Hydrocarbons, C9-C11, n-alkanes, <2% aromatics

Endpoint summary

Description of key information

Repeated Dose Oral 90d - NOAEL ≥ 500 mg/Kg bw/day for rats (OECD 408); BMDL = 1857 mg/Kg bw/day.

Repeated Dose Inhalation 90d - NOAEC (Rat): ≥ 10400 mg/m³

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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. 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

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

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

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).

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

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

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 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: 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

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).

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 408: 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

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

According to EPA guideline 82-1

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 238-295g (males); 180-236g (females)
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1990-12-17 To: 1991-07-13

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was mixed with corn oil to ensure a 10ml/kg dose volume at all dose levels.

Test material mixtures were administered by oral gavage at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as main test and allowed to recover for 28 days post-treatment.

VEHICLE
- Amount of vehicle (if gavage): 10ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test material in corn oil were analyzed by Gas Chromatography for concentration verification, stability and uniformity analysis. Concentration verification analysis showed the values to be within 5.6% of the target levels over a three month period. Samples of the 5% and 50% nominal concentration levels (500 and 5000 mg/kg/day, respectively) were kept under conditions of room temperature and refrigeration, prior to analyzing aliquots of these samples on days 0, 5 and 8. Sample aliquots were stable for up to 8 days under both conditions. To evaluate uniformity, triplicate aliquots of the 5% and 50% nominal concentration levels were analyzed. Mean values of triplicate aliquots were 5.25% ± 0.13 and 52.5% ± 0.51, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days/week

Dose / conc.:

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

Remarks:

Group 1 (Control)

Dose / conc.:

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

Remarks:

Group 2 (Low Dose)

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Group 3 (Mid Dose)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 (High Dose)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group 5 (Satellite Group)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Test material mixtures were administered by oral gavage at three different doses at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as the main test and allowed to recover for 28 days post-treatment.

- Post-exposure recovery period in satellite groups: 28 days post-treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily monday-friday and once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, the day of dose initiation, and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
at study initiation and during the final week of the main study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals:all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following parameters were statistically analyzed for significant differences: mean hematology parameters, serum chemistry parameters, organ weights, organ to body weight ratios, body weights, mean food consumption. Comparisons were limited to within sex analysis. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test of ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differ significantly from control. In addition to ANOVA, a standard regression analysis for liner response in the dose groups and linear lack of fit were preformed.

For the nonparametric procedure the test of equality of means was performed using the Kruskal-Wallis test. If significant differences among the means was indicated, Dunn’s Summed Rank test was used to determine which treatment group differ significantly from control. In addition, Jonckheere’s test for monotonic trend in the dose response was performed.

The statistical t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. In addition, the t-test was used to compare the satellite group's and the control group's relative organ weights. The t-test was also used to compare the high dose and satellite groups to ensure similar results in order to accurately evaluate the recovery effects.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of animals in the control, low and mid-dose groups appeared normal. Very low sporadic incidences of scabs, alopecia, fur staining, dry/wet rales, dyspnea, dried red nasal discharge and hypoactivity were observed across all dose groups, but particularly in the high dose and satellite groups. The frequencies of these observations notably decreased over time during the satellite recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of fourteen unscheduled deaths were recorded across all dose groups for the duration of the study. With the exception of one 2500 mg/Kg female, for which the cause of death was not determined, all other unscheduled deaths were attributed to dosing trauma and/or incidental aspiration of test material based on post-mortem and histopathological findings. The animal deaths associated with the dosing procedures appeared to be related to physical characteristics of the test material and high dosage volume.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrements in mean body weights were observed for mid dose males from week 11 and high dose males from week 8 (p≤ 0.05 and p ≤0.01 significance level, respectively). Body weights for male rats in the satellite group were similar to those in the high dose groups, although there was a trend towards recovery following main study termination. Statistically significant body weight differences in treated female rats were small (≤10% difference) and restricted to mid and high dose groups at week 13. Mean body weights for females in the satellite group were similar to controls, suggesting that slight changes observed in the mid and high dose groups were not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of blood samples from rats at study termination showed a statistically significant, dose-dependent increase in platelet counts in all treated males and high dose females. In addition, red blood cell counts, hematocrit, hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin were statistically significantly decreased in mid dose males compared to controls. Although the cause of these decreases could not be ascertained, the lack of similar effects in the high dose males suggested these changes were not treatment related. With the exception of the increased platelet counts, all other effects were reversed in recovery group rats.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Examination of serum chemistry values revealed a statistically significant increase in blood urea nitrogen (BUN) and gamma glutamyl transferase (GGT) for high dose males and also mid dose males for BUN. Cholesterol levels were dose dependently increased relative to control in both males and females, with statistically significant increases occurring at the mid and high dose groups. Glucose values were significantly lower than the control values at the p ≤ 0.01 level for both males and females in the mid and high dose groups and for the male low dose group at the p ≤0.05 significance level. Statistically significant increases in alanine aminotransferase (ALT) levels of 2- and 2.4-fold were observed in mid and high dose males, respectively. In the females, the high dose group showed a slight but not statistically significant increase in GGT compared to controls. Also noted was a statistically significant increase in total bilirubin (TBIL) in the high dose groups for both sexes. With the exception of small decreases in chloride levels in mid and high dose females, no statistically significant changes were noted for serum levels of calcium, phosphate, sodium and potassium (data not shown). All changes reported as statistically significant at study termination showed evidence of recovery trends in satellite rats held for 28 days post last exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences in mean kidney weights, compared to vehicle control rats were observed for all treated male rats. Liver weights for all treated female rats and low and mid dose males were statistically significantly increased relative to respective vehicle controls. Although liver weights for high dose males showed an increasing trend relative to controls, they were not significantly different from control values. Mean adrenal weights were also significantly increased for high dose males, including mid and high dose females. With respect to changes in organ/body weight ratios, relative kidney weights were statistically significantly increased for all treated male rats. Similar changes were also observed for mid and high dose rat livers and adrenal glands for high dose males.
Relative testes weights for high dose males were statistically significantly increased (p ≤0.05); however the difference was small and may have been related to the differences in body weights. In females, statistically significant differences in relative liver and adrenal weights were observed for mid and high dose groups. Similar to male rats, relative kidney weights were also statistically significantly increased for all treated female rats. No changes were observed in relative ovary weights. All changes showed trends towards almost complete recovery in high dose rats held without treatment for 28 days post last exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic changes were observed in the kidneys of all treated male rats, livers of all treated male and female rats, and the stomach and/or anus of male and female rats in the mid and high dose groups. Microscopic evaluation of the stomach revealed a dose-dependent increase in the incidence and severity of thickening of the non-glandular mucosa due to hyperplasia and hyperkeratosis of the squamous epithelium. Edema and inflammatory cell infiltrations in the submucosa and focal necrosis of the superficial glandular mucosa were also noted, although at a lower incidence. These changes showed reversibility in the severity of the hyperplasia and hyperkeratosis of the mucosa.

