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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a GLP facility to OECD guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
no
GLP compliance:
yes
Test type:
up-and-down procedure
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study and identified by a number written on a cage card. At the start of the study the animals were
eight to twelve weeks of age. The bodyweights fell within an interval of ±20% of the mean weight of any previously dosed animals.
The animals were individually housed in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the
study. The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
For the purpose of the 1110 mg/kg dose level, the test item was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. For the purpose of the 35, 111 and 350 mg/kg dose levels, the test item was freshly prepared, as required, as a solution at the appropriate concentration in distilled water. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration. All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time (at least 48 hours) was allowed between each individual animal to confirm the outcome of the previously dosed animals.
Doses:
Using information available on the test item an estimate of the LD50 value was made. This value (111 mg/kg) was entered into the AOT425 Statistical Program with the slope of the dose-response curve set to the default value (sigma = 0.5). The statistical program gave a recommended dose progression of 1.11, 3.5, 11.1, 35, 111, 350, 1110 and 2000 mg/kg.
No. of animals per sex per dose:
1 Animal per dose
Control animals:
no
Details on study design:




Animal Dose Level
number mg/kg
Dose vol

1
35 0
2
111 0
3
350 0
4
1110 X*
5
350 X
6
111 0
7
350 0
8
1110 X
9
350 X
10
111 0

X* = animal humanely killed, X= animal died, 0= Animal survived
Statistics:
The oral LD50 was calculated by the maximum likelihood method. Data evaluations also included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects. Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was calculated by the statistical program
Sex:
female
Dose descriptor:
approximate LD50
Effect level:
350 mg/kg bw
95% CL:
174.2 - 885
Mortality:
2/2 mortalities at 1110 mg/kg, 2/3 at 350 mg/kg, 0/3 at 111 mg/kg and 0/1 at 35 mg/kg
Clinical signs:
Signs of systemic toxicity noted were hunched posture, lethargy, ataxia, pilo-erection, ptosis, occasional body tremors, pallor of the extremities abdominal discomfort, increased salivation and splayed gait. Additional signs of systemic toxicity noted in the animal treated at a dose level of 1110 mg/kg that was humanely killed were prostration, emaciation, hypothermia, laboured respiration and decreased respiratory rate. There were no signs of systemic toxicity noted in animals treated at dose levels of 35 and 111 mg/kg or one animal treated at a dose level of 350 mg/kg.
Body weight:
Surviving animals showed expected gains in bodyweight over the study period, except for one animal treated at a dose level of 111 mg/kg which showed expected gain in bodyweight during the first week but slight bodyweight loss during the second week.
Gross pathology:
Abnormalities noted at necropsy of animals that died or were humanely killed during the study were patchy pallor or dark liver, dark or pale kidneys and haemorrhage and epithelial sloughing of the gastric mucosa. No abnormalities were noted at necropsy of animals that were killed at the end of the study.
Interpretation of results:
toxic
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The acute oral median lethal dose (LD50) and 95% confidence limits of the test item in the female Wistar strain rat were calculated to be 350 (174.2 – 885.0) mg/kg bodyweight (based on an assumed sigma of 0.5).
Executive summary:

A total of ten female animals were dosed individually in sequence with sufficient time (at least 48 hours) between each animal, at dose levels ranging from 35 mg/kg bodyweight to 1110 mg/kg bodyweight. The test item was administered orally undiluted at a concentration of 1110 mg/kg or as a solution in distilled water at concentrations of 35, 111 or 350 mg/kg. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. One day after dosing two animals treated at a dose level of 350 mg/kg and both animals treated at a dose level of 1110 mg/kg were found dead or killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted were hunched posture, lethargy, ataxia, pilo-erection, ptosis, occasional body tremors, pallor of the extremities abdominal discomfort, increased salivation and splayed gait. Additional signs of systemic toxicity noted in the animal treated at a dose level of 1110 mg/kg, that was humanely killed were prostration, emaciation, hypothermia, laboured respiration and decreased respiratory rate. There were no signs of systemic toxicity noted in animals treated at dose levels of 35 and 111 mg/kg or one animal treated at a dose level of 350 mg/kg. Surviving animals showed expected gains in bodyweight over the study period, except for one animal treated at a dose level of 111 mg/kg which showed expected gain in bodyweight during the first week but slight bodyweight loss during the second week. Abnormalities noted at necropsy of animals that died or were humanely killed during the study were patchy pallor or dark liver, dark or pale kidneys and haemorrhage and epithelial sloughing of the gastric mucosa. No abnormalities were noted at necropsy of animals that were killed at the end of the study. The acute oral median lethal dose (LD50) and 95% confidence limits of the test item in the female Wistar strain rat were calculated to be 350 (174.2 – 885.0) mg/kg bodyweight (based on an assumed sigma of 0.5).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
300 mg/kg bw
Quality of whole database:
Oral toxicity data consists of two modern acute oral toxicity tests and two tests in rodents that were conducted in a reputable laboratory to the standards of the time. The second modern study is in finalization and will be added via update in early June 2013. The results of this test are identical to those of the key study.

