Nonane

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1995 - October 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

only males tested; not all parameter measured/observed

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nonane

EC Number:

203-913-4

EC Name:

Nonane

Cas Number:

111-84-2

Molecular formula:

C9H20

IUPAC Name:

nonane

Details on test material:

- Name of test material (as cited in study report): n-Nonane
- Physical state: liquid
- Analytical purity: 99%
- Impurities (identity and concentrations): no measurable impurities
- Purity test date: before test initiation
- Lot/batch No.: 5921EL, obtained from Aldrich Chemical Co., Milwaukee, WI, USA
- Stability under test conditions: yes
- Storage condition of test material: stored in chemical storage cabinet

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: C57BL/6 [C57BL/6NCrlBR, Lot E42]
- Source: Charles River Breeding Laboratories, Raleigh, NC, USA (6 weeks old)
- Age at study initiation: 9 weeks
- animals were examined for ecto- and endoparasites
- Weight at study initiation: 24 ± 0.3 g mean per group of 10 animals
- Fasting period before study: none
- Housing: housed in the AL/OEVM vivarium upon receipt and subjected to a two-week quarantine; Throughout study, animals were housed individually in plastic cages with hardwood chip laboratory bedding (Sanichips). Cages were changed twice per week.
- Diet (e.g. ad libitum): ad libitum (Purina Formulab #5002, powdered)
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 25°C
- Humidity (%): 40 - 60%
- Photoperiod (hrs dark / hrs light): 12 / 12 hrs

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Neat n-nonane was administrated orally (via gavage) on a daily basis throughtout the study. Dosages were administrated on the basis of weight of test substance (using a density correction of 0.72 g/mL for n-nonane) per animal body weight (not to exceed a volume of 1.0 mL/100 g body weight). Controls received an equivalent volume (1 mL/100 g body weight) of distilled water.
Using a glass syringe, the test substance or distilled water was administered by stomach intubation through a commercial 18-gauge ball-end stainless steel needle.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

dosed orally via gavage. Purity of test material was analysed by gas chromatography and mass spectrography.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.1, 1, 5.0 g/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 males per dose

Control animals:

other: yes, concurrent distilled water

Details on study design:

- Dose selection rationale: 7-day dose range-finding study was performed; 5 males per group were given oral (gavage) doses of n-nonane of 0, 0.7, 1.8, and 3.6 g/kg bw for 7 consecutive days. Clinical signs, neurobehavioral tests, body weights, gross necropsy, and organ weights were monitored. At conclusion of the study mice of the 3.6 g/kg group had increased liver and spleen weights in comparison to control group. The only indication of toxicity in the lower dose groups was an increase in liver weights in mice of the 1.8 g/kg group.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: determined and recorded immediately prior to initiation of the study and weekly thereafter. Body weight gains were computed


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: determined and recorded weekly on an individual animal basis.


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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 12 hours after conclusion of 90 days study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 27
- Parameters checked in table were examined: see "remarks and results including table and figures"


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 12 hours after conclusion of 90 days study
- Animals fasted: No
- How many animals: 27
- Parameters checked in table were examined: see "remarks and results including table and figures"


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-exposure (week -1), 4 weeks into the exposure period, and near the conclusion of the exposure period (week 12)
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

- Necropsy: samples of tissue (fat, muscle, liver) were taken for analysis at necroscopy; additionally gross necroscopy included examination of the external surface, all orifices, and the cranial, thoracic, and abdominal cavity, including their contents.

- Organ weights: included liver, kidneys (pair), adrenals (pair), gonads (pair), spleen, lungs, and brain

- Histopathology: tissues and organs from animals of the control and high-dose groups, "target tissues" from lower dose groups. Gross lessions identified at necropsy and animals that died during study were also subjected to histopathologic examinations; organs examined: liver, kidneys, adrenals, pancreas, spleen, pituitary, thyroid/parathyroid, thymus, testes, ovaries, heart, trachea, nasopharyngeal tissues, accessory genital organs (epididymis, prostate, seminal vesicles), representative aorta, brain, spinal cord, peripheral nerve, representative lymph nodes, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, uterus, lungs, sternum with bone marrow, salivary glands.

