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Administrative data

Description of key information

Repeated Dose Oral 90d - NOAEL = 100 mg/kg bw/day for female rats (OECD 408)

Repeated Dose Oral 90d - NOAEL = 100 mg/kg bw/day for male mice (OECD 408)

Repeated Dose Inhalation 90d – NOAEC ≥ 24300 mg/m3 for rats (similar to OECD TG 413)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 422: GLP
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
Males were treated from day 14 prior to the mating phase until the end of the mating phase and then killed, Females were treated from day 14 prior to mating, through day 4 of lactation and then killed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males were treated from day 14 prior to the mating phase until the end of the mating phase and then killed, Females were treated from day 14 prior to mating, through day 4 of lactation and then killed.
Frequency of treatment:
7days/week
Remarks:
Doses / Concentrations:
0, 25, 150, or 1000 mg/kg/day (10 ml/kg dosing volume)
Basis:
other: gavage
No. of animals per sex per dose:
10 male, 10 female per group
Control group: 10 male, 10 female, 0.5% methylcellulose
Control animals:
yes
Observations and examinations performed and frequency:
Effects on general toxicity, neurobehavioral activity, clinical chemistry, and hematology were evaluated. Gross necropsies and histopathologic examination of tissues were conducted with emphasis on the male reproductive tract.
Sacrifice and pathology:
All surviving animals were sacrificed following dosing
Statistics:
Adult body and organ weight, food consumption, clinical chemistry, open field activity and hematologic data (raw or transformed) were compared using either parametric or nonparametric (Kruskal-Wallis) ANOVA depending on whether the data were found to be homogeneous or nonhomogeneous using Bartlett's homogeneity of variance procedure. If ANOVA analysis indicated significant differences, Dunnett's test and Mann Whitney's U test, for parametric and nonparemetric data, respectively, were used to analyze for differences between the various dose groups.
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):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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:
no effects observed
Details on results:
No deaths or clinical signs of toxicity or behavioral changes were noted. No significant differences in body weights or feed consumption were observed. Startle reflex, open field test, and forelimb grip reflex performance data also revealed no treatment-related findings.
There were also no treatment-related changes in hematology or blood chemistry parameters, organ weights or gross pathology. An apparent treatment-related, slight to moderate hyperplasia of the non-glandular mucosa of the stomach, associated with degeneration, hyperkeratosis and submucosal subacute inflammation and, in a few cases, with erosion, was seen in animals of all treated groups. This effect was considered an artifact of the dosing method and not directly related to the toxicity of the test material. No other treatment related histological changes were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No treatment-related mortality or significant adverse clinical effects occurred.
Critical effects observed:
not specified
Conclusions:
Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >= 1000 mg/kg/day, the highest dose tested.
Executive summary:

Groups of 10 male and 10 female Sprague Dawley rats were dosed with decane daily by gavage at exposure levels of 0, 25, 150, or 1000 mg/kg/day. Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation.  Oral dosing of decane produced no evidence of any adverse effects on clinical observations, organ weights, gross pathology, neurobehavioral activity, clinical chemistry or hematology endpoints. Evidence of irritation of the nonglandular mucosa of the stomach was observed, but was considered an artifact of the dosing method and not attributed to the inherent toxicity of the test material.  Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >=1000 mg/kg/day, the highest dose tested. 

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
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
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
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
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
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.
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.
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
Key result
Critical effects observed:
not specified

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

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).

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
only females tested; not all parameter measured/observed
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Fischer 344 [CDF (F-344/CrlBR, Lot T68]
- Source: Charles River Breeding Laboratories, Raleigh, NC, USA (6 weeks old)
- Age at study initiation: 9 weeks
- Females were nulliparous and nonpregnant
- animals were examined for ecto- and endoparasites
- Weight at study initiation: 131.3 ± 1.4 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
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 administrated 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
Remarks:
Doses / Concentrations:
0, 0.1, 1, 5.0 g/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 females per dose (12 in the high dose due to unexpected mortality)
Control animals:
other: yes, concurrent distilled water
Details on study design:
- Dose selection rationale: 7-day dose range-finding study was performed; 5 females 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 rats of the 3.6 g/kg group had decreased body weights in comparison to control group and signs of irritation in the perianal area
Positive control:
none
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: 34
- 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: 35
- 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 consistent 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.
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):
effects observed, treatment-related
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 (6 of 9 rats). 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.

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
Food consumption values of the 5.0 and 1.0 g/kg groups were lower than the control values for the first two weeks of the study. However, no further decreases from control means were observed, except for the 1.0 g/kg group on study days 43 and 50.