Most rats in the high dose groups exhibited anal swelling with thickened skin and mucosa around the anus due to hyperplasia and hyperkeratosis. Areas of necrosis, neutrophilic inflammatory cell infiltrations and pustular formations in the superficial mucosa and epidermis of the anus and surrounding skin were also observed. All other microscopic changes were considered to have occurred spontaneously and to have been unrelated to treatment.

Microscopic examination of the kidneys of male rats showed changes that are typical of male rat-specific hydrocarbon nephropathy. Renal changes consisted of accumulations of hyaline droplets in the cytoplasm of the proximal convoluted tubules, dilatation and granular cast formations in the medullary tubules and increased basophilia of cortical tubules. Affected basophilic cortical tubules showed changes consistent with both degeneration and regeneration. The renal changes were observed only in male rats, and there were no differences in the incidence and/or severity of
the lesions across treatment groups. Microscopic examination of the kidneys in the satellite group male rats necropsied after the 28-day recovery period showed no evidence of hyaline droplets in the cortical tubules. However, there was a 50% incidence of dilated tubules with granular casts in the medulla and a 30% incidence of focal chronic nephritis in rats in the recovery group. There was no difference in the incidence of cortical basophilic tubules between the control and recovery group male rats, indicating that the renal changes were reversible with discontinuation of exposure to test material.

Treatment-related effects in the liver consisted of hepatocellular hypertrophy, predominantly in centrilobular areas. The incidence and severity of hepatocellular hypertrophy was dose-related, consistent with the increased liver weights seen in all treated rats irrespective of sex. The liver lesions were completely absent in recovery rats.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
One male and 1 female died in the control group, 2 females died in the 2500 mg/kg dose group, 4 females died in the 5000 mg/kg dose group, 2 males and 3 females died in the satellite group. With the exception of one 2500 mg/kg female, all of the other 13 listed spontaneous deaths appear to be a result of dosing trauma and/or aspiration of test material (due to physical characteristics of test material and the high dosage volume).

The majority of animals in the control, low and mid dose groups displayed no observable abnormal clinical signs. Observations included but are not limited to scabs, maloccluded incisors, alopecia and staining of fur, dry/wet rales, dyspnea, nasal discharge. The type and incidence of abnormal clinical signs were similar between the high dose and satellite groups with a dramatic increase in incidence when compared to mid dose group. Clinical signs most frequently noted included swollen anus, ano-genital staining, emaciation, and alopecia. During the satellite recovery period, the incidence of abnormal signs decreased over time with an increase in the number of animals exhibiting no observable abnormalities.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases from controls at the p<=0.05 level of significance were noted for mid dose males on days 77, 84, 91 and termination and for the high dose males on Day 42. A statistically significant decrease (p<=0.01) was noted for the high dose group males on Day 49 and continued through the end of the treatment period. Statistically significant decreases were noted for mid dose females (p<=0.05) on day 91 and for high dose females on days 77 and 91. At termination both mid and high dose females displayed a statistically significant decrease in body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings.

HAEMATOLOGY
A statistically significant increase in platelets which was linearly related to dose in both the males and females was observed. In addition the male animals displayed a linear dose related increase in white blood cells. The mid dose male values were noted to differ significantly from those of controls for hematocrit and hemoglobin at the p<=0.01 level of significance and mean corpuscular volume and mean corpuscular hemoglobin at the p<=0.05 level of significance.

CLINICAL CHEMISTRY
Statistically significant increases in males (p<=0.01) for urea nitrogen and gamma glutamyl transpeptidase for the high dose males and also the mid dose males for urea nitrogen. An increase for cholesterol was noted for the mid and high dose groups of both sexes (p<=0.01). An increase in alanine aminotransferase was also noted for the mid and high dose males (p<=0.01). Glucose levels were significantly lower than the control values (p<=0.01) for both sexes in the mid and high dose and for the male low dose (P<=0.05). A statistically significant increase in bilirubin in the high dose of both sexes was observed. Other parameters showing statistically significant differences from controls included creatinine, chloride, tryglycerides.

ORGAN WEIGHTS
Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg. Testes weights were elevated in male rats at 5000 mg/kg. Both the male and female relative kidney weights for all treated groups were significantly different from the control value (p<=0.01).

GROSS PATHOLOGY
Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).

Key result

Dose descriptor:

other: BMDL

Effect level:

1 857 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Critical effects observed:

no

Table 2. Mean Hematology Values after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbons solvent

 

Parameter	Exposure dose (mg/kg/day)
	0 (control)	500	2500	5000	5000 (Recovery)^a
Males	N=8	N=10	N=10	N=10	N=8
WBC (×10^3/mm^3)	7.6±2.7	9.2±2.3	10.4±2.8	10.8±2.3	7.7±1.7
RBC (×10^6/mm^3)	8.72±0.27	8.66±0.36	8.53±0.34	8.78±0.21	8.48±0.29
HGB (g/dl)	15.6±0.6	15.2±0.4	14.6±0.6 **	15.2±0.5	15.6±0.5
HCT (%)	45.0±1.3	44.0±1.3	41.9±1.8 **	44.0±1.7	43.0±1.3
MCV (fL)	52.0±2.0	51.0±2.0	49.0±1.0 *	50.0±2.0	51±1
MCH (pg)	17.9±0.7	17.6±0.5	17.1±0.5 *	17.4±0.6	18.4±0.5
MCHC (g/dl)	34.7±0.4	34.6±0.4	34.9±0.4 *	34.6±0.4	36.2±0.7
PLT (×10^3/mm^3)	770±40	877±45 *	1008±73 **	984±115 **	1050±96
Females	N=8	N=10	N=8	N=10	N=3
WBC (×10^3/mm^3)	5.4±2.5	4.4±1.0	4.2±0.8	6.8±1.6	5.1±1.9
RBC (×10^6/mm^3)	7.43±0.32	7.35±0.31	7.21±0.57	7.72±0.44	8.16±0.12
HGB (g/dl)	14.1±0.6	14.1±0.4	13.5±1.2	14.5±0.7	15.4±0.3
HCT (%)	39.6±1.5	40.0±1.2	38.0±3.4	40.8±1.9	42.8±0.9
MCV (fL)	53.0±1.0	54.0±1.0	53.0±2.0	53.0±1.0	53±2
MCH (pg)	19.0±0.5	19.2±0.6	18.7±0.7	18.8±0.4	18.9±0.4
MCHC (g/dl)	35.6±0.3	35.2±0.5	35.5±0.3	35.6±0.4	36.0±0.3
PLT (×10^3/mm^3)	792±116	846±81	892±89	1023±110 **	1052±154

