Adiponitrile

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 31, 1981 through October 30, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to current guideline study performed under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

The number of animals per group was different, some clinical pathology parameters were not measured, some organs were not preserved for histopathological examination.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Adiponitrile

EC Number:

203-896-3

EC Name:

Adiponitrile

Cas Number:

111-69-3

Molecular formula:

C6H8N2

IUPAC Name:

hexanedinitrile

Details on test material:

As cited in study report: Adiponitrile (Hexanedinitrile, ADN, CN(CH2)4cw, M.W. 108.14, B.P. 2950C at 760 mm Hg, C.P. No. 016191, EHL T810038) was supplied by the sponsor and was identified as Decatur, Alabama Plant Production material Lot No. 40. The sponsor reported the test material was 99% pure and had a specific gravity of 0.958 at 250C. Test material samples were submitted before the study for purity analyses and throughout the study for stability determinations. Stability data are located in the study data package. All test material analyses indicated > 99.5% adiponitrile.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

As cited in study report: One hundred twenty (60 males, 60 females) Sprague-Dawley rats (Crl:COBS® (CD)(SD)BR) were obtained from Charles River Breeding Laboratories, Inc. (Portage, MI). All animals were considered healthy prior to release from a ten-day quarantine. Three days prior to the start of the study, animals to be exposed were 43-48 days of age and weighed 185-217 g. for males and 147-171 g. for females. Each rat was uniquely identified by an eartag and a barcoded cage card. Animals of each sex were randomly assigned via a computer program on the basis of body weight to either treatment or control groups. This randomization precluded significant differences among group mean body weights and variability of individual body weights within groups. Rats were individually housed in stainless steel mesh cages suspended over absorbent paper bedding. Animal rooms were maintained routinely at approximately 72 + 2°F and 35-60% relative humidity with a 12-hour light/12-hour dark cycle each day. Ralston Purina Rodent Chow 5002® was provided ad libitum and water (City of St. Louis tap water) was supplied via an automatic watering system. Both food and water were unavailable during the six-hour exposure intervals.

Administration / exposure

Route of administration:

other: Inhalation: Vapor-Aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: Conditioned Air

Remarks on MMAD:

MMAD / GSD: MMAD 3.36-4.19 microns
GSD Range: 1.70 - 2.08

Details on inhalation exposure:

As cited in study report: Four 10-cubic meter Rochester-style inhalation chambers were used for the exposures. Airflow from the central ventilation supply was maintained at approximately 9.8 to 12.0 chamber volumes per hour. The test material for the mid- and high levels was metered from a tank using a capillary restrictor to a Laskin-style nebulizer which was used to generate the test atmosphere. The test material for the low exposure level was delivered using a Harvard Apparatus Syringe Pump (Harvard Apparatus Company Inc., 150 Dover Road, Millis, Mass. 02054) to the Laskin-style nebulizer. The concentration of test material in the inhalation chamber was controlled either by regulating the pressure in the tank headspace or in the syringe pump, and consequently, the flowrate of test material into the nebulizer. The nebulizer was positioned in the side of a vertical particle-size separator which was connected to the air inlet of the inhalation chamber. One nebulizer was used in each of the generatioh systems. The particle-size separator prevents most of the large, non-respirable aerosol particles from entering the chamber and the large particles collect in the bottom of the separator. The nominal concentration for the exposure was calculated as the net amount of test material (amount delivered to the nebulizer minus amount recovered in the bottom of the separator) entering the air inlet of the inhalation chamber per unit time divided by the total airflow through the chamber per unit time. Chamber airflow and temperature were measured at a minimum of four times each day on each chamber. Relative humidity measurements were routinely recorded on each chamber, a minimum of once per week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As cited in study report: Ten liters of test chamber atmosphere were drawn at approximately two liters/minute through an impinger (Bendix 7202) containing 15 ml of methanol. These samplings were performed four times per exposure day for the first 27 exposure days and at least twice per exposure day thereafter. The concentration of adiponitrie (ADN) in the impinger solution was determined by gas chromatography. Additional atmosphere samples were collected at 9 different locations in each chamber during the study period to demonstrate the uniformity of distribution of the ADN vapor and aerosol. Particle-size of the aerosol was determined for only the high concentration level using a non-viable, nine-stage Andersen impactor (Andersen Samples Inc., 4215-C Wendell Dr., Atlanta, GA 30336). The concentration in the chamber of ADN particles smaller than 10 microns was determined by summing the weights of test material collected on each of the nine stages and dividing it by the total airflow for the time in which the sample was collected. The concentration of ADN particles greater than 10 microns collected in the pre-separator was determined by performing a gas chromatographic analysis on the methanol washes of test material and dividing it by the total airflow. The total concentration of ADN aerosol, not vapor, is represented by the sum of the concentrations of particles smaller and greater than 10 microns.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 12.9, 30.6, and 99 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before treatment, 2nd and 5th hour of treatment, and following treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On each of 3 days following each 2 days of exposure during week 13 of the study
- Anaesthetic used for blood collection: Yes (chloroform)
- Animals fasted: Yes
- How many animals: 15