FOOD EFFICIENCY
not examined

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

OPHTHALMOSCOPIC EXAMINATION
not examined

HAEMATOLOGY
In the 0.1 and 5.0 g/kg groups, the white blood cell count was increased compared to the control value. The mean percentages of neutrophils and basophils were also increased in the 5.0 g/kg group, but the lymphocyte percentage was decreased.

CLINICAL CHEMISTRY
In rats of the 5.0 g/kg group there were decreases in mean values of cholesterol, triglycerides, and albumin and an increase in alanine aminotransferase (ALT). Rats of the 1.0 g/kg group had lower albumin and total protein concentrations compared to the control group.

URINALYSIS
not examined

NEUROBEHAVIOUR
Grip Strength: No statistical significant differences related to nonane exposure in female rats. 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: In female rats, there was a pattern of decreased motor activity during the first half of the week 12 in the high dose group, however, the overall effect of n-nonane was not statistically significant. There were isolated cases of statistically significant treatment group differences at four weeks, but no consistent pattern emerged. Measures of clockwise rotations, counterclockwise rotations, and the ratio of the two were also analyzed. There was no indication of rotational behavior in any of the treatment groups during any of the three test sessions.

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. Rats of the 5.0 g/kg group had increased liver, lung and adrenal weights, but decreased spleen and ovary weights. The increase in adrenal weights and decrease in ovary weights were also observed in the 1.0 g/kg rats, but there were no differences in 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. Five high dose group rats were found dead between days 1 and 10, with gross and histologic lesions suggestive of dosing accidents (pulmonary hemorrhage; severe transmural hemorrhagic gastritis). Another high dose rat died during week 11, and an intermediate dose rat died during week 13. These deaths also were attributed to dosing accidents.

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 (intracornal abscesses). The glandular stomach was histologically normal in all animals. Eleven high dose rats were examined. Ten had marked forestomach hyperplasia and hyperkeratosis. Seven of these animals had gastric inflammation, and two had mild inflammation of the proximal duodenal mucosa. In nine of the high-dose rats, perianal epidermal hyperplasia and hyperkeratosis, often with mild inflammation, was noted. Of ten medium dose rats, six had mild and four had marked forestomach squamous hyperplasia and hyperkeratosis. Eight of the animals had gastric inflammatory changes; in four changes were marked. Five medium dosed rats exhibited mild perianal squamous hyperplasia; however, inflammation was noted in only one animal. In low dose rats, mild forestomach hyperplasia and hyperkeratosis were present in eight of ten animals. Seven had accompanying mild gastric inflammation; perianal lesions were not noted. Control rats were normal.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: increased adrenal weights and decreased ovary weights in the 1 g/kg/day dose group increased liver, lung and adrenal weights, but decreased spleen and ovary weights in the 5 mg/kg/day dose group
Key result
Critical effects observed:
not specified

Tab.: Mean hematologic values and standard deviation of female rats

Parameters

Dose (g/kg/day)

 

0.0

0.1

1.0

5.0

N

10

10

9

5

WBC (10^3)

6.8 ± 0.8

8.7(a) ± 1.1

7.9 ± 1.5

9.6(a) ± 1.2

RBC (10^6)

8.9 ± 0.4

8.8 ± 0.5

8.7 ± 0.6

8.9 ± 0.8

HGB (g/dL)

15.4 ± 0.6

15.5 ± 0.8

15.2 ± 1.0

15.5 ± 1.2

HCT (%)

47.2 ± 1.7

46.9 ± 2.5

46.6 ± 2.9

47.2 ± 3.8

MCV (fL)

53.2 ± 0.6

53.3 ± 0.2

53.5 ± 0.6

52.8 ± 0.6

MCH (pg)

17.4 ± 0.3

17.6 ± 0.2

17.4 ± 0.3

17.3 ± 0.2

MCHC (g/dL)

32.7 ± 0.6

33.0 ± 0.2

32.6 ± 0.3

32.8 ± 0.3

Platelets (10^3)

886 ± 69

955 ± 86

908 ± 77

965 ± 97

Neutrophils (%)

21.1 ± 3.2

22.1 ± 3.9

24.4 ± 3.5

29.5(a) ± 7.3

Lymphocytes (%)

73.8 ± 3.2

73.2 ± 4.0

70.9 ± 3.6

65.3(a) ± 6.9

Monocytes (%)