Other examinations:

- Blood and tissue sampling for test substance analysis: blood samples taken in weeks 5, 10, and 13; two samples taken during each collection period, one immediately prior to dosing, a second blood sample two hours after gavage dosing; blood samples were drawn via the lateral vein.
Observations: Blood concentrations increased with dose and were considerably lower in value prior to dosing compared to post-dosing. Blood concentrations were consitent between study weeks at each dose level. At the conclusion of the study, concentrations of n-nonane were the highest in fat tissue compared to muscle or liver. Though inter-animal variability was large, tissue concentrations consistently increased with dose.

Statistics:

Body weights and food consumption were intercompared using a repeated measures analysis of variance. Other continuous variables (e.g., organ weights, hematology and serum chemistry) were intercompared using an analysis of variance. Homogeneity of variance was tested using Levene's test. For significant F-values, multiple comparisons were conducted using a Bonferroni correction of t-tests.
Nonparametric data were transformed and, if normal in distribution, parametric tests were performed. If the transformed data were not normal, appropriate nonparametric tests were carried out. Frequency data were compared using chi-squared tests and multiple comparisons were made using Bonferroni-corrected Fisher's Exact Test. The fiducial limit of 0.005 (two-tailed) was used as the criterion for significance when assumptions for homoscedasticity and normality were not violated.
Grip strength scores from the five trials were averaged to produce one score per animal each test day. The scores were subsequently analyzed in a repeated measures ANOVA. In the locomotor activity test, each 20 minute session was divided into 10 two-minute blocks for the purpose of statistical analyses. The different measures of motor activity were seperately analyzed in repeated measures ANOVAs. Since the data were highly variable and not normally distributed, a Kruskal-Wallis analysis of variance was used for each test session, time block and dependent measure.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Results of pre-study quality control procedures were negative. Deaths, attributed to oral gavage trauma, were observed: one death at middle dose, 6 at high dose. Except for an occasional incidence of dry red material around the eyes of rats in the 0.0, 0.1 and 1.0 g/kg groups, clinical signs of irritancy and/or toxicity were observed only in the high dose (5.0 g/kg) groups (7 of 10 mice). The clinical findings included wet urigenital/perianal areas, matted fur in the anal area, perianal alopecia (hair loss), perianal/hindlimb erythema, dark-colored urine, diarrhea, erythema/excreta at base of tail, hunched posture, dry red material around the eyes and nose, lower jaw alopecia, and matted rough body fur. Mice of the 5.0 g/kg group had occasional redness and swelling of the penis and scrotal area. Mice of the 0, 0.1, and 1.0 g/kg groups were normal in appearance.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in mean body weights between control and treated groups throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no statistically significant differences in mean food consumption between control and treated groups throughout the study.

FOOD EFFICIENCY
not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
not examined

OPHTHALMOSCOPIC EXAMINATION
not examined

HAEMATOLOGY
In mice, decreases in red blood cell count, hemoglobin concentration, hematocrit percentage and percent lymphocytes were observed in the 5.0 g/kg group. Neutrophil percentage was increased compared to control in the 0.1, 1.0 and 5.0 g/kg groups.

CLINICAL CHEMISTRY
Decreases in the mean values of chloride, aspartate aminotransferase (AST), alkaline phosphatase, total bilirubin, and albumine were observed in mice of the 5.0 g/kg group. Mice of the 1.0 g/kg group had lower chloride, alkaline phosphatase and albumin values compared to control mice. Alkaline phophatase was also lower in the 0.1 g/kg group.