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On each of 3 days following each 2 days of exposure during week 13 of the study
- Animals fasted: Yes
- How many animals: 15

URINALYSIS: Yes
- Time schedule for collection of urine: On each of 3 days following each 2 days of exposure during week 13 of the study
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table MSL-2947-1)
HISTOPATHOLOGY: Yes, control and high dose group (see table MSL-2947-1)
CLINICAL PATHOLOGY: Yes (see table MSL-2947-2)

Statistics:

As cited in study report: Body weight, organ weight, hematology (excluding WBC differentials), blood chemistry and urine thiocyanate data for animals of each sex/group were evaluated by a two-tailed Dunnett's Test to detect significant differences between the means of treated groups and those of the respective control groups. Statistical analyses of organ weight/terminal body weight ratios were performed using the Mann Whitney Test (3) with the Bonferoni Inequality Modification. Group differences in the incidence of microscopic lesions were tested using the Fisher's exact test with the Bonferoni Inequality Procedure.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

HAEMATOLOGY: Statistically significant decreases were observed in the RBC, Hct, and Hb values of high exposure level females. The high exposure males had a statistically significant increase in MCHC values.

URINALYSIS: Urinary thiocyanate levels were significantly elevated in all exposure groups. This effect is expected in animals exposed to organonitriles, and therefore it is not considered an adverse effect.

OTHER FINDINGS: Alopecia , fur discoloration and secretions about the nose and eyes were observed before and after exposure. Most of the signs were either inconsistently or infrequently demonstrated or equally observed among exposure groups, therefore these were not considered adverse dose-related effects.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

30.6 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose.

2

Dose descriptor:

LOAEC

Effect level:

99 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Decreases in RBC, Hct, and Hb were observed in females exposed at this dose.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

Analytical Determination of Concentration
Target:	Concentration (mg/m3)
	Low (13)	Mid (30)	High (100)
Analytical Mean+S.D.	12.9+2.2	30.6+3.2	99+13
Nominal+S.D.	29.9+6.0	64.8+8.4	146+18

  

Summary of Weekly Occurrences of Signs of Adiponitrile Toxicity (No. of Animals)
Week	No./sex /group	Fur Discoloration				Alopecia
		Males/Females				Males/Females
		N	Low	Mid	High	N	Low	Mid	High
1	15	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
2	15	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
3	15	0/0	0/0	0/0	0/0	0/0	1/0	0/0	0/0
4	15	0/0	0/0	0/0	0/0	0/0	1/0	0/0	0/0
5	15	0/0	0/0	0/0	0/0	0/0	1/0	0/0	0/0
6	15	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
7	15	0/0	0/0	0/0	0/0	0/1	2/0	0/2	0/0
8	15	0/0	0/0	0/0	0/0	1/1	2/0	0/3	0/1
9	15	0/0	0/0	0/0	0/0	2/0	2/1	0/2	0/1
10	15	0/0	5/2	7/7	10/10	1/2	2/0	0/3	1/2
11	15	0/0	1/0	5/2	7/9	1/2	2/0	1/3	1/2
12	15	0/0	0/0	0/0	0/0	1/2	4/0	1/3	0/2
13	15	0/0	0/0	4/1	3/2	2/2	4/0	1/3	0/2

  

Summary of Initial and Final Body Weights (g)
Group (15/sex/group)		Males		Female
		Initial	Final	Initial	Final
N	Mean	200.4	510.8	158.6	300.9
	Std. Dev.	10.04	40.04	7.04	18.05
Low	Mean	200.0	508.7	158.7	300.3
	Std. Dev.	10.10	53.82	6.84	22.78
Mid	Mean	200.4	511.4	158.9	294.5
	Std. Dev.	9.72	28.62	7.11	21.19
High	Mean	199.9	511.5	158.5	301.5
	Std. Dev.	10.04	38.89	7.46	37.03