4.2 ± 0.9

3.6 ± 1.2

3.9 ± 1.2

3.7 ± 1.1

Eosinophils (%)

0.9 ± 0.3

0.8 ± 0.2

0.7 ± 0.4

0.7 ± 0.4

Basophils (%)

0.1 ± 0.1

0.3 ± 0.2

0.2 ± 0.2

0.8 ± 0.7

(a) p<0.01 compared to control

Tab.: Mean serum chemistry values and standard deviation of female rats

Parameters

Dose (g/kg/day)

 

0.0

0.1

1.0

5.0

N

10

10

9

6

BUN (mg/kg)

18.7 ± 1.7

19.2 ± 2

17.0 ± 1.7

17.2 ± 1.5

Creatine (mg/dL)

0.5 ± <0.1

0.5 ± <0.1

0.5 ± 0.1

0.5 ± <0.1

Chloride (mmol/L)

98.7 ± 1.3

98.8 ± 1.9

99.7 ± 0.9

98.3 ± 2.1

Calcium (mg/dL)

11.3 ± 0.2

11.3 ± 0.4

11.0 ± 0.3

10.9 ± 0.4

Phosphorous (mg/dL)

10.1 ± 1.0

9.6 ± 0.8

9.4 ± 0.5

10.0 ± 9.8

Total Protein (g/dL)

6.3 ± 0.2

6.3 ± 0.2

5.9(a) ± 0.2

6.1 ± 0.3

AST (IU/L)

85.6 ± 23.0

78.5 ± 8.2

83.1 ± 15.0

97.3 ± 15.3

ALT (IU/L)

48.8 ± 4.1

47.9 ± 7.2

44.7 ± 5.8

61.8(a) ± 5.0

Alkaline phosphatase (IU/L)

127 ± 13

124 ± 21

113 ± 11

157 ± 53

Glucose (mg/dL)

131 ± 22

141 ± 20

142 ± 23

151 ± 19

Sodium (mmol/L)

147 ± 2

147 ± 2

146 ± 2

146 ± 1

Triglycerides (mg/L)

62.2 ± 14.8

69.4 ± 18.3

49.8 ± 6.7

37.2(b) ± 11.4

Magnesium (mg/dL)

2.8 ± 0.2

2.6 ± 0.2

2.7 ± 0.2

2.7 ± 0.2

Potassium (mmol/L)

5.4 ± 0.5

5.2 ± 0.5

5.5 ± 0.3

5.3 ± 0.2

Cholesterol (mg/dL)

77.9 ± 7.5

79.1 ± 5.1

72.7 ± 3.2

64.5(a) ± 6.6

Total Bilirubin (mg/dL)

0.3 ± 0.1

0.3 ± 0.1

0.3 ± 0.1

0.3 ± 0.1

Albumin (g/dL)

3.6 ± 0.1

3.6 ± 0.1

3.2(a) ± 0.2

3.2(a) ± 0.2

Globulin (g/dL)

2.8 ± 0.1

2.8 ± 0.1

2.7 ± 0.1

3.0 ± 0.3

(a) p<0.05 compared to control, (b) p<0.01 compared to control

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

Endpoint:
sub-chronic toxicity: oral
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The ‘justification for the read across’ is provided in the ‘Attached justification’ section below.
Species:
rat
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
other: female rats and male mice
Quality of whole database:
2 key, sub-chronic, substance-specific studies available and 1 key, short-term study from a structural analogue available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Male and nulliparous, nonpregnant female Sprague-Dawley rats (VAF/ Plus Crl:CD BR)
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approximately 5 wk old when purchased, acclimated for approximately 2 wk prior to the initiation of the study
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages in air-conditioned rooms
- Diet (e.g. ad libitum): Certified rodent diet 5002 (PMI Feeds, Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water from an automated watering system was available ad libitum
- Acclimation period: 2 weeks. All animals were assigned a temporary identification number at receipt and examined by the staff veterinarian during the acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12 during the acclimation and all nonexposure periods
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: nitrogen
Details on inhalation exposure:
Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Exposure levels were determined three times daily by gas chromatography. The highest concentration was approximately 75% of the lower explosive limit. Animals’ positions in the cages were rotated for each exposure to ensure uniform exposure of every animal.

