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Description of key information

There is a reliable acute oral LD50 value in mice, which are among the most sensitive species tested, of 617 mg/kg.  There is a reliable 4-hr inhalation LC50 value in mice of 6022 mg/m3.  Reliable studies show an acute 24-hour dermal LD50 value in rabbits of >2000 mg/kg.

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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD-1 (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI.
- Age at study initiation: 6-8 weeks of age.
- Weight at study initiation: 24-33g (m); 19-28g (f)
- Housing: The animals were housed 3-4 per cage for the first few days of the acclimation period in order to become accustomed to the automatic watering system, then were housed individually in stainless-steel cages.
- Diet: Certified Rodent Chow® #5002, PMI Feeds, Inc., St. Louis, Missouri was available ad libitum, except during designated fasting periods (3-4 hours prior to dosing and 1-2 hours after dosing).
- Water: Water was available ad libitum.
- Acclimation period: 8-19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 66-77°F
- Humidity: 34-73%
- Photoperiod: 12 hours flourscent light
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
- Rationale for the selection of the starting dose: The 2000 mg/kg dose is specified by regulatory agencies for the limit test. The other doses were selected to produce partial mortalities in order to calculate an LD50.
Doses:
Single dose 300, 500, 650, 900, 1200, 2000 mg/kg.
No. of animals per sex per dose:
6 groups of 5 males and 5 females each.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed approximately 1, 2, and 4 hours after dosing on the day of test article administration (study day 1). They were then observed twice daily (morning and afternoon) for 13 additional days and once on the day of necropsy. Individual body weights were obtained just prior to test article administration, on study day 8, at study termination (study day 15), or when an animal was found dead.
- Necropsy of survivors performed: yes, A gross necropsy was performed on all animals dying on study as well as those surviving until study termination (study day 15). For animals surviving until study termination, euthanasia was by carbon dioxide inhalation followed by exsanguination from the abdominal aorta. External abnormalities including palpable masses were examined. Subcutaneous masses were identified and correlated with antemortem findings. Organs were removed and examined and the tissues and carcasses discarded.
Statistics:
The median lethal dose (LD50) and its 95% confidence limits were calculated by the method of Bliss, CI (1938), The determination of the dosage-mortality curve from small numbers. Quarterly Journal of Pharmacy and Pharmacology, 11: 192-216.
Sex:
male
Dose descriptor:
LD50
Effect level:
469 mg/kg bw
95% CL:
>= 163 - <= 699
Sex:
female
Dose descriptor:
LD50
Effect level:
765 mg/kg bw
95% CL:
>= 539 - <= 1 104
Sex:
male/female
Dose descriptor:
LD50
Effect level:
617 mg/kg bw
95% CL:
>= 450 - <= 787
Mortality:
Test article-related combined sex mortalities were 10, 30, 60, 80, 90, and 90% for dose levels 300, 500, 650, 900, 1200, and 2000 mg/kg of Acetonitrile (HPLC Grade), respectively. With the exception of the 650 mg/kg group, mortalities were approximately equal for both sexes. No mortalities occurred after study day 2 for any group.
Clinical signs:
Significant clinical signs observed during the study included death, tremors, prostration, decreased activity, impaired righting reflex, labored breathing, convulsions, gasping, and increased salivation. All surviving animals were judged normal by study day 4, with the exception of a single 300 mg/kg group animal that exhibited increased salivation on study day 8.
Body weight:
Body weight gains for survivors were similar for both sexes. The 300 and 500 mg/kg groups were the only groups with survivors from both sexes. Body weight gains were 2-3 grams for each sex.

Gross pathology:
At necropsy, there were no test article-related findings in any animal.
Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, the oral LD50 of Acetonitrile (HPLC Grade) was calculated to be 617 mg/kg for male and female mice combined (with 95% confidence limits of 450-787 mg/kg).
Executive summary:

In a guideline (OECD 401 equivalent) and GLP study by MPI Research (1998), the acute oral LD50 of Acetonitrile (HPLC Grade) was calculated to be 617 mg/kg for male and female mice combined (with 95% confidence limits of 450-787 mg/kg). Significant clinical signs observed during the study included death, tremors, prostration, decreased activity, impaired righting reflex, labored breathing, convulsions, gasping, and increased salivation. All surviving animals were judged normal by study day 4, with the exception of a single 300 mg/kg group animal that exhibited increased salivation on study day 8.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
617 mg/kg bw
Quality of whole database:
A guideline-comparable study in the mouse is supported by published and unpublished data in various species

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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD-1® (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
Individually identified by unique ear tag.