WBC – white blood cell

RBC – red blood cell

HGB – hemoglobin

HCT – hematocrit

MCV – mean cell volume

MCH – mean corpuscular hemoglobin

MCHC – mean corpuscular hemoglobin concentration,

PLT – platelet count

^a Measurements on day 125, 2 of 10 and 2 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table3. Mean Clinical Chemistry values after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

Parameter	Exposure dose (mg/kg/day)
	0 (control)	500	2500	5000	5000 (Recovery)^a
Males	N=8	N=10	N=10	N=10	N=8
GGT (lU/L)	1.80.18±0.83	1.60±0.97	2.90±0.99	4.90±1.91 **	0.88±0.83
Albumin (g/l)	4.2±0.1	4.2±0.1	4.1±0.2	4.1±0.1	3.9±0.2
Glucose (mg/dl)	128.6±11.3	113.8±10.5 *	94.9±12.5 **	93.8±12.6 **	117.5±13.8
Chol (mg/dl)	39.3±3.8	46.8±8.8	65.0±14.6 **	66.8±15.0 **	35.5±4.4
TBIL (mg/dl)	0.49±0.04	0.48±0.06	0.62±0.15	0.61±0.12 *	0.48±0.07
BUN (mg/dl)	9.8±1.5	10.4±1.3	13.3±3.4 **	14.5±2.4 **	18.8±2.5
ALT (IU/il)	37.5±3.9	42.5±8.0	75.0±18.3 **	90.7±26.0 **	31.8±6.2
AST IU/l)	94.8±18.4	88.0±15.2	91.7±18.3	106.0±10.0	119.4±19.4
Crea (mg/dl)	0.5±0.1	0.6±0.1	0.6±0.1	0.6±0.1	0.5±0.1
Females	N=9	N=10	N=8	N=10	N=3
GGT (lU/L)	1.0±0.87	0.60±0.70	1.38±0.92	1.90±1.60	2.33±1.15
Albumin (g/l)	4.6±0.3	4.8±0.3	5.0±0.2	4.8±0.4	4.3±0.1
Glucose (mg/dl)	107.2±6.4	104.0±13.6	89.5±9.7 **	81.4±6.8 **	118.0±18.7
Chol (mg/dl)	48.3±8.4	63.6±9.8	95.0±18.2 **	81.9±16.1 **	66.3±17.2
TBIL (mg/dl)	0.54±0.07	0.58±0.08	0.63±0.07	0.68±0.15 *	0.50±0.10
BUN (mg/dl)	12.8±2.6	12.2±1.9	11.6±1.8	13.2±1.9	17.7±0.6
ALT (IU/il)	60.7±48.2	38.0±39.5	51.5±22.4	69.3±16.0	26.7±5.5
AST IU/l)	113.9±49.0	93.6±38.3	97.3±13.6	115.9±13.9	88.7±23.7
Crea (mg/dl)	0.6±0.1	0.7±0.1 *	0.6±0.1	0.7±0.1	0.6±0.1

TBIL – total bilirubin

ALT – alanine amino transferase

AST – aspartate amino transferase

Chol – cholesterol

BUN – blood urea nitrogen

GGT – gamma glutamyl transferase

Crea – creatinine

^a Measurements on day 125, 2 of 10 and 3 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table 4. Mean Absolute and Relative Organ Weights after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

Parameter	Exposure dose (mg/kg/day)
	0 (control)	500	2500	5000	5000 (Recovery)^a
Males	N=8	N=10	N=10	N=10	N=8
Mean absolute (g)
Kidney	3.32±0.46	4.43±0.58 **	4.38±0.57 **	4.11±0.40 **	3.48±0.32
Liver	14.69±2.93	18.50±2.51 *	19.85±2.98 **	18.01±2.90	12.18±1.52
Adrenals	0.048±0.008	0.048±0.009	0.061±0.009	0.078±0.15 **	0.055±0.013
Testes	3.8519±0.3234	3.7200±0.3770	3.4243±0.7249	3.5136±0.3797	3.7671±0.2455
Mean relative (g)
Kidney	0.0063±0.0005	0.008±0.0011 **	0.01±0.0007 **	0.0102±0.0015 **	0.0078±0.0006
Liver	0..028±0.0001	0.033±0.001	0.045±0.004 **	0.044±0.004 **	0.027±0.001
Adrenals	0.0001±0.00001	0.00009±0.00001	0.00014±0.00001	0.0019±0.00003 **	0.00012±0.00002
Testes	0.0074±0.001	0.0067±0.0008	0.0078±0.0017	0.0087±0.0004 *	0.0084±0.0011
Females	N=9	N=10	N=8	N=10	N=3
Mean absolute (g)
Kidney	2.18±0.28	2.40±0.17	2.34±0.18	2.42±0.27	2.41±0.23
Liver	8.57±1.12	10.33±0.89 *	14.71±1.51 **	13.80±2.19 **	8.85±0.71
Adrenals	0.067±0.019	0.074±0.010	0.094±0.010 **	0.102±0.012 **	0.075±0.007
Testes	0.081±0.020	0.082±0.033	0.067±0.024	0.072±0.033	0.077±0.025
Mean relative (g)
Kidney	0.0072±0.0007	0.0084±0.0006 **	0.0089±0.0007 **	0.0091±0.0008 **	0.008±0.0002
Liver	0.028±0.002	0.036±0.002	0.056±0.005 **	0.052±0.008 **	0.029±0.001
Adrenals	0.00022±0.00006	0.00026±0.00004	0.00036±0.00004 **	0.00039±0.00006 **	0.00025±0.00004
Testes	0.00027±0.00007	0.00029±0.00012	0.00025±0.00008	0.00027±0.00012	0.00026±0.00009

^a Measurements on day 125, 2 of 10 and 3 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table 5. Incidence and Degree of Severity of Treatment-related Histopathological findings in the Kidney and Liver after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

 