Chamber Operation: Animals were housed individually in wire mesh, stainless steel cages within a 1000-L glass and stainless steel exposure chamber. Chamber temperature and humidity were monitored every half hour during exposure and maintained, to the extent possible, within the ranges of 20–24°C temperature and 40–60% relative humidity. Animals did not receive food or water during the exposure period. Exposure chambers were operated dynamically at a calibrated airflow rate of 200 L/min (lpm). Recordings of airflow and static pressure were made every half hour. All animals remained in the chamber for a minimum of 30 min at the end of exposure while the chamber was operated using clean air only. Chambers were exhausted through a system of coarse filter, HEPA filter, and charcoal filter.

Atmosphere Generation: LAND-2 was pumped directly from the 5-gal container, housed within a freezer constantly flushed with nitrogen, using a laboratory pump, equipped with a piston, that was insulated within a styrofoam container. An ice bag was placed on top of the piston to keep the piston chamber cold to inhibit volatilization of LAND-2 in the pump and delivery lines. LAND-2 was delivered onto the central glass helix of a countercurrent volatilization chamber (one generator per chamber). The glass helix was heated by an internal nichrome wire inserted in the center of the glass tube that supported the helix (external to the volatilization chamber) and was controlled by a variable autotransformer. House-line nitrogen delivered from a regulator with a backpressure gauge was divided with a stainless steel T into the generation flow system and a purge flow system. Purge nitrogen was delivered to the bottom of the tube containing the nichrome wire to continuously purge the area surrounding the wire, protecting it from oxidation. Nitrogen for the generation system was directed through a flowmeter to the ball-and-socket joint at the bottom of the volatilization chamber, flowed up the chamber, passed over the coil, and volatilized the test material. The LAND-2-laden nitrogen flowed through a T tube at the top of the volatilization chamber into the turret of a 1-m3 glass and stainless steel exposure chamber, where it mixed with room air to appropriate exposure concentrations as it was drawn into the chamber (flow rate of 200 lpm). Control animals were sham-exposed to nitrogen alone introduced into the turret and mixed with air in the chamber.

Exposure Chamber Monitoring: Samples for determination of analytical exposure levels and the major components of LAND-2 vapor were withdrawn by vacuum pump from the breathing zone in the exposure chambers three times per exposure for treated groups and once per exposure for controls. Samples were pulled through Teflon lines into the multipositional control module and directed to a Hewlett Packard 5890II gas chromatograph, equipped with a flame ionization detector for analysis by ASTM method D5134-92 (ASTM, 1992). Composition and stability of the test material were evaluated by characterizing neat LAND-2 and comparing the major components in the neat and generated atmospheres at the beginning and end of the study. Particle size distribution measurements of any background aerosol were performed once during each exposure for chambers and room air using a TSI Aerodynamic Particle Sizer. Samples were drawn for 20 s at a rate of 5 L/min. Mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD), and total mass concentration (TMC) were calculated. Nominal concentrations (mg/m3) were calculated from the loss of weight from the generation apparatus divided by the total air flow through the chamber during exposure. This value was converted to parts per million (ppm) using an average molecular weight for this mixture of 89.2.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determined three times daily by gas chromatography.
Composition and Uniformity Chamber Gas Chromatographic Results (% Weight): n-Butane: 3.21; iso-Pentane: 34.343; 2,3-Dimethylbutane: 12.977, 2-Methylpentane: 4.096; 2,4-Dimethylpentane: 5.663; 2,3-Dimethylpentane: 2.680; 2,2,4-Trimethylpentane: 16.885; 2,3,4-Trimethylpentane: 3.578; 2,3,3 –Trimethylpentane: 4.505; 2,2,5-Trimethylhexane: 2.499.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 668, 2220, and 6646 ppm (0, 2.4, 8.1, and 24.3 g/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed
Details on study design:
Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures.

Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically.
Observations and examinations performed and frequency:
Animals were evaluated twice daily for mortality and gross signs of toxicological or pharmacological effects. During each exposure, rats were observed as a group once for abnormal behavior. Detailed physical examinations were performed twice pretest and weekly during the study. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at wk 14 (terminal) and 18 (recovery).

Body Weights and Food Consumption. All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period.

Animals were transported from the room in which they were housed during non-exposure hours to the neurotoxicity laboratories. Behaviors were evaluated pretest and during wk 5, 9, 14, and 18 (recovery groups). Temperature, humidity, and illumination were measured and recorded to minimize variation in environmental conditions during evaluations. Noise levels were not recorded. Testing was staggered over 8 sessions within a 4-d period, each session consisting of approximately 2 rats/sex/treatment group.

