Necropsy of survivors performed: yes

Statistics:

Particle size characteristics and respirable mass fraction were determined by calculation of MMAD and GSD employing probit analysis and linear regression as necessary. Means and single standard deviations of body weights are calculated. Body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA was used. Rectal temperature measurements were statistically evaluated using the ANOVA procedure.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 513 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: highest technically achievable concentration

Mortality:

There were no mortalities during the study.

Clinical signs:

other: Nonspecific clinical signs, piloerection (2/5) and ungroomed hair coat (3/5) were transiently observed in cyprosulfamide-treated female rats for 1 - 2 days.

Body weight:

There were no treatment-related differences in bodyweight gain between treated animals and controls.

Gross pathology:

No observable findings were made for both controls and treated animals including the respiratory tract.

Other findings:

Reflex measurements:
A battery of reflex measurements was made on the first post-exposure day. In comparison to the rats of the control group, none of the rats in the treated group exhibited changes in the reflex behavior.

Rectal temperature:
Statistical comparisons between the control and the exposure group revealed that there was no toxicologically significant effect on the body temperature (although a statistically significant decreased body temperature was observed).

Table 2: Summary of acute inhalation toxicity - 4 hour exposure to aerosolized testsubstance (powder)

Test Group	Measured concentration [mg/m^3]	Toxicological Result	Onset and Duration ofSigns	Onset of Mortality	Rectal Temperature (°C)
Male
Control		0 / 0 / 5	-	-	37.9
Test Substance		0 / 0 / 5	-	-	37.3*
Female
Control		0 / 0 / 5	-	-	38.5
Test Substance		0 / 3 / 5	1d – 2d	-	37.5**

* = p < 0.05, ** = p < 0.01

Toxicological Result:

First Number = Number of dead animals

Second Number = Number of animals with signs after cessation of exposure

Third Number = Number of animals exposed

Interpretation of results:

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

Conclusions:

The study was performed in accordance to OECD TG 403 under GLP conditions and is considered reliable. The LC50 was determined to be > 3513 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 3 513 mg/m³ air

Physical form:

inhalation: aerosol

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Feb - 17 Feb 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted 12 May 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

Version / remarks:

adopted 9 Oct 2017

Deviations:

not applicable

Remarks:

The study was correctly conducted in accordance with an old version of the guideline as a standard acute method. The current requirements of the Fixed Dose Procedure in the current guideline version do not apply.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Wistar HsdCpb:Wu

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan/Winkelmann GmbH, Borchen, Germany.
- Age at study initiation: Approx. 9 -13 weeks
- Body Weight at study initiation: 245 - 281 g for males and 205 - 223 g for females.
- Housing: Caged individually in polycarbonate cages on low dust wood granulate bedding.
- Diet: Standard diet for rats and mice "Provimi Kliba 3883.0.15 Maus/Ratte Haltung, Kaiseraugst Switzerland", ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 2005-02-03 to 2005-02-17

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: Back and flanks.
- % coverage: Approx. 10% (6.0 cm x 5.0 cm = 30.0 cm²)
- Type of wrap if used: Test material was placed on a wet gauze-layer of a "Cutiplast® steril" coated with air-tight "Leukoflex®" gauze strip which was placed on the rat's back and secured in place using "Peha®-Haft" cohesive stretch tape (8 cm x 23 cm) and additionally covered with a "Lomir biomedical Inc rat jacket" and connnected with a safety pin to the stretch tape.

REMOVAL OF TEST SUBSTANCE
- Washing: Rinsed with tepid water using soap and gently patting the area dry.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied: 2000 mg/kg bw; 21.8 - 25 mg/cm² for males and 18.2 - 19.8 mg/cm² for females.
- Constant volume or concentration used: yes
- For solids, paste formed: No, test substance was applied via a wet gauze layer.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical signs and mortality rates were determined several times on the day of application and subsequently at least once daily thereafter. Body weight and bodyweight gain were checked weekly until the end of the study.
- Necropsy of survivors performed: Yes

Statistics:

Means and standard deviations were calculated for bodyweights
The LD50 value was calculated with the aid of a software program according to Spearman, Karber (D. J. Finney; Statistical method in biological assay, 2nd Edition, Griffin, London, 524-530; 1971). The algorithm was taken from L. Sachs (Angewandte Statistik, 6th Edition 1984, pp. 178 ff.).
Where calculation of the LD50 using the software program was not possible or meaningful, an assessment was made based on the applied dose and dose-response curve, respectively.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred during the study.

Clinical signs:

other: Clinical signs of toxicity were not observed during the 14-day observation period.

Gross pathology:

The necropsies performed at the end of the study revealed no specific findings.

Interpretation of results:

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

Conclusions:

The study was performed in accordance to OECD TG 402 under GLP conditions and is considered reliable. The LD50 was determined to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

The acute oral toxicity study was performed in accordance with OECD 423 (M-249800-01-1, 2005). The substance formulated in tap water with 2% Cremophor EL was administered by gavage to six female Wistar HsdCpb:Wu rats at a dose of 2000 mg/kg bw. There were no deaths or signs of toxicity during the study. Gross examination of organs and tissues at necropsy did not reveal any abnormalities. According to OECD 423, the LD50 cut-off value of the test substance was ≥ 5000 mg/kg bw.

 

According to OECD 402, 2000 mg/kg bw of the test substance was dermally applied to the intact skin of five male and five female Wistar HsdCpb:Wu rats (M-247221-01-1, 2005). The test substance was placed on a wet gauze patch and applied to the skin for 24 h under semi-occlusive conditions. After the exposure period, the gauze patch was removed and the treated skin was rinsed and dried. There were no deaths or signs of toxicity during the study. Gross examination of organs and tissues at necropsy did not reveal any abnormalities. The LD50 of the test substance was > 2000 mg/kg bw.

 

The acute inhalation toxicity of the test substance on five male and five female Wistar Hsd Cpb:WU (SPF) rats was conducted in accordance with OECD 403 (M-076020-02-1, 2004). The rats were nose-only exposed to a mean solid aerosol concentration (powder) of 3513 mg/m³. The aerosol generated was respirable to the rats (MMAD 3.2 µm, GSD 1.8). The maximum achievable concentration of 3513 mg/m³ did not cause mortality. Non-specific clinical signs were observed in some females without indication of a treatment-relationship. There were no effects on body weight development. Gross examination at necropsy did not reveal any abnormalities. The LC50 of the test substance was > 3513 mg/m³.

Justification for classification or non-classification

The available data on acute toxicity (oral, inhalation and dermal) of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.