Tissue/lesions	Male (mg/kg/day)					Female (mg/kg/day)
	0 (veh)	500	2500	5000	5000(Rec)^a	0 (veh)	500	2500	5000	5000(Rec)^a
Liver
No. examined	10	10	10	10	8	10	10	10	14	3
No. normal	3	4	4	2	2	4	4	1	2	1
Hypertrophy, hepatocellular, centrilobular
Minimal	0	3	4	1	0	0	3	5	6	0
Slight	0	0	1	3	0	0	0	4	4	0
Kidneys
No. examined	10	10	10	10	8	10	10	10	14	3
Normal	8	0	0	0	2	8	10	9	13	3
Basophilia, cortical tubules, multifocal
Minimal	1	3	3	2	2	0	0	0	1	0
Slight	0	2	3	6	0	0	0	0	0	0
Moderate	0	3	3	3	0	0	0	0	0	0
Dilated tubules/granular casts, medulla
Minimal	0	0	3	1	4	0	0	0	0	0
Slight	0	0	1	4	0	0	0	0	0	0
Moderate	0	3	3	3	0	0	0	0	0	0
Hyaline droplets, cortical tubules
	0	10	10	10	0	0	0	0	0	0

^a2 male and 3 female recovery rats died prior to the 90 day necropsy. 4 female recovery rats were also transferred to the female high dose group

Table 6. Incidence and Degree of Severity of Treatment-related Gastritis and Peri-anal Irritation after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

 

Tissue/lesions	Male (mg/kg/day)					Female (mg/kg/day)
	0 (veh)	500	2500	5000	5000(Rec)^a	0 (veh)	500	2500	5000	5000(Rec)^a
Stomach
No. examined	10	10	10	10	8	10	10	10	14	3
No. normal	10	9	3	1	5	10	9	2	3	2
Edema/inflammation, sub mucosa
Slight	0	0	1	0	0	0	0	0	0	0
Moderate	0	0	1	1	0	0	0	1	0	0
Hyperplasia/hyperkeratoasis, non-glandular mucosa
Minimal	0	0	4	0	2	0	0	6	0	0
Slight	0	0	1	3	1	0	0	2	2	0
Moderate	0	0	1	6	0	0	0	0	9	0
Marked	0	0	1	0	0	0	0	0	0	0
Anus
No. examined	0	0	0	8	0	0	0	0	14	0
No. normal	0	0	0	0	0	0	0	0	0	0
Hyperplasia/hyperkeratosis
Slight	0	0	0	1	0	0	0	0	2	0
Moderate	0	0	0	4	0	0	0	0	14	0
Infiltration, neutrophilic/pustules
Minimal	0	0	0	2	0	0	0	0	2	0
Slight	0	0	0	2	0	0	0	0	9	0
Moderate	0	0	0	3	0	0	0	0	3	0
Marked	0	0	0	1	0	0	0	0	0	0
Necrosis, mucosa
Slight	0	0	0	1	0	0	0	0	0	0
Moderate	0	0	0	1	0	0	0	0	0	0
Marked	0	0	0	1	0	0	0	0	0	0

^a2 male and 3 female recovery rats died prior to the 90 day necropsy. 4 female recovery rats were also transferred to the female high dose group

Table 7. Benchmark Dose Estimation of the Point of Departure for ALT Responses in Male Rats.

Model	BMD (mg/kg)	BMDL (mg/kg)	GOF^ap-value	AIC	Scaled residual for dose group
Default BMR (1SD)
Exponential M4	271.174	163.321	0.2286	233.7707	-1.084
BMR – (adverse effect as 100% above concurrent control mean)
Polynomial	2588.94	1857.37	0.2902	233.439874	0.577

^aGoodness of fit (p-value). The chosen model is considered as an acceptable model fit to the data when the p-value is greater than 0.1.

Conclusions:

Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg.

Executive summary:

MRD-89-582 was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/kg, 7 days a week for 13 weeks to assess the subchronic toxicity.  An additional group of animals, dosed at 5000 mg/kg/day, was held for 4 weeks to assess reversibility.  No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/kg dose groups.  Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day.  Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg.  Testes weights were elevated in male rats at 5000 mg/kg.  Kidney effects occurred in males at all dose levels, and 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.

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver.  Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups.  These findings are believed to have been a compensatory response and not an indication of toxicity.  Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans.  Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance.  These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity.  All treatment-related effects were reversible within the 4-week recovery period. Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided

for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg/day.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to OECD guideline 408: 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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Males: 156.2-223.2 g; Females: 136.2-170.9 g
- Housing: Individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): maintained range of 68-76
- Humidity (%): maintained range of 40-70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: October 24, 1990 To: September 27, 1991

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was diluted in vehicle at the following concentration to ensure a 5ml/kg dose volume at all dose levels:
-Group 2=0.1g/kg (2.0% w/v)
-Group 3= 0.5g/kg (10% w/v)
-Group 4 and 5= 1.0g/kg (20.0% w/v)

VEHICLE
- Amount of vehicle (if gavage): 5ml/kg

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

The test material mixtures and control were administered by oral gavage at a dose volume of 5ml/kg, 7 days per week for a period of 13 weeks.

Remarks:

Doses / Concentrations:
0.1g/kg (2.0w/v)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0.5g/kg 10% w/v)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1.0g/kg (20% w/v)
Basis:
actual ingested

No. of animals per sex per dose:

All groups consisted of 20 mice (10males; 10 females)
Group 1=Control group (
Group 2= 0.1g/kg (2.0% w/v)
Group 3= 0.5g/kg (10% w/v)
Groups 4=1.0g/kg (20% w/v).
Group 5 (satellite)= 1.0g/kg (20% w/v).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: observed for reversibility, persistence or delayed occurrence of toxic effects
- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were made daily. Clinical laboratory studies were performed on all animals pre-dose, interim (day 32 for males, and day 33 for females), and at main study termination. For the satellite animals, clinical laboratory studies were also performed on the day of recovery sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing (pretest), on the day of dose initiation (Day 0), and weekly thereafter. Body weights were also recorded at sacrifice or death.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initation and during the final week of the main study
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-dose, interim (day 32 for males, and day 33 for females), and at main study termination. Also on day of recovery sacrifice for the satellite animals.
- Anaesthetic used for blood collection: Yes-methoxyflurane
- Animals fasted: yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-dose, interim (day 32 for males, and day 33 for females), and at main study termination. Also on day of recovery sacrifice for the satellite animals.
- Animals fasted: yes
- How many animals: all animals

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The necropsy included an examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. The kidneys, liver, ovaries, tested, adrenals, and brain were weighed prior to fixation.