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 27-37g (m); 21-27g (f)
- Housing: Animals were individually housed in stainless-steel wire mesh cages.
- Diet: Certified Rodent Chow #5002, PMI Feeds Inc., St. Louis, Missouri available ad libitum, except during the exposures.
- Water: Available ad libitum, except during the exposures.
- Acclimation period: 7 - 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 64-74°C
- Humidity: 37-57%
- Photoperiod: 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Test article was delivered from a Fluid Metering Incorporated (FMI) pump, equipped with a 1/8 inch pump head through 1/8 inch Teflon tubing at a constant rate to a glass chromatography column containing glass beads. Compressed air, metered by a flowmeter at approximately 0.21-0.32 (Group 1), 0.40 (Group 2), 0.32(Group 3) or 0.28 (Group 4) mL/min flowed counter current, vaporizing the test article. The test article vapor passed through a condensation trap prior to being introduced into the chamber through a turret inlet.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass whole-body exposure chamber
- Exposure chamber volume: 160 liter
- Source and rate of air: Minimum chamber air flow rate of 32 L/min resulting in 12 air changes/hour.
- Temperature, humidity, pressure in air chamber: The chamber was maintained to the maximum extent possible at a temperature between 20 to 24degrees C and a relative humidity between 40 to 60%. Chamber temperature, percent relative humidity, and air flow rate were monitored continuously and recorded at 30 minute interval during the exposure period.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: A single particle size distribution was performed once during each exposure using a cascade impactor to determine the mass median aerodynamic diameter and geometric standard deviation of any aerosol present. After completion of the last animal exposure, the presence of any aerosols was re-evaluated using a Sibata, Model P-5H, light scattering digital dust indicator at the high concentration level (5000 ppm). The P-5H was placed in line, downstream from the generation system, between the aerosol trap and chamber inlet (refer to Figure 2). In this design, all of the generated test atmosphere passed through the P-5H before entering the exposure chamber. Output from the P-5H was recorded with a strip chart recorder. Light scattering readings were recorded during a 20 minute control period, a 30 minute test period, and a 20 minute recovery period.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
continuously by infrared spectrometry
Duration of exposure:
4 h
Concentrations:
3202, 5499, 4653, 3747 ppm (nominal); 3039, 5000, 4218, 3568 ppm (analytical).
No. of animals per sex per dose:
4 groups of 5 males and 5 females.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days during which all animals were observed once daily for clinical signs. The examination included, but was not limited to, observations of the general condition, skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration and palpation (post-exposure) of tissue masses.
- Necropsy of survivors performed: yes, Euthanasia of animals surviving to study termination was by intraperitoneal injection of sodium pentobarbital anesthesia. The trachea was exposed ad clamped such that the lungs were removed and examined in an inflated state. All major organ systems in the thoracic and abdominal cavities were observed for gross abnormalities by a veterinary pathologist. No tissues were preserved.
- Other examinations performed: All animals were observed at least twice a day for morbidity, mortality, injury, and availability of food and water. The use of a computerized data collection system (Xybion) required observations and body weights to be defined in such a manner that day 1 was the day of exposure and days 2-15 were post-exposure days 1-14. Body weights were recorded just prior to the exposure, on days 8 and 15 (post-exposure days 7 and 14), and when an animal was found dead.
Statistics:
The concentration mortality data were statistically analyzed for the LC50 and its confidence limits by the method of Bliss, CI (1938), The determination of the dosage-mortality curve from small numbers. Quarterly Journal of Pharmacy and Pharmacology, 11: 192-216.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
3 587 ppm
95% CL:
> 2 938 - < 4 039
Exp. duration:
4 h
Mortality:
Combined sex mortalities were 20, 80, 90, and 50% for Groups 1- 4, respectively. Male mortality was slightly greater than the female mortality in each exposure group. All mortalities occurred on the day of exposure, except for a single Group 1 male that died on post-exposure day 1 (study day 2).
Clinical signs:
other: Clinical signs observed during the exposure and up to 4 hours post-exposure included death, decreased activity, abnormal gait, loss of righting reflex, slow respiration, labored breathing, rapid respiration, gasping, cold to touch, limbs splayed, leaning
Body weight:
Surviving animal body weights from Groups 1 and 3, and the Group 4 males remained at pre-exposure levels during the post-exposure observation period. Of the 2 surviving females from Group 2, 1 gained weight and 1 remained at the pre-exposure level during the first post-exposure week, but both lost weight (1 or 2 grams) during the second post-exposure week. Three of the surviving 4 Group 4 females gained weight during the post-exposure period. The remaining Group 4 female lost weight (1 gram) during the first post-exposure week and regained its pre-exposure weight by the end of the second post-exposure week.
Gross pathology:
At necropsy, no test article-related macroscopic findings were observed in male or female mice.
Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, the 4-hour LC50 of Acetonitrile (HPLC Grade) in mice (via whole-body exposure) was calculated to be 3587 ppm, with 95% confidence limits of 2938-4039 ppm.