Motor Activity. Locomotor activity was monitored as a function of the number of beam breaks in an activity box, using an automated photo- beam activity system. Sessions were 60 mm in length divided into twelve 5-mm intervals. Rats were evaluated at pretest, at 3 time points during the treatment period (wk 5, 9, and 1 4), and at the end of the recovery period. Treatment groups were counter balanced across test times. Three sets of analyses were performed. The first analysis was conducted using the pre-dose data with animals nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals. The second analysis used data from the 3 treatment time points, and animals were nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals nested within the 3 time periods. The third analysis was similar to the first, using the recovery data. All three analyses tested for treatment effects, sex differences, treatment group by sex interactions, and these effects crossed with periods (second analysis only) and intervals. Analyses were repeated using transformed rank data to achieve a normal distribution of the residuals. Residuals from the models were tested for normality by the Shapiro-Wilk W-test or the Kolomogorov D-test.

Functional Operational Battery. The battery was comprised of home- cage evaluations (posture, vocalizations, and palpebral closure), handling evaluations (reactivity to general stimuli, signs of autonomic function), open-field behavior (arousal level and gait, urination and defecation frequency, convulsions, tremor, abnormal behaviors, piloerection, and exophthalmos), and reflex assessments (response to visual and auditory stimuli, tail pinch, pupillary function). Animals were also evaluated for forelimb and hindlimb grip strength, landing foot splay, and air righting ability. For landing foot splay, a small dot of paint was applied to each hindpaw. The rat was dropped from a height of 2 ft above a flat surface and the distance between the marks left by the hindpaws was measured in centimeters. To evaluate air righting reflex ability, the rat was held upside down, dropped from a height of 2 ft above a container of bedding, and the landing position was observed. Treatment groups were counterbalanced across test times. The observer performing the evaluation did not know the identity of the animal’s dose group.
Sacrifice and pathology:
All animals were sacrificed by intraperitoneal injection of sodium pentobarbital, and tissues were preserved in situ by transcardial perfusion with phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde/1 % glutaraldehyde in the same buffer. Animals were killed at termination of 13 wk of exposure (week 13 terminal sacrifices performed during week 14) or at the completion of the 4-wk recovery period (control and high-dose groups only). A complete macroscopic examination was performed on all animals and 12 organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus, and uterus. The length and width of the brain of each rat was measured.

Thirty-nine tissues were preserved from all animals in all dose groups. Tissues from all animals in the control and high-dose groups were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological examination. The kidneys of selected animals were also stained with Mallory-Heidenhain stain. In addition, tissues of the nervous system were fixed for all animals. Brain, spinal cord, ganglia, and spinal nerve roots were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin-eosin, Luxol fast blue, and Sevier-Munger stains. Peripheral nerve sections (sciatic, tibial, sural, and optic) were embedded in glycol methacrylate and stained with toluidine blue. Slides of nervous system tissues were examined from animals (6/sex/group) designated through random selection for neuropathology, in the control and high-dose groups sacrificed at the end of 13 weeks of exposure. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons, and medulla.
Statistics:
Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett’s test at 1 % significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1 % significance, two- sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett’s test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn’s summed rank test was used to determine differences from controls.

A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal orJonckheere’s test for monotonic trend in nonparametric cases.
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
All animals survived the treatment period and were sacrificed according to study design at the end of 13 wk or at 18 wk (recovery groups). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. No LAND-2-related ocular disease was observed. All groups showed similar mean body weights, body weight gains, and food consumption.

At wk 13 terminal sacrifice, there was a statistically significant decrease, relative to control values, in hemoglobin (5%), hematocrit (5%), and erythrocytes (7%) in blood of high dose males (data not shown). The hemoglobin was still decreased (4%) after the 28-d recovery period. However, because these differences were small and within the historical range for control animals in this laboratory, they are not considered toxicologically relevant. Clinical chemistry results showed a statistically significant decrease in aspartate aminotransferase (AST 32%) and alanine aminotransferase (ALT 46%) in females of the high-dose group (data not shown) compared to controls. However, several control female rats had elevated AST and ALT relative to the other nine female rats in the group and historical controls. Comparison of ALT and AST values from high-dose females with these elevated concurrent control values produced a statistical significance that is not toxicologically relevant. These results are not considered LAND-2 related.