URINALYSIS
not examined

NEUROBEHAVIOUR
Grip Strength: No statistical significant differences related to nonane exposure in male mice. There were reliable differences across test sessions, such that grip strength was greater during the week 12 test than during the pre-exposure or week 4 test. There was no interaction of treatment with test session indicating that increased grip strength was similar across all test groups and was likely an effect of age and experience with the test.
Locomotion activity: There was an overall decrease in the amount of motor activity in the high dose group during the first half of the week 12 locomotor activity test. These group differences are found in the measures of distance traveled, time resting, and time ambulatory where the activity in the high dose group is reliably lower than in the control group. There are some spurious results in other measures; however, the group differences are transient and are not indicative of a dose-response effect. No evidence of rotational behavior was found.

ORGAN WEIGHTS
Mean organ weight values between control and treated groups were similar for final body weights and absolute brain weights. Statistically significant
differences in absolute organ weight values agreed, in the majority of cases, with statistically significant diffferences in relative organ weight values. In male mice, liver weights were increased and kidney weights were decreased in the 5.0 and 1.0 g/kg groups. There were no statistically significant differences in mean organ weights between the control and 0.1 g/kg groups.

GROSS PATHOLOGY
Observations: Mild to moderate perianal alopecia and inflammation were observed in a majority of the rats of the 5.0 g/kg group.Two high dose mice died on days 19 and 74; lesions again were suggestive of dosing accidents. Two intermediate dose mice and one control mouse died as well, presumbly to dosing accidents. No other treatment-related lesions were observed in the remaining groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Observations: Lesions in the alimentary tract were present in all n-nonane treated groups, but not in controls. Most lesions were in the non-glandular stomach. These lesions consisted of varying degrees of hyperplasia and hyperkeratosis of the squamous epithelium, often accompanied by infiltrates of neutrophils, eosinophils, lymphocytes, and lesser macrophages in the lamina propria and submucosa. Occasionally, erosion and ulceration of the mucosa were present. In the most severe manifestations, the squamous epithelium was thickened up to 6-fold, often producing pronounced invaginating folds. The keratinized layer was similarly thickened, occasionally with dense aggregates of degenerating neutrophils (intracornual abscesses). The glandular stomach was histologically normal in all animals. In the high dose (5.0 g/kg) mice, seven of eight had marked forestomach squamous hyperplasia and hyperkeratosis, four with inflammation; one had moderate hyperplasia and hyperkeratosis with no inflammation. Eight of ten high dose mice had hyperplasia, hyperkeratosis, and inflammation in the perianal epithelium. In medium dose mice, six of eight had moderate to marked forestomach hyperplasia and hyperkertosis, four with inflammation, and one had mild hyperplasia (no inflammation); perianal lesions were not noted in these animals. In low dose mice, five of ten had moderate to marked forestomach squamous hyperplasia and hyperkeratosis, four accompanied by inflammation, and one animal had mild hyperplasia (no inflammation); perianal lesions were not noted. Control mice were normal.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: increased white blood cell count, increased alkaline phosphatase increased liver weights and decreased kidney weights in the higher dose groups

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Tab.: Mean hematologic values and standard deviation of male mice