HISTOPATHOLOGY: Yes
-erythrocyte count
-hematocrit
-hemoglobin
-leukocyte count
-mean corpuscular volume
-mean corpuscular hemoglobulin
-mean corpuscular hemoglobulin concentration
-platelets
-reticulocyte count

SERUM CHEMISTRY:
-total bilirubin
-albumin
-blood urea nitrogen
-calcium
-cholesterol
-creatinine
-electrolytes
-glucose
-total protein
-triglycerides
-phosphorous
-gamma glutamyl transferase
-serum aspartate aminotransferase
-serum alanine aminotransferase

Statistics:

The following parameters were statistically analyzed for significant differences:
-mean hematology parameters
-mean serum chemistry parameters
- mean organ weights
- mean organ to body weight ratios
- mean body weights
- mean food consumption.
Comparisons were limited to within sex analysis.

Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. Bartlett's test was performed first. If the dose groups had equal variance, a parametric method was used. Otherwise, nonparametric techniques were used.

Parametric procedures involved a standard one way ANOVA using the F distribution. If significant differences among the means were indicated, Dunnett’s test was used to determine significant differences from control. In addition, a standard regression analysis for linear response in the dose groups and linear lack of fit were performed.

Nonparametric procedures involved the test of equality of means using the Kruskal-Wallis test. If significant differences were indicated, Dunn’s Summed Rank test was used. In addition, Jonckheere’s test for monotonic trend in the dose response was performed.

The statistical t-test was used to compare the satellite group’s main study termination and recovery termination values. In addition, the t-test was used to compare the satellite group’s and the high dose group's relative organ weights. The t-test was also used to evaluate recovery.

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 specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic changes were observed in the kidneys and liver of male rats and in the livers of female rats. The treatment-related effects in the kidney were characterized predominantly by accumulations of hyaline droplets in the cytoplasm of the proximal tubules of the cortex. An increased incidence of multifocal cortical tubular basophilia with changes consistent with both degeneration and regeneration of the tubular epithelium also was present as well as dilated medullary tubules with granular casts. These renal changes were observed only in males in all doses necropsied immediately after the 90-day treatment period. The incidence and severity of these changes generally occurred in a dose-related manner. After the reversibility period, there were still residual changes but of a lesser degree. Dilated tubules with granular casts in the medulla and an increased incidence of multifocal cortical tubular basophilia were noted. No-treatment related microscopic changes were observed in the kidneys of the female rats.

Changes in the liver consisted of a minimal to slight centrilobular hepatocellular hypertrophy in the high dose male rats and in the female rats of the mid and high dose groups. The centrilobular areas were more prominent and the hepatocytes were larger with an increased amount of eosinophilic cytoplasm. Centrilobular hepatocellular hypertrophy was not observed in any of the satellite group rats.

OTHER FINDINGS
Two control female rats died prior to study termination. One rat had diffuse fibrinopurulent pleuropneumonia and pericarditis. These changes are believed to be related to a dosing accident with perforation of the esophagus in the thoracic cavity. The cause of death in the second control female included bacterial nephritis and an associated mucosal hyperplasia and distention of the ureter and urinary bladder.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No treatment-related clinical in-life signs of toxicity and no mortality were observed at the highest dose tested.

Critical effects observed:

not specified

Microscopic changes in the male kidneys characterized by hyaline droplet accumulation in the cytoplasm of the proximal tubules of the cortex and an increased incidence of multi-focal cortical tubular basophila with changes consistent with both degeneration and regeneration of the tubular epithelium and dilated medullary tubules with granular casts are typical of a syndrome that occurs specifically in male rats and is unlikely to have a correlation to humans.  The syndrome, Alpha-2u-Globulin Nephropathy or Light Hydrocarbon Nephropathy is related to the accumulation of alpha-2u globulin in the lysosomes of the kidney.

 

Treatment-related microscopic change in the liver (centrilobular hepatocellular hypertrophy) of the mid-dose females and the high dose of both sexes in the absence of necrosis is typical of an adaptive change probably related to the livers' metabolism of large volumes of test material.  This observation is supported by the increase in relative liver weights in these animals which is typical of an adaptive change.

Conclusions:

Oral administration via gavage for 90 days produced no treatment-related clinical in-life signs of toxicity and no mortality at the highest dose tested. The no observable adverse effect level for MRD-90-868 is > 1000 mg/kg.

Executive summary:

A 90-day subchronic study was conducted in rats to assess the toxicity of MRD-90-868. The test mixture was administered by oral gavage at a dose of 0, 100, 500, or 1000 mg/ kg 7 days per week for a period of 13 weeks.  The control animals received a carrier (corn oil) dose and a satellite group was dosed at 1000 mg/ kg, 7 days/week for 13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment.  Observations were made as to the nature, onset, severity, and duration of toxicological signs. There were no deaths attributed to the oral administration of MRD-90-868 (two control group females died prior to termination).  The majority of animals in all groups displayed no observable abnormalities during the test period.  The most frequently noted observations included broken/maloccluded incisors, alopecia, and scabs, all of which were considered incidental.  Body weight, food consumption, and hematology data displayed no biologically significant trends for either males or females during the test period.  The most remarkable finding was a treatment-related microscopic change in the liver of the mid-dose females and the high dose of both sexes.  This change was minor and is typical of an adaptive change probably related to the livers metabolism of large volumes of test material and was reversible upon microscopic evaluation of the tissues from the satellite recovery group.  Microscopic changes were also observed in the male kidneys at all doses.  These changes are characteristic of kidney changes produced in male rats by hydrocarbons and are considered to be a male rat specific phenomenon without human significance.  Based on the data recorded in this study, the NOAEL for MRD-90 -868 is >1000mg/kg.

Reference 6

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

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Four key read across sub-chronic toxicity studies from structural analogues available for assessment. BMDL level determined and presented in the additional information.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 413.

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Species:

rat

Strain:

other: albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory Breding Unit
- Age at study initiation: 10-13 weeks
- Housing: three of one sex per cage
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum

During the period of the test the laboratory temperature varied between 19.4°C and 26.1°C and the relative humidity between 37% and 74%.
Barometric pressure was within the range 753 to 768 mm Hg

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Details on inhalation exposure:

The atmospheres were generated by completely evaporating the solvent into the streams of ventilating air entering the chambers using micrometering pumps and vaporizers. The vaporizers consisted of electrically heated quartz tubes whose surface temperatures were adjusted during preliminary experiments to the minimal for complete evaporation of the solvent.