NEUROBEHAVIORAL STUDIES

Motor Activity. Shapiro-Wilk analysis of data from the predose period indicated that the only statistically significant effects on response pattern occurred in the low-dose group due to inactivity among females for intervals 6—8, and increased activity of males during interval 10 (data not shown). Data from the treatment intervals and the recovery period were analyzed based on the Blom transformed data because the residuals from the model were not normally distributed by the Shapiro—Wilk statistic at the .01 level. There were statistically significant differences in the number (ct < .04) and relative pattern (cx. < .02) of beam breaks among the dose groups over the treatment testing periods. There were expected differences between sexes, and pattern differences across the 1 2 measuring intervals. In the recovery period in which only the room air controls and the high-dose animals were evaluated, there were no dose-group-related differences in response. Overall, dose-group differences did not occur in a dose-related pattern. Although statistically significant, the magnitudes of the differences were not large, and none of the treatment-group differences were larger than differences seen during the pre-dose period.

Functional Operational Battery. No differences were detected in the distance between foot splay for male or female rats in any dose group over any time interval evaluated. Grip strength of both fore- and hindlimbs in general increased from pretest through wk 14 for both sexes in all treatment groups. Values for control and high-dose recovery animals were lower at wk 18 than in previous treatment weeks but similar between the groups. There was no test-material-related effect on any endpoint evaluated within the functional operational battery of tests.

Pathology. At the wk 13 terminal sacrifice there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex, based on positive staining reaction by the Mallory-Heidenhain method, and increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific and are not considered relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at wk 13 sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Lung and brain weights were comparable to controls. Lungs were macroscopically and microscopically comparable to controls. Brain length and width measurements showed no test-material-related effects. There were no microscopic findings in the brain, spinal cord, or peripheral nerves that could be attributable to exposure to LAND-2.
Key result
Dose descriptor:
NOAEC
Effect level:
24 300 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: no effects except adaptive response of liver weight to exposure equivalent to 6646 ppm
Critical effects observed:
not specified
Conclusions:
The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
Executive summary:

The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. Only male rats tested, organ weights not determined, no recovery period.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Only males tested, no recovery period, organ weights not determined
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Harlan-Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 216.1 ± 35.7 - 227.8 ± 37.7 g (mean values)
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable, vapour
Details on inhalation exposure:
TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber air samples, collected with airtight syringes, were injected within 30 seconds after capture into a gas chromatograph, samples were taken twice a day. Actual doses received: 1.9, 3.1, 8.4 mg/L (nominal exposure levels: 2.5, 5.0, 10 mg/L).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
1.9, 3.1, 8.4 mg/L (corresponding to 360, 590, 1600 ppm)
Basis:
analytical conc.
No. of animals per sex per dose:
25 males
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period in satellite groups: no
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: overall appearance, behaviour


DETAILED CLINICAL OBSERVATIONS: Yes: 3, 3, and 8-9 rats/group, respectively
- Time schedule: at 3, 8 and 13 weeks of exposure (19, 38 and 63 days actual exposure, respectively): prior to sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: weekly


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 8 and 13 weeks of exposure (19, 38 and 63 days actual exposure, respectively): prior to sacrifice
- Anaesthetic used for blood collection: No (tail vein)
- How many animals: 3, 3, and 8-9 rats/group, respectively (sacrificed)
- Parameters examined: hematocrit, total erythrocyte counts, reticulocyte counts, total and differential leukocyte counts


CLINICAL CHEMISTRY: Yes (from severed cervical vessels)
- Time schedule for collection of blood: at 3, 8 and 13 weeks of exposure (19, 38 and 63 days actual exposure, respectively)
- How many animals: 3, 3, and 8-9 rats/group, respectively (sacrificed)
- Parameters examined: serum alklaline phosphatase, SGOT, SGPT, blood urea nitrogen


URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 8 and 13 weeks of exposure (19, 38 and 63 days actual exposure, respectively): prior to sacrifice
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, but organ weights not determined.
HISTOPATHOLOGY: Yes: brain, respiratory tract, heart, thyroid, liver, kidney, adrenal, spleen, pancreas, stomach and intestine, skeletal muscle, bone marrow and peripheral nerve. Reproductive organs were not examined
Statistics:
Results of quantitative continuous variables (e.g. body weight changes) were evaluated using Bartlett's homogeneity of variance, analysis of variance and rank sum. Duncan's multiple range test was used if F for ANOVA was significantly high. If Bartlett's test indicated heterogeneous variances, the F-test was used for each group vs controls. If these F-tests were not significant, Student's t-test was used; if significant, means were compared by Cochran's t-test or rank sum test. Frequency data (e.g. mortality, micropathological conditions) were compared between groups by Chi square with Yates correction for continuity. Critical level of significance was 0.05.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1.9 mg/L: 2 animals died during the 13 week exposure period, one during the 46th exposure and the other after the 52nd exposure. These deaths were determined to be not dose or treatment related.
3.1 mg/L: All animals survived through study termination. There were no signs of distress throughout the 13-week study. 
8.4 mg/L: Two rats died during the first day of exposure.