Parameters	Dose (g/kg/day)
	0.0	0.1	1.0	5.0
N	4	9	6	8
WBC (10^3)	5.6 ± 1.3	7.1 ± 2.6	5.9 ± 1.4	7.6 ± 2.0
RBC (10^6)	11.5 ± 1.2	11.4 ± 0.6	10.9 ± 0.9	9.7(a) ± 1.5
HGB (g/dL)	16.7 ± 0.7	16.0 ± 0.3	15.5 ± 0.5	14.5(b) ± 2.0
HCT (%)	53.7 ± 5.1	52.4 ± 4.1	49.4 ± 2.9	44.1(b) ± 6.9
MCV (fL)	46.8 ± 1.3	46.1 ± 2.8	45.5 ± 1.8	45.4 ± 3.2
MCH (pg)	14.6 ± 1.6	14.1 ± 0.7	14.3 ± 1.0	14.9 ± 1.2
MCHC (g/dL)	31.2 ± 2.9	30.7 ± 2.6	31.5 ± 1.5	33.1 ± 4.8
Platelets (10^3)	1326 ± 240	1596 ± 295	1612 ± 344	1322 ± 468
Neutrophils (%)	9.6 ± 5.5	10.4(b) ± 8.1	10.7(b) ± 5.5	25.8(b) ± 10.9
Lymphocytes (%)	85.6 ± 4.4	86.2 ± 6.8	85.5 ± 4.2	70.1(b) ± 14.5
Monocytes (%)	3.5 ± 2.0	2.7 ± 3.2	2.7 ± 0.7	3.5 ± 4.1
Eosinophils (%)	0.1 ± 0.1	0.1 ± 0.1	0.3 ± 0.3	0.1 ± 0.1
Basophils (%)	1.2 ± 2.0	0.6 ± 1.0	0.9 ± 1.4	0.5 ± 0.6

(a) p<0.05 compared to control

(b) p<0.01 compared to control

Tab.: Mean serum chemistry values and standard deviation of male mice

Parameters	Dose (g/kg/day)
	0.0	0.1	1.0	5.0
N	4	9	6	8
BUN (mg/kg)	19.6 ± 1.7	19.6 ± 3.3	21.7 ± 4.6	18.6 ± 5.4
Creatine (mg/dL)	0.2 ± 0.1	0.2 ± <0.1	0.2 ± 0.1	0.2 ± 0.1
Chloride (mmol/L)	115.0 ± 4.0	115.0 ± 2.0	113.0(a) ± 1.0	111.0(b) ± 2.0
Calcium (mg/dL)	9.9 ± 0.8	9.6 ± 0.3	9.5 ± 0.3	9.8 ± 0.2
Phosphorous (mg/dL)	8.7 ± 0.4	8.5 ± 1.0	8.5 ± 0.9	8.6 ± 0.8
Total Protein (g/dL)	5.0 ± 0.3	4.8 ± 0.3	4.8 ± 0.1	4.6 ± 0.3
AST (IU/L)	64.1 ± 4.4	52.3 ± 5.4	54.5 ± 13.5	50.6(a) ± 5.6
ALT (IU/L)	16.8 ± 6.0	15.8 ± 7.3	24.5 ± 14.5	19.3 ± 9.1
Alkaline phosphatase (IU/L)	98.3 ± 15.0	96.0(a) ± 17.4	89.5(b) ± 7.7	65.5(b) ± 18.0
Glucose (mg/dL)	223 ± 57	193.0 ± 36.0	193.0 ± 28.0	202.0 ± 33.0
Sodium (mmol/L)	154 ± 3.0	155 ± 2.0	155 ± 3.0	153 ± 2.0
Potassium (mmol/L)	7.3 ± 0.6	7.0 ± 0.7	7.0 ± 1.1	7.6 ± 0.8
Total Bilirubin (mg/dL)	0.4 ± 0.1	0.3 ± 0.1	0.3 ± <0.1	0.2(a) ± 0.1
Albumin (g/dL)	2.6 ± 0.2	2.5 ± 0.1	2.4(a) ± 0.1	2.2(b) ± 0.2
Globulin (g/dL)	2.3 ± 0.2	2.3 ± 0.2	2.4 ± 0.1	2.4 ± 0.2

(a) p<0.05 compared to control

(b) p<0.01 compared to control

Applicant's summary and conclusion

Conclusions:

In conclusion a NOAEL was found at the low dose level (0.1 g/kg) in mice, for all lesions except the proliferative and inflammatory lesions in the non-glandular forestomach (species-specific target organ).

Executive summary:

In conclusion a NOAEL was found at the low dose level (0.1 g/kg) in mice, for all lesions except the proliferative and inflammatory lesions in the non-glandular forestomach (species-specific target organ).