Each chamber was constructed of aluminum, with a volume of 1 m3 and was ventilated by air drawn from the laboratory through dust filters. The exhaust ducts from each chamber entered a common exhaust duct through which the air was drawn by a fan situated on the roof of the laboratory.

The total air flow rate through the main duct exhausting all four chambers was recorded continuously throughout the test by means of an electro—anemometer mounted in the duct. Slight adjustments were made as required to compensate for the effects of wind at the efflux point. The total flow rate was maintained at 2.0 + 0.03 m3 ∙min- 1. The individual flow rates through each chamber were balanced before the exposures began but were not checked further throughout the test since any significant changes would have been detected by the resulting changes in toxicant concentration. The flow rates were adjusted to 0.50 m3 ∙min- 1.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmospheres were analyzed sequentially by means of a total hydrocarbon analyzer fitted with a flame-ionisation detector (Beckman 109A). The analyzer was calibrated during the test by means of known concentrations of SHELLSOL TD, prepared in a Teflon FEP gas sampling bag.

The recorder traces from the analyser were examined daily and a ‘daily mean concentration’ value was estimated by visual inspection. The daily mean concentrations for each of the test atmospheres were then ‘pooled’ to give weekly mean concentrations. The overall means of the weekly mean concentrations are given below:
Nominal concentration Observed concentration
(mg/m3) (mg/m3) (ppm)
10400* 10186 SD 327 1444
5200 5200 SD 207 737
2600 2529 SD 116 359
*83% saturated.

The desired concentrations of solvent in the test atmospheres were reached within 10 mm of the start of each exposure period. They then stayed remarkably constant throughout the 6 h exposure period.

Duration of treatment / exposure:

Six hours/day

Frequency of treatment:

five days/week for 13 weeks

Remarks:

Doses / Concentrations:
0, 2600, 5200, 10400 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

6 animals/sex/dose (total of 12 animals/dose)

Control animals:

yes, sham-exposed

Details on study design:

The start and finish of the experiment was staggered in order that the optimum number of animals could be examined histopathologically after exposure. On each of four consecutive days, four male and four female rats per chamber were started on the experiment. The remaining two males and two females were started the next day. Thirteen weeks later, four male and four female rats per chamber were removed from the experiment for pathological examination on each of four consecutive days. The remaining two males and two females were removed the next day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations: daily

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined weekly: Yes


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: Yes / No / No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly


OPHTHALMOSCOPIC EXAMINATION: No



HAEMATOLOGY: Yes
- Time schedule for collection of blood: 18h after the last 13 week exposure
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 18h after the last 13 week exposure
- How many animals: all



URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No



OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes for all animals exposed to the high and medium concentrations, plus the control animals. Kidneys of low concentration males were also examined.

Other examinations:

Organ weights
After post-mortem examinations the following organs were weighed:
Brain
Liver
Heart
Spleen
Kidneys
Testes

Histopatholgy. Tissues taken for histological examination were:

Mammary gland (posterior site with skin)
Mesenteric lymph node
Pancreas
Stomach
Intestine at 5 levels
Caecum
Spleen
Liver (middle, left and triangular lobes)
Adrenals
Kidneys
Ovaries or testes
Uterus or prostate
Seminal vesicles
Urinary bladder
Thyroid (with oesophagus and trachea)
Trachea (mid course and bifurcation)
Heart
Lungs
Nasal cavity
Thymus
Eye and lacrimal glands
Salivary gland (submaxillary)
Brain
Spinal cord (thoracic)
Pituitary
Tongue
Sciatic nerves
Muscle (femoral)
Knee joint and femur
Plus any other macroscopic lesion in any tissues.
The samples marked were held in 4% neutral formalin and only processed for histological examination if indicated by clinical or other pathological findings.

Statistics:

Body and organ weights were analysed by covariance analysis using initial body weight as the covariate. Reported means were adjusted for initial body weight if a significant covariance relationship existed: where no significant covariance relationship was found, unadjusted means were reported.

Organ weights were further examined by covariance analysis using the terminal body weight as the covariate. The organ weight means are reported as adjusted for terminal body weight if a significant covariance relationship existed. Although not a true covariance analysis (because the terminal body weights are dependent upon treatment), the analysis does provide an aid to the interpretation of organ weights when there are differences in terminal body weights. The analysis attempts to predict what the organ weights would have been, had all the animals had the same terminal body weight.
Clinical, chemical and haematological parameters were examined using analysis of variance.

The analysis allowed for the fact that animals were multihoused. Differences in response can be affected by cage environment as well as by treatment but this effect is minimal in a study of this duration.
The significance of any difference between treated and control group means was tested using the Williams t test (1971, 1972). However, if a monotonic dose response could not be assumed Dunnett’s test (1964) was used.

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

No deaths were recorded and clinical signs of toxicity were absent in the low and medium exposure groups; the high exposure groups were slightly lethargic when examined up to one hour after cessation of exposure. Body weight gain was slightly reduced in all female groups and in high exposure males. Water intake was increased in the high exposure males only.

Female aspartate amino transferase and alanine amino transferase were decreased in all female groups exposed to SHELLSOL-TD. No pathological changes were detected which could explain the observed decreases in these enzymes. In view of this lack of supporting evidence and the fact that the control values for these two parameters were high when compared with historical controls in the laboratory, these changes were not considered toxicologically significant.

Male alkaline phosphatase, potassium, chloride and albumin were increased at the high exposure level. These were considered to represent biological variation in the rat and were not considered treatment-related.

Male kidney weights were increased at all exposure levels. Hyaline intracytoplasmic inclusions and an increased incidence of tubular degeneration and/or dilatation were seen in the cortical tubules of all exposed males. These are a common effect observed in repeated-dose animal studies with hydrocarbon solvents. These kidney changes have been identified to result from an alpha2u-globulin-mediated process that because of its sex and species specificity, is not regarded as relevant to humans.

A low grade anemia was evident in all males exposed to SHELLSOL TD, characterized by slight reductions in haemoglobin, packed cell volume and total erythrocyte counts. Splenic weight was increased in the high concentration males. These changes were not seen in females and were not considered dose-related and therefore considered not toxicologically relevant.

Male and female liver weights were increased at the high and medium exposures, and male liver weights at the low exposures also. No lesions were identified histologically in the livers of treated animals that could account for the increased weight. This change was considered a physiological response to exposure rather than a toxic response and as such is not of toxicological significance.