Acute toxicity: via inhalation route

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Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted under GLP. However, as this was in the early days of GLP, the study report is lacking in detail. We have confirmed with the manager of the testing laboratory that during this time period, all work was conducted to the GLP standards of the day, even though the study reports do not specifically call it out.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes
Test type:
other:
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female rats (CRL:CD8(SD)BR) were obtained from Charles River Laboratories, Inc., Wilmington, MA. Rats were chosen for this study
because they are a common representative species for toxicity studies. Animals were received on September 10, 1986, and were isolated for five days. On September 15, 1986 the animals were released in good health from isolation. Twenty males and twenty females approximately seven and eight weeks old respectively, were divided into four equal groups of five male and five females each, weighing 182 to 217g (males) and 171 to 194g (females) at the start of the study (Day 0). Housing Temperature and relative humidity were 70-77°F and 47-58%, respectively, during the isolation period. Animals were group housed in stainless steel suspended cages during the isolation period, but subsequently singly housed in multicompartmented stainless steel mesh cages designed for inhalation studies. Males and females in each group were housed on the same rack. A 12 hour (6 a.m. to 6 p.m.) light/dark cycle was maintained. Cages and racks were washed once per week. Absorbent paper under the cages was changed three times per week. Feed and Water Certified feed [Agway Prolab Animal Diet (RKH 3000, pellets)] was available ad libitium during non-exposure periods. Water (Monroe County Water Authority) was available ad libitium during nonexposure periods through an automatic watering system. No known contaminants in feed or water were expected to be present which would interfere with the outcome of this study.


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposures were to target concentrations of 2000, 4000, 6000, or 8000 ppm and were conducted in a 420 L stainless steel and glass inhalation chamber. Animals were exposed for one hour and then observed for 14 days. For each exposure concentration, males and females were exposed in the same inhalation chamber. The exposures were run sequentially in the same chamber on the same day, except for the 2000 ppm group which was added to the experiment on the third day in order to better define the LC10 level. The chamber was maintained under negative pressure (0.5" water) and at 12 air changes per hour. Vapors were generated by metering the test material dropwise into a heated glass bead-packed column supplied with metered dried oil-free compressed air from the Kodak Park Utilities.. Chamber vapor concentrations were determined four or five times per hour by a HIIA infrared analyzer equipped for automated sampling and analysis. In addition, temperature and humidity were determined twice per hour and nominal vapor concentration for the exposure was calculated. Once during exposure, concentration of background nongaseous material was measured in the 6000 ppm chamber relative to that of a chamber containing air in order to insure that exposures were to vapor and not aerosol.

Distribution of test material was determined by measurement from ten positions throughout the chamber and was compared to a fixed reference position. Subsequently, determination of chamber vapor concentration as done remotely from the fixed reference position.

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
1 h
Concentrations:
2000, 4000, 6000 or 8000 ppm
No. of animals per sex per dose:
5 per sex per dose
Control animals:
no
Details on study design:
Rats were exposed to the test article via whole body inhalation for 1 hour, followed by a 14-day observation period.
Statistics:
Mortality data were evaluated by probit analysis. The LC10 value and its 95% confidence limits were
estimated by probit analysis using a computer generated statistical analysis software package
(Version 5, 1985, SAS Institute Inc., Cary, NC).
Sex:
male
Dose descriptor:
other: LC10
Effect level:
> 2 000 - < 4 000 ppm
Exp. duration:
1 h
Sex:
female
Dose descriptor:
other: LC10
Effect level:
> 4 000 - < 6 000 ppm
Exp. duration:
1 h
Mortality:
1/5 males and 0/5 females at 2000 ppm
5/5 males and 2/5 females at 4000 ppm
5/5 males and 4/5 females and 6000 and 8000 ppm
Clinical signs:
other: .All males and females exposed to 4000, 6000, and 8000 ppm exhibited minor to severe central nervous system depression in a concentration-dependent manner during the exposure, while the 2000 ppm groups had no clinical signs of toxicity during the exposure
Body weight:
All males exposed to 4000, 6000, and 8000 ppm died within 24 hours of exposure. The four males in
the 2000 ppm group which survived the exposure exhibited sustained weight gain. Four females
exposed to 8000 ppm, four females exposed to 6000 ppm, and two females exposed to 4000 ppm died
within 24 hours after exposure. No mortality was seen in females exposed to 2000 ppm. All survivors
exhibited comparable weight female gains.
Gross pathology:
No treatment-related changes were observed on gross examination. No tissue was collected for microscopy. No cause of death was determined.
Interpretation of results:
toxic
Remarks:
Migrated information Criteria used for interpretation of results: US CPSC / US OSHA
Conclusions:
The LC10 value for males and females combined was 1848 ppm with a 95% confidence interval of 695 to 2656 ppm.
Executive summary:

Exposure of rats to vapor concentrations of 2000, 4000, 6000, or 8000 ppm of NBN for one hour resulted in acute central nervous system (CBS) depression and death. The severity of the CNS depression and the incidence of mortality were concentration-dependent. Mortality was greater among males than females. Survivors exhibited minimal signs of toxicity which resolved within 24 hours of exposure. No treatment-related changes were observed upon gross examination of animals found dead, or of those examined at the end of the study. No cause of death was identified in animals found dead. A no-observed-effect-level was not determined. The LC10 value for males and females combined was 1848 ppm with a 95% confidence interval of 695 to 2656 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
5 223 mg/m³
Quality of whole database:
Acute inhalation toxicity testing was done in the 1980's in a GLP facility to curent guidelines. The testing conducted consisted of a single concentration limit test followed by a full inhalation study.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted at a GLP facility under OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Five male and five female Wistar (RccHan™:WIST) strain rats were supplied by Harlan
Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to
cages. The females were nulliparous and non-pregnant. After an acclimatisation period
of at least five days the animals were selected at random and given a number unique
within the study by indelible ink-marking on the tail and a number written on a cage card.
At the start of the study the animals weighed at least 200 g, and were eight to twelve
weeks of age.
The animals were housed in suspended solid-floor polypropylene cages furnished with
woodflakes. The animals were housed individually during the 24-Hour exposure period
and in groups of up to four, by sex, for the remainder of the study. Free access to mains
drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan
Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking
water and bedding were routinely analysed and were considered not to contain any
contaminants that could reasonably be expected to affect the purpose or integrity of the
study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to
70% respectively. Any occasional deviations from these targets were considered not to
have affected the purpose or integrity of the study. The rate of air exchange was at least
fifteen changes per hour and the lighting was controlled by a time switch to give twelve
hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered
not to contain any contaminant of a level that might have affected the purpose or integrity
of the study.
Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
For each animal the calculated volume of test item was applied as evenly as possible to
an area of shorn skin (approximately 10% of the total body surface area) using a
graduated syringe. A piece of surgical gauze was placed over the treatment area and
semi-occluded with a piece of self-adhesive bandage. The animals were caged
individually for the 24-Hour exposure period and for the remainder of the test. Shortly
after dosing the dressings were examined to ensure that they were securely in place.
After the 24-Hour contact period the bandage was carefully removed and the treated skin
and surrounding hair wiped with cotton wool moistened with distilled water to remove any
residual test item.
Duration of exposure:
24 hours
Doses:
500, 1000 or 2000 mg/kg
No. of animals per sex per dose:
1 for the rangefinding study and 4 for the limit test.
Control animals:
no
Details on study design:
On the day before treatment the back and flanks of each animal were clipped free of hair. Using available information on the toxicity of the test item, a single group of animals was initially treated as follows:

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (ml/kg) Male/Female
======================================
500 50 10 1/1

In the absence of mortality at a dose level of 500 mg/kg, an additional group of animals was
treated as follows:

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (ml/kg) Male/Female
======================================
1000 0.778 1.27 1/1


In the absence of mortality at a dose level of 1000 mg/kg, an additional group of animals
was treated as follows:

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (ml/kg) Male/Female
======================================
2000 0.778 2.54 1/1

In the absence of mortality at a dose level of 2000 mg/kg, an additional group of animals
was treated as follows:

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (ml/kg) Male/Female
======================================
2000 0.778 2.54 1/1