Executive summary:

In a guideline (OECD 403 equivalent) and GLP study, the 4-hour vapor LC50 of Acetonitrile (HPLC Grade) in mice (via whole-body exposure) was calculated to be 3587 ppm (6022 mg/m3), with 95% confidence limits of 2938-4039 ppm (4933 - 6781 mg/m3). Clinical signs observed during the exposure and up to 4 hours post-exposure included death, decreased activity, abnormal gait, loss of righting reflex, slow respiration, labored breathing, rapid respiration, gasping, cold to touch, limbs splayed, leaning to the right, and yellow body surface staining. Surviving animals from Groups 2-4 (5000, 4218, and 3568 ppm) were judged normal by study day 2. Clinical signs observed during the 14-day observation period for Group 1 (3039 ppm) included death, decreased activity, and decreased defecation. Group 1 survivors were judged normal by study day 5.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
6 022 mg/m³
Quality of whole database:
A guideline-comparable study in the mouse is supported by published and unpublished data in various species

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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. Kalamazoo, MI.
- Age at study initiation: 3.5 months of age.
- Weight at study initiation: 2721 - 3035g (m); 2477 - 2884g (f)
- Housing: Individually in stainless-steel cages.
- Diet: Certified Rabbit Chow® # 5322 ad libitum
- Water: Ad libitum
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 66 - 70°F
- Humidity: 46 - 80%
- Photoperiod: 12 hours a day light

IN-LIFE DATES: From: August 11, 1997 To: September 18, 1997
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: The test article was applied to the shaven intact skin on the back of each rabbit.
- % coverage: approximately 15% of body surface.
- Type of wrap if used: The trunk of each rabbit was wrapped with gauze bandaging and secured with Dermiform® tape.

REMOVAL OF TEST SUBSTANCE
- Following the exposure period, the bandaging materials and collars were removed and the test sites wiped with disposable toweling moistened with water to remove any residual material.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dosage volume was 2.6 mL/kg based on a test article density of 0.777 g/mL.
Duration of exposure:
24 hours.
Doses:
Single 2000 mg/kg dose.
No. of animals per sex per dose:
5 males and 5 females.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: yes. A gross necropsy was performed on all animals at study termination (study day 15). Euthanasia was by an overdose injection of sodium pentobarbital.
Statistics:
No data were analyzed statistically.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
All animals survived to study termination.
Clinical signs:
No signs of toxicity or ill health were observed in the males during the conduct of this study. With the exception of decreased defection observed in 3 females for 1 day during the 14-day observation period ( which was described as a probable test article-related change), no other signs of ill health or toxicity were observed in the females during the study.
Body weight:
Mean group body weights for each sex increased at each observation interval. All animals gained weight at each observation interval with the exceptions of 1 male that lost 23 grams at study day 8 and another that lost 2 grams at study termination.
Gross pathology:
At necropsy, no visible abnormalities were observed at the application site or in other tissues.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, the acute dermal LD50 value of Acetonitrile (HPLC Grade) is greater than 2000 mg/kg for male and female rabbits.
Executive summary:

In a guideline (OECD 402 equivalent) and GLP study by MPI Research (1998), the acute dermal LDLo (24 -hour exposure) of Acetonitrile (HPLC Grade) was found to be greater than 2000 mg/kg for male and female rabbits. No signs of toxicity or ill health were observed in the males during the conduct of this study. With the exception of decreased defection observed in 3 females for 1 day during the 14-day observation period (which was described as a probable test article-related change), no other signs of ill health or toxicity were observed in the females during the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
A guideline-comparable study in the rabbit is supported by other rabbit studies.

Additional information

Acute oral toxicity

The range of acute oral LD50 values reported for acetonitrile in experimental animals is between 139-6690 mg/kg bwt. The mouse and guinea pig seem to be the most sensitive species. A study showed that acetonitrile was more toxic to immature rats (14-day-old) than to older rats given oral doses of 160-3500 mg/kg bw. Another study reported that using male or female Wistar or Nelson albino rats, the males were more susceptible than the females; LD50 values of 6762 mg/kg are reported for females and 1327 mg/kg for males. In a reliable study conducted in mice, the oral LD50 of acetonitrile was calculated to be 617 mg/kg bw. The main signs of toxicity in animals appear to be prostration followed by seizures and convulsions. Animals exposed to acetonitrile via different routes of dosing always showed respiratory symptoms: rapid and irregular respiration, laboured or difficult breathing and intense dyspnea. Co-administration of sodium thiosulphate or sodium nitrite has been shown to reduce acetonitrile mortality.