Key result

Dose descriptor:

NOAEC

Effect level:

> 10 400 mg/m³ air (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:

The NOAEC for SHELLSOL TD is 10186 mg/m3 (actual) (1444 ppm) under the test conditions of this study.

Executive summary:

SHELLSOL TC was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 10400 mg/m³, 5200 mg/m³, and 2600 mg/m³ to assess inhalation toxicity.  No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed.  Liver and kidney weights were increased in male rats at all exposure levels, male heart weights were increased at the highest exposure level and liver and kidney weights were increased in female rats at 10400 mg/m3.  In addition, the male rats exposed to SHELLSOL TC at all concentrations showed tubular degeneration and hyaline inclusion-droplets in the epithelium.  There was also scattered degeneration of the proximal renal tubules which showed cytoplasmic pallor and shrinkage. Occasionally the degenerate tubules were surrounded by a lymphocyte infiltrate. Many tubules also showed dilatation of the cortico-medullary junction, the dilated tubule being filled with a flocculent eosinophilic material. 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 weights mentioned above 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 10400 mg/m³.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 413.

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Species:

rat

Strain:

other: Albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory
- Age at study initiation: 10-13 weeks
- Fasting period before study:
- Housing: three of one sex/cage
- Diet (e.g. ad libitum): ad libitum, removed during exposure
- Water (e.g. ad libitum): ad libitum

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The atmospheres were generated by completely evaporating the solvent into the streams of ventilating air entering the chambers using micro metering pumps and vaporizers. The vaporizers consisted of electronically heated quartz tubes.
- Exposure apparatus: aluminum with a volume of 1m3
- Source and rate of air: exhausted duct through which air was drawn by a fan situated on the roof of the laboratory
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 1.8-2.0 m3/min
- Treatment of exhaust air: dust filters


TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed continuously by means of a total hydrocarbon analyzer fitted with flame-ionization detectors.
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed continuously by means of a total hydrocarbon analyzer fitted with flame-ionization detectors.

Duration of treatment / exposure:

6h/day, 5days/week for 13 weeks

Frequency of treatment:

6h/day, 5days/week for 13 weeks

Remarks:

Doses / Concentrations:
1500mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3000 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
6000mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

18 rats /sex/dose

Control animals:

yes

Details on study design:

The start and finish of the experiment was staggered in order that the optimum number of animals could be examined at necropsy after exposure. On each of 6 consecutive days, 3 male and 3 female rats per dose were started on the experiment. Thirteen weeks later, 3 male and 3 female rats per dose were removed from the experiment for pathological examination on each of 6 consecutive days.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g/cage of 3 rats/week: Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of experiment
- Anaesthetic used for blood collection: No
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of experiment
- Animals fasted: No
- How many animals: all rats


URINALYSIS: Yes
control, low and high dose groups only
- Time schedule for collection of urine: following last exposure


NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body and organ weights were analyzed by covariance analysis using the initial body weight as the covariate. Reported means were adjusted for initial body weight if a significant covariance relationship existed. Organ weights were further examined by covariance analysis using the terminal body weight as the covariate. The organ weight means were reported as adjusted for terminal body weight if a significant covariance relationship existed. Clinical chemical and hematological parameters were examined using analysis of variance. The significance of any difference between treated and control group means was tested using the Williams t-test. However, if a monotonic dose response could not be assumed, Dunnett’s test was used.

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths were recorded during the experiment and the general health of the animals remained good throughout the period of exposure.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in male body weights due to exposure. Female body weights, however, were significantly reduced from control at the high dose.

FOOD CONSUMPTION
Female food intake showed and early significant reduction from control at the high exposure level.


WATER CONSUMPTION
Some significant increases were seen in both males and females at the highest dose.


HAEMATOLOGY
Significant decrease in male reticulocytes at the high dose, all other red and white cell parameters were normal

CLINICAL CHEMISTRY
Significant decrease in female ALT values at all exposure levels. Male chloride was significantly reduced at the medium and high dose levels and female beta protein was increased at the high level.

URINALYSIS
Glucose and protein were present in the urine of many of the rats, but animals were fed during collection period, therefore this is not unexpected. Very low incidence of exposed rats with glycosuria compared with control rats.

ORGAN WEIGHTS
Increase in male kidney weights at all exposure levels and an increase in male liver weights at the medium and high dose exposures. Female liver weights were also increased compared with controls at the high dose level. The differences were slightly enhanced when results were adjusted for terminal body weight.

GROSS PATHOLOGY
Increased incidence of renal pallor and subcapsular granularity in the male rats exposed to high concentrations. No other changes observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys of male rats exposed to all concentrations of BRIGHTSOL contained multiple, hyaline, intracytoplasmic, inclusion-droplets in the epithelium of the proximal convoluted tubules and showed an increased incidence of focal cortical, tubular basophilia.

A low grade catarrhal inflammatory reaction was evident in the nasal cavities of the majority of rats exposed to the medium concentration. The lesions were confined to the olfactory epithelium and comprised mild mucosal and submucosal edema, focal congestion and diffuse low grade inflammatory cell infiltrates. Unilateral and bilateral lesions occurred.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 6 000 mg/m³ air (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:

The NOAEC for BRIGHTSOL vapor following a 13-week inhalation exposure is greater than or equal to 6000mg/m3

Executive summary:

BRIGHTSOL was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 1500 mg/m³ and 3000 mg/m³, and 6000 mg/m³ to assess inhalation toxicity.  No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed.  Liver and kidney weights were increased in male rats at all exposure levels, and liver weights were increased in female rats at 6000 mg/m³.  In addition, the male rats exposed to BRIGHTSOL at all concentrations contained multiple, hyaline, intracytoplasmic, inclusion-droplets in the epithelium of the proximal convoluted tubules and showed an increased incidence of focal cortical, tubular basophilia.  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 tubules 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 liver weight to body weight ratios mentioned above 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 6000 mg/m³.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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.

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

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

10 400 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Three key read across sub-chronic toxicity studies from structural analogues available for assessment.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available for Hydrocarbons, C9-C11, n-alkanes, <2% aromatics. However, data is available for structural analogues, Nonane; Decane; Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics, and presented in the dossier. 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.

Oral

Nonane

 

A 90-day oral toxicity study with nonane was conducted in female rats and male mice (AFRL, 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 non-glandular 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 OECD Guideline 422 screening reproductive/developmental toxicity 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. 