  

Summary Incidence of Individual Gross Necropsy Alterations
Tissue	Male/Female
	15 animals/sex/group
	N	Low	Mid	High
Brain (small)	0	0	1	0
Eye (Focal Corneal Opacity)	0	0	0	0
Kidney, Left
- Cysts(s)	0	0	1	0
- Dilated Pelvis (Hydronephrosis)	0	0	0	1
Kidney, Right
- Cysts(s)	0	0	0	0
- Dilated Pelvis (Hydronephrosis)	1	0	0	0
Thymus (multiple, purple-red foci)	0	1	0	0
Uterus (hydrometra)	0	0	0	0

  

Summary Incidence of Microscopic Findings
Tissue	Male/Female
	15 animals/sex/group
	N	Low	Mid	High
Brain	0/0			0/0
Heart
- Inflammatory Cell Infiltration/Myocarditis	1/0			0/0
- Myocyte Proliferation	0/0			2/0
- Myocardiolysis	0/0			1/0
Kidney
- Mononuclear Cellular Infiltr.(Chron. Interst. Nephritis)	5/2			5/2
- Tubular Epithelial Regeneration	3/0			1/0
- Fibrosis, Intertubular	1/0			0/0
- Hydronephrosis (Uni- or Bi-Lateral)	0/0			0/1
Liver
- Periportal Mononuclear Cellular Infiltration	3/8			4/4
- Mononuclear Cell Infiltration (Random)	6/2			7/4
- Hepatocytic Mecrosis / Lysis	0/1			0/1
Lung
- Medial Hypertrophy of Medium-Sized Arteries	2/1			5/1
- Emphysema	0/0			0/1
- Peribroncial Lymphoid Hyperplasia / Cuffing	3/0			5/1
- Perivacular Lymphocytic Infiltration	1/0			0/0
Nose/Turbinates
- Acute Inflammation	1/0			0/0
- Chronic Inflammation	4/0			2/0
Pancreas
- Inflammation	1/0			1/0
- Islet Cell Hyperplasia	1/0			0/0
Pituitary - Cysts	1/0			0/0
Prostate - Inflammation (Males Only)	0			2
Parathyroid-Fibrosis	0/1			0/0
Spleen - Presence of Excessive Hemosiderin	0/0			10/
Thyroid - Ultimobranchial Cyst(s)	3/3			2/1
Thymus - Hemorrhage	2/0			1/0
Urinary Bladder	0/0			0/1
Uterus (Females Only)
- Dilation of the Uterine Lunen (Hydrometra)	1			3
- Cystic Dilation of Endometrial Glands	0			2

  

Hematology - Summary Data (Mean Values)
	Males/Females
Group	WBC	RBC	HGB	HCT	MCV	MCH	MCHC
N	12.8/10.6	7.85/7.49	16.5/16.8	41.6/42.1	53/56	21.1/22.5	39.5/39.9
Low	11.2/9.1	7.75/7.30	16.5/16.4	41.0/40.9	53/56	21.3/22.5	40.0/40.0
Med	9.9/9.6	7.78/7.43	16.2/16.6	40.5/41.3	52/56	20.8/22.4	39.8/40.1
High	11.3/9.6	7.67/7.02	16.3/15.7	40.3/39.6	53/57	21.3/22.5	40.3/39.6

 

Applicant's summary and conclusion

Conclusions:

The results of this study indicate there was no substance-related toxicological effects of adiponitrile aerosol and vapor at 30.6 mg/m3 or less, on male and female rats exposed six hours per day each workday for 66 days.

Executive summary:

Four groups each consisting of 15 male and 15 female Sprague-Dawley rats were each exposed to an adiponitrile vapor/aerosol atmosphere in 10 M3 inhalation chambers six hours a day, five days per week for 13 weeks (66 exposure days, 92 total days). Exposure levels were analytically determined as 0.00 (Control), 12.9, 30.6 and 99 mg adiponitrile per cubic meter of air. No gross signs of toxicity relating to compound exposure were noted, and no deaths occurred during the course of the study. High exposure level females had mildly decreased RBC, hemoglobin and hematocrit levels. Males and females of all exposure groups had increased urine thiocyanate values. Significant necropsy findings were non-existent and there were no compound-related microscopic lesions. The results of this study indicate that other than an expected elevation in urinary thiocyanate levels there was no real effect of adiponitrile aerosol and vapor at 30.6 mg/m3 or less on male and female rats exposed six hours per day each workday over a 13 week exposure period.