A total of ten animals were therefore treated at a dose level of 2000 mg/kg in the study.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
None
Clinical signs:
Red/brown staining around the eyes or snout was noted in the male treated at a dose
level of 1000 mg/kg and three animals treated at a dose level of 2000 mg/kg. There
were no signs of systemic toxicity noted in the remaining animals
Body weight:
One animal treated at a dose level of 1000 mg/kg and five animals treated at a dose
level of 2000 mg/kg showed bodyweight loss during the first week but expected gain in
bodyweight during the second week. The remaining animals showed expected gains in
bodyweight over the study period.
Gross pathology:
No abnormalities were noted at necropsy.
Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The acute dermal median lethal dose (LDso) of the test item in the Wistar strain rat was
found to be greater than 2000 mg/kg bodyweight
Executive summary:

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat. The method was designed to be compatible with the following: OECD Guidelines for the Testing of Chemicals No. 402 "Acute Dermal Toxicity" (adopted 24 February 1987) ; Method 83 Acute Toxicity (Dermal) of Commission Regulation (EC) No. 440/2008. Initially, three groups, each of two animals (one male and one female), were given single, 24 hour, semi-occluded dermal applications of the test item to intact skin at dose levels of 500, 1000 or 2000 mg/kg bodyweight. Based on the results of the initial test a further group of eight animals (four males and four females) was similarly treated with the undiluted test item at a dose level of 2000 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths. Red/brown staining around the eyes or snout was noted in the male treated at a dose level of 1000 mg/kg and three animals treated at a dose level of 2000 mg/kg. There were no signs of systemic toxicity noted in the remaining animals. Very slight erythema was noted at the test site of the female treated at a dose level of 500 mg/kg. There were no signs of dermal irritation noted in the remaining animals. One animal treated at a dose level of 1000 mg/kg and five animals treated at a dose level of 2000 mg/kg showed bodyweight loss during the first week but expected gain in bodyweight during the second week. The remaining animals showed expected gains in bodyweight over the study period. Necropsy. No abnormalities were noted at necropsy. Conclusion. The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Dermal toxicity data consists of two modern acute dermal toxicity tests and a test from the 1960's that was conducted in a reputable laboratory to the standards of the time. The second modern test is being finalized and will be added by update in early June. The results of this test are identical to those of the key study. The study from the 1960's has a much lower LD50, but the study was poorly documented ( a 1-line report), and there is no data to review. Thus, we have decided to base the dermal LD50 on the pair of modern studies that were conducted at different facilities, and should be the most reliable data.

Additional information

The existing acute oral and dermal data for n-Butyronitrile prior to 2012 was a set of 1960's studies that were poorly documented (one line reports). While the veracity of the data was not in question, there was simply no experimental detail to review. Based on the potential need for Annex IX testing proposals, and the high toxicity shown in these acute studies, a new set of oral and dermal toxicity studies were commissioned to standardize the test methods and results. However, the results of the new studies (Sanders, 2012) indicated LD50 values for oral and dermal toxicity that were much higher (less toxic) than the results from the 1960's. Analysis of the test article retain sample showed that the test article was in specification, but there was no analysis of the dosing solution, so a mistake could have occurred in dosing. Considering that the nitrile functionality indicates the potential for significant toxicity, the studies were repeated in a different testing facility. Results from the follow-up studies were identical to the first set done in 2012 (Sanders), and thus it is unlikely that the results obtained were due to a procedural error. Thus, our belief is that the pair of current acute oral and dermal studies describe the appropriate toxicity of n-butryonitrile, although we can not determine why the testing done in the 1960's indicates a much higher level of toxicity, due to a lack of study documentation. The second set of 2012 acute studies are completed in-life but the draft report has not been issued. IUCLID will be updated when the final report is available.

 


Justification for selection of acute toxicity – oral endpoint
The key study (Sanders, 2012) was done to guidelines and GLP. The 2 studies from the 1960's were done at a reputable lab to the standards of the time, but are one line reports with essentially no details provided. As the results from 2012 showed much less toxicity than those from the 1960's, the 2012 acute oral study was repeated at a second testing laboratory, and identical results were obtained. Note that IUCLID will not allow a range to be entered here, but the estimated LD50 is 50-300 mg/kg. The second study from 2012 is completed in-life and the results are available, but the report is not yet available. IUCLID will be updated when the report is finalized.

Justification for selection of acute toxicity – dermal endpoint
The key study (Sanders, 2012) was done to guidelines and GLP. The study from the 1960's was done at a reputable lab to the standards of the time, but is a one line reports with essentially no details provided. As the results from 2012 showed much less toxicity than those from the 1960's, the 2012 acute dermal toxicity study was repeated at a second testing laboratory, and identical results were obtained. The second study from 2012 is completed in-life and the results are available, but the report is not yet issued. IUCLID will be updated when the report is finalized

Justification for classification or non-classification