 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

 

In a key repeated dose oral toxicity study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/Kg, 7 days a week for 13 weeks to assess the subchronic toxicity. An additional group of animals, dosed at 5000 mg/Kg/day, was held for 4 weeks to assess reversibility. No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/Kg dose groups. Liver weights were elevated in male and female rats at 2500 and 5000 mg/Kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/Kg. Testes weights were elevated in male rats at 5000 mg/Kg. Kidney effects occurred in males at all dose levels, and 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.

 

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver. Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups.These findings are believed to have been a compensatory response and not an indication of toxicity. Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans. Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance. These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity. All treatment-related effects were reversible within the 4-week recovery period. Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

 

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg/day.

 

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics

 

A 90-day sub-chronic study (ExxonMobil, 1991), was conducted in rats to assess the toxicity of the test material (Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, < 2% aromatics). The test mixture was administered by oral gavage at a dose of 0, 100, 500, or 1000 mg/ kg 7 days per week for a period of 13 weeks. The control animals received a carrier (corn oil) dose and a satellite group was dosed at 1000 mg/ kg, 7 days/week for 13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment.  Observations were made as to the nature, onset, severity, and duration of toxicological signs. There were no deaths attributed to the oral administration of the test material (two control group females died prior to termination). The majority of animals in all groups displayed no observable abnormalities during the test period.  The most frequently noted observations included broken/maloccluded incisors, alopecia, and scabs, all of which were considered incidental. Body weight, food consumption, and hematology data displayed no biologically significant trends for either males or females during the test period. The most remarkable finding was a treatment-related microscopic change in the liver of the mid-dose females and the high dose of both sexes. This change was minor and is typical of an adaptive change probably related to the livers metabolism of large volumes of test material and was reversible upon microscopic evaluation of the tissues from the satellite recovery group.  Microscopic changes were also observed in the male kidneys at all doses. These changes are characteristic of kidney changes produced in male rats by hydrocarbons and are considered to be a male rat specific phenomenon without human significance.  Based on the data recorded in this study, the NOAEL for the test material is >1000mg/Kg.

Additionally, in order to comply with standard information requirements for Annex X substances, OECD Guideline 90-day sub-chronic (OECD 408) toxicity tests are proposed for structural analogues Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics (EC# 920-134-1) and Hydrocarbons,C14-C19, isoalkanes, cyclics, <2% aromatics (EC# 920-114-2). The testing proposals for the same have been presented in the lead registrant dossiers for these substances already submitted to ECHA. These studies will be conducted subsequent to ECHA's approval and this endpoint will be updated upon completion of the above studies.

Inhalation

Nonane

 

In a study conducted similar to OECD 413, groups of male rats were exposed by whole body inhalation to 0, 360, 590, or 1600 ppm nonane for 6 hours/day, 5 days/week, for 13 weeks (Carpenter et al., 1978). 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 sub-chronic inhalation toxicity 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).

 

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

 

In a key sub-chronic toxicity study (Shell, 1980), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 10400 mg/m3, 5200 mg/m3, and 2600 mg/m3 to assess inhalation toxicity. No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed. Liver and kidney weights were increased in male rats at all exposure levels, male heart weights were increased at the highest exposure level and liver and kidney weights were increased in female rats at 10400 mg/m3. In addition, the male rats exposed to the test material at all concentrations showed tubular degeneration and hyaline inclusion-droplets in the epithelium. There was also scattered degeneration of the proximal renal tubules which showed cytoplasmic pallor and shrinkage. Occasionally the degenerate tubules were surrounded by a lymphocyte infiltrate. Many tubules also showed dilatation of the cortico-medullary junction, the dilated tubule being filled with a flocculent eosinophilic material. 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 weights mentioned above were judged to have been compensatory rather than toxic effects. Based on these results, the No Observed Adverse Effect Concentration (NOAEC) was determined to be greater than or equal to 10400 mg/m3.

 

In a supporting sub-chronic toxicity study (ExxonMobil Corp., 1978), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by inhalation to rats at vapor concentrations of 300 or 900 ppm for 6 hours/day, 5 days/week for 12 weeks. No treatment-related effects on mortality were observed and there were no significant alterations in hematology or clinical chemistry parameters. Body weights were decreased and kidney weights were elevated in male rats at 300 and 900 ppm. Relative mean liver weights were elevated in males at 900 ppm, but no changes were noted in histopathology. Under the conditions of this study, the No Observed Adverse Effect Concentration (NOAEC) was determined to be greater than 900 ppm (> 5220 mg/m3).

 

In a supporting short-term toxicity study (Chevron Phillips, 1969), four rhesus monkeys were exposed to 4.2 mg/L of the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) for 6 hours/day, for 3 days a week for 4 weeks. A total of 13 treatments were completed. There were no noted changes in behavior, clinical chemistry, hematological, or histopathological parameters. The NOAEC in primates was determined to be > 4.2 mg/L (> 4200 mg/m3).

 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

 

In a supporting study (Chevron, 1969), test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics) vapor was introduced into a stainless steel and glass exposure chamber (volume‑85. cubic feet) at a rate of 4.25 cubic feet per minute. The final concentration of test material in the chamber was calculated to be 4.2 mg/L (654 ppm). An equilibrium period of 15 minutes was allowed prior to introduction of the test animals into the chamber. The exposure lasted for six hours per day, three days per week (Monday, Wednesday, and Friday), for a total of 13 exposures. Observations throughout the one month exposure period were conducted for behavioral patterns, body weight, food consumption, hematology, clinical chemistry, urine analysis, gross pathology, and microscopic pathology. All four rhesus monkeys survived the study. Observations for behavioral patterns, body weight, food consumption, clinical chemistry, urine analysis, gross pathology, and microscopic pathology were unremarkable. A slight lymphocytopenia and a slight neutrophilia were noted in the differential leucocyte count at the mid point of the study and after the last exposure.

 

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics

 

In a key sub-chronic repeated dose toxicity study (Shell, 1980), the test material (Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics ) was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 1500 mg/m3 and 3000 mg/m3, and 6000 mg/m3 to assess inhalation toxicity. No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed. Liver and kidney weights were increased in male rats at all exposure levels, and liver weights were increased in female rats at 6000 mg/m3. In addition, the male rats exposed to the test material at all concentrations contained multiple, hyaline, intracytoplasmic, inclusion-droplets in the epithelium of the proximal convoluted tubules and showed an increased incidence of focal cortical, tubular basophilia. 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 tubules 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 liver weight to body weight ratios mentioned above 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 6000 mg/m3.

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 from structurally related substances, Hydrocarbons, C9-C11, n-alkanes, <2% aromatics 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).