BODY WEIGHT AND WEIGHT GAIN
1.9 mg/L: Both deceased animals showed weight gains during the week before they died. 
3.1 mg/L: No effects reported.
8.4 mg/L: The mean body weights or mean body weight changes of rats were statistically significantly lower than controls when compared at 3, 17, 32, 46 and 61 exposure days.


HAEMATOLOGY
No effects reported.


CLINICAL CHEMISTRY
8.4 mg/L: Serum glutamic pyruvic transaminase value for blood taken from rats after 4 weeks was statistically significantly greater than that of controls. However, the increases were not observed after 8 or 13 weeks suggesting a transient effect. 
Other dose groups: No effects reported.


URINALYSIS
No effects reported.


GROSS PATHOLOGY
1.9 mg/L: Gross and micropathological examination of the lung tissue of both deceased animals revealed suppurative bronchopneumonia. These deaths were determined to be not dose or treatment related.
3.1 mg/L: No effects reported.
8.4 mg/L: Two rats died during the first day of exposure. Lung congestion and hemorrhage were noted at necropsy and no other significant lesions were found upon histopathological examination.

 
HISTOPATHOLOGY: NON-NEOPLASTIC
8.4 mg/L: Micropathological evaluation of tissues after 4, 8, and 13 weeks revealed only common sporadic lesions that were not considered to be treatment related.
Other dose groups: No effects reported.
Key result
Dose descriptor:
NOAEC
Effect level:
8 400 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: clinical signs and body weight
Critical effects observed:
not specified

Under the conditions of this inhalation study in rats, the NOAEC was 8.4 mg/L.

Conclusions:
Under the conditions of this inhalation study in rats, the NOAEC was 8.4 mg/L.
Executive summary:

Under the conditions of this inhalation study in rats, the NOAEC was 8.4 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
24 300 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 key, sub-chronic, substance-specific study available and 2 sub-chronic studies (1 key and 1 supporting) available from structural analogues.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Short-term and sub-chronic repeated dose toxicity data is available for Nonane. Additionally, short-term data is available for structural analogue Decane and sub-chronic data is available for structural analogues Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics and light alkylate naphtha distillate.

Oral:

 

Nonane

A 90-day oral toxicity study with nonane was conducted in female rats and male mice (Dodd et al., 2003). This study was conducted to support site remediation activities. Animals were administered 0, 100, 1000, 5000 mg/kg nonane via oral gavage 7 days per week for 90 days. The authors concluded that there were no statistical differences in mean body weights between control and treated groups of both species. Nonane was clearly irritating to the nonglandular stomach in both rats and mice at all dose levels with no apparent dose-response. In female rats, increased liver, lung and adrenal weights and decreased spleen and ovary weights were noted in the high dose group (5000 mg/kg) and ovary weights were also slightly decreased in the mid dose group (1000 mg/kg). Mice in the 1000 and 5000 mg/kg dose groups showed an increase in liver weight and a decrease in kidney weight. Although significant hematologic and serum chemistry alterations were seen primarily in the high dose group of both rats and mice, they were within the normal limits for the species. Hematologic alterations, increases in neutrophils, and decreases in lymphocytes were consistent with normal physiologic response to stress and minor inflammation and correlated with the histopathologic findings in the alimentary tract and nasal passages. Histopathological lesions in both species of all dose groups were primarily in the alimentary tract, primarily hyperplasia and hyperkeratosis, and perianal epidermal hyperplasia and hyperkeratosis often with mild inflammation and multifocal minimal to mild necrosis. Suppurative inflammation of the nasal turbinates was observed in both species. In rats these lesions were present with pulmonary effects consistent with aspiration of foreign material. The authors reported a NOAEL of 100 mg/kg for both species for all lesions except the proliferative and inflammatory lesions in the non-glandular forestomach, which were ruled to be a species-specific response of no clinical significance to humans.

 

Decane

In a key study (Sasol, 1995) groups of 10 male and 10 female Sprague Dawley rats were dosed with decane daily by gavage at exposure levels of 0, 25, 150, or 1000 mg/kg/day. Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation.  Oral dosing of decane produced no evidence of any adverse effects on clinical observations, organ weights, gross pathology, neurobehavioral activity, clinical chemistry or hematology endpoints. Evidence of irritation of the nonglandular mucosa of the stomach was observed, but was considered an artifact of the dosing method and not attributed to the inherent toxicity of the test material.  Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >=1000 mg/kg/day, the highest dose tested.