Acute dermal toxicity

A 24-hour LD50 value of 2000 mg/kg bw was obtained in a reliable acute dermal toxicity guideline study in rabbits (MPI, 1997). Early studies reported similar as well as significantly lower values, but the purity of the test material and the details of the methods used are not clear. For these reasons the recent well-conducted guideline study is considered to be the most reliable result available for acetonitrile acute dermal toxicity.

Acute inhalation toxicity

The acute inhalation toxicity of acetonitrile vapour varies across experimental species. 4 -hour inhalation LC50 values ranging from 26.9 – 33.5 mg/L have been reported in the rat. Comparative studies showed the dog to be least sensitive, with rats, guinea pigs, and rabbits following in decreasing order. In a reliable study an LC50of 3587 ppm (6022 mg/m3) was obtained in mice. As in the case of oral exposure, co-administration of inorganic cyanide antagonists sodium thiosulphate and sodium nitrite has been shown to reduce acetonitrile mortality.

Acute median lethal doses of acetonitrile by the intraperitoneal and intravenous routes have also been reported in the literature. These are listed in the table below.

Summary of Acute Toxicity Data for Acetonitrile in Experimental Animals

Route

Species

LD50/ LC50

Reference

oral

Mouse CD-1

617 mg/kg

MPI Research 1998

oral

Mouse ddY

269 mg/kg

Tanii and Hashimoto (1984)

oral

Rat (SD) (14-day old)

158 mg/kg

Kimura et al, 1971

oral

Rat (SD) (Young adult)

3081 mg/kg

Kimura et al, 1971

oral

Rat (SD) (Older adult)

3476 mg/kg

Kimura et al, 1971

oral

Rat

6500 mg/kg

TSCATS / DuPont, 1968

oral

Rat (Wistar)

>200 mg/kg

<2000 mg/kg

BASF AG, 1989

oral

Rat (Wistar-Nelson)

1320-6690 mg/kg

Pozzani et al, 1959

oral

Rat

3800 mg/kg

Smyth and Carpenter, 1948

oral

Guinea pig

139 mg/kg

Pozzani et al, 1959

Inhalation

Mouse CD-1

6022 mg/m3(4h)

MPI Research 1998

Inhalation

Mouse CD-1

4521 mg/m3(1h)

Willhite, 1981

Inhalation

Rat

33,495 mg/m3(4h)

TSCATS/ Monsanto, 1979

Inhalation

Rat

28,710 mg/m3(4h)

TSCATS / DuPont, 1968

Inhalation

Rat Nelson

26863 mg/m3(4h)

Pozzani 1959

Inhalation

Rat Nelson

12678– 20878 mg/m3(8h)

Pozzani 1959

Inhalation

Rabbit

4748 mg/m3 (4h)

Pozzani 1959

Inhalation

Guinea pig

9494 mg/m3(4h)

Pozzani 1959

Dermal

Rabbit

>2000 mg/kg (24h)

MPI Research 1998

Dermal

Rabbit

980 mg/kg undiluted

Pozzani et al, 1959

Dermal

Rabbit

392 mg/kg aqueous solution

Pozzani et al, 1959

Dermal

Rabbit

3750 mg/kg (4d)

Smyth and Carpenter 1948

i.p.

Rat (Wistar or Nelson)

666-6240 mg/kg undiluted

Pozzani et al, 1959

i.p.

Rat (Wistar or Nelson)

3890 -5620 mg/kg saline

Pozzani et al, 1959

i.p.

Mouse (CD-1)

175 mg/kg

Willhite of Smith (1981)

i.p.

Mouse

250 mg/kg

Pozzani et al, 1959

i.p.

Mouse (NMRI)

400 mg/kg

Zeller et al, 1969

i.p.

Mouse

521 mg/kg

Yoshikawa (1968)

i.v.

Rat-Wistar or Nelson

1320 mg/kg

Pozzani et al, 1959



Justification for selection of acute toxicity – oral endpoint
Guideline-comparable study

Justification for selection of acute toxicity – inhalation endpoint
Guideline-comparable study

Justification for selection of acute toxicity – dermal endpoint
Guideline-comparable study

Justification for classification or non-classification

Acetonitrile has a harmonised classification according to the CLP Regulation as Acute Tox. 4 (H302, H312, H332). The available studies do not contradict this classification; no change is proposed.