Additionally, an OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) test is proposed for structural analogue, Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13. This endpoint will be updated subsequent to ECHA's approval of the testing proposal and availability of data upon completion of the study.

 

Inhalation:

 

Nonane

In a key study conducted similar to OECD 413 (Carpenter et al., 1978), groups of male rats were exposed by whole body inhalation to 0, 360, 590, or 1600 ppm n-nonane for 6 hours/day, 5 days/week, for 13 weeks. Clinical signs included salivation, mild loss of coordination and fine tremors throughout the first 4 days of exposure in the high dose group. Salivation and lacrimation were observed during the remaining exposure periods. Mean body weights or mean body weight changes of rats at 1600 ppm were statistically significantly lower than controls throughout the study. Histopathological evaluation conducted on weeks 4, 8, and 13 revealed only common sporadic lesions that were not considered to be treatment-related. No effects were noted in the 360 or 590 ppm dose groups throughout the study. The NOAEC for this study was determined to be 8400 mg/m³ corresponding to 1600 ppm.

 

Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics

In a supporting study (ExxonMobil, 1978), the test material (Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics) was administered by inhalation to Sprague-Dawley rats for 6 hours/day, 5 days/week for 12 weeks at nominal vapor concentrations of 300 ppm and 900 ppm to assess subchronic inhalation toxicity.  Ten animals per sex per group were examined at 4 weeks, 8 weeks and all survivors were sacrificed and examined at 12 weeks. Male body weight gain was significantly decreased at 900 ppm.  There were no treatment-related effects in any of the hematology and serum chemistry values.  Liver and kidney weights were increased in male rats at 900 ppm, and adrenal weights were increased in female rats at 900 ppm.  The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.  Histopathological examination did not reveal any abnormalities that were considered treatment related.  As there were no pathologic changes, changes in organ weight to body weight ratios were judged to have been compensatory rather than toxic effects.  Based on these results, the No Observed Adverse Effect Concentration (NOAEC) was greater than or equal to 900 ppm (>=5220 mg/m3).

 

Light alkylate naphtha distillate

A key 13-week inhalation toxicity study (Schreiner et al., 1998), was conducted using wholly vaporized light alkylate naphtha distillate (LAND-2) generated in nitrogen. Male and female rats were exposed by inhalation in whole-body exposure cages 6 hours/day, 5 days/week for 13 weeks at analytical concentrations of 0, 668, 2220, and 6646 ppm. All animals survived the treatment period and were sacrificed according to study design at the end of week 13 or 18 (recovery group). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. At week 13, there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex as well as an increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific. Therefore, these effects are not considered to be relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at week 13 after sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Thus, the NOAEC for systemic toxicity was 24,300 mg/m³ corresponding to 6646 ppm.

 

Other routes:

 

Nonane

Enzyme activity and potential for hepatotoxicity have been studied in female rats using octane and nonane by daily intraperitoneal injection of 1 mL (corresponding to ca. 703 and 722 mg/kg bw) for 2 or 7 days. Octane and nonane showed similar patterns of responses: Reduction in specific activity of the enzymes, benz(a)pyrene hydrolase and glutathione -S-transferase as well as decreases in cytochrome P450 and free sulfhydryl content were reported. Liver lipid peroxidation increased with exposure for 2 or 7 days (Khan et al., 1980). In another study, body weight and protein per gram liver values were decreased and liver wet weight relative to body weight increased significantly after 7 days of treatment. Decreased activity in microsomal enzymes (amino-N-methylase, aniline hydroxylase) and glucose-6-phosphatase and prolonged pentobarbital-induced sleeping time were also observed. These findings were indicative of inhibition of biotransformation activity in the liver despite the increase in wet liver weight ratio (Khan and Pandya, 1980). Administration of octane or nonane also induced increased alkaline phosphatase activity in liver, spleen and bone marrow. Increases in spleen alkaline phosphatase from a single dose of either n-alkane lasted up to 42 days post-exposure (Pandya and Khan, 1982). Decreases in serum acetylcholinesterase and carboxylesterase, increased aldolase levels, and decreased albumin, protein, and total and esterified cholesterol as well as increased free cholesterol levels, have also been reported (Khan and Pandya, 1985).

Justification for classification or non-classification

Based on available read across data, Nonane does not meet the criteria for classification for repeated dose toxicity (STOT-RE) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).