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EC number: 203-892-1 | CAS number: 111-65-9
Endpoint summary
Administrative data
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 ≥ 24300 mg/m3 for rats (similar to OECD TG 413)
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: According to or similar to guideline study OECD 422: GLP
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- yes
- Limit test:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Details on oral exposure:
- Males were treated from day 14 prior to the mating phase until the end of the mating phase and then killed, Females were treated from day 14 prior to mating, through day 4 of lactation and then killed.
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Males were treated from day 14 prior to the mating phase until the end of the mating phase and then killed, Females were treated from day 14 prior to mating, through day 4 of lactation and then killed.
- Frequency of treatment:
- 7days/week
- Remarks:
- Doses / Concentrations:
0, 25, 150, or 1000 mg/kg/day (10 ml/kg dosing volume)
Basis:
other: gavage - No. of animals per sex per dose:
- 10 male, 10 female per group
Control group: 10 male, 10 female, 0.5% methylcellulose - Control animals:
- yes
- Observations and examinations performed and frequency:
- Effects on general toxicity, neurobehavioral activity, clinical chemistry, and hematology were evaluated. Gross necropsies and histopathologic examination of tissues were conducted with emphasis on the male reproductive tract.
- Sacrifice and pathology:
- All surviving animals were sacrificed following dosing
- Statistics:
- Adult body and organ weight, food consumption, clinical chemistry, open field activity and hematologic data (raw or transformed) were compared using either parametric or nonparametric (Kruskal-Wallis) ANOVA depending on whether the data were found to be homogeneous or nonhomogeneous using Bartlett's homogeneity of variance procedure. If ANOVA analysis indicated significant differences, Dunnett's test and Mann Whitney's U test, for parametric and nonparemetric data, respectively, were used to analyze for differences between the various dose groups.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- No deaths or clinical signs of toxicity or behavioral changes were noted. No significant differences in body weights or feed consumption were observed. Startle reflex, open field test, and forelimb grip reflex performance data also revealed no treatment-related findings.
There were also no treatment-related changes in hematology or blood chemistry parameters, organ weights or gross pathology. An apparent treatment-related, slight to moderate hyperplasia of the non-glandular mucosa of the stomach, associated with degeneration, hyperkeratosis and submucosal subacute inflammation and, in a few cases, with erosion, was seen in animals of all treated groups. This effect was considered an artifact of the dosing method and not directly related to the toxicity of the test material. No other treatment related histological changes were observed. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: No treatment-related mortality or significant adverse clinical effects occurred.
- Critical effects observed:
- not specified
- Conclusions:
- Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >= 1000 mg/kg/day, the highest dose tested.
- Executive summary:
Groups of 10 male and 10 female Sprague Dawley rats were dosed with decane daily by gavage at exposure levels of 0, 25, 150, or 1000 mg/kg/day. Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation. Oral dosing of decane produced no evidence of any adverse effects on clinical observations, organ weights, gross pathology, neurobehavioral activity, clinical chemistry or hematology endpoints. Evidence of irritation of the nonglandular mucosa of the stomach was observed, but was considered an artifact of the dosing method and not attributed to the inherent toxicity of the test material. Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >=1000 mg/kg/day, the highest dose tested.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- 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
- 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.
- 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
Referenceopen allclose all
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 (×103/mm3) |
7.6±2.7 |
9.2±2.3 |
10.4±2.8 |
10.8±2.3 |
7.7±1.7 |
RBC (×106/mm3) |
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 (×103/mm3) |
770±40 |
877±45 * |
1008±73 ** |
984±115 ** |
1050±96 |
Females |
N=8 |
N=10 |
N=8 |
N=10 |
N=3 |
WBC (×103/mm3) |
5.4±2.5 |
4.4±1.0 |
4.2±0.8 |
6.8±1.6 |
5.1±1.9 |
RBC (×106/mm3) |
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 (×103/mm3) |
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) |
GOFap-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.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- 1 key short-term read across study and 1 key sub-chronic read across study available from structural analogues. BMDL level determined and presented in the additional information section.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Male and nulliparous, nonpregnant female Sprague-Dawley rats (VAF/ Plus Crl:CD BR)
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approximately 5 wk old when purchased, acclimated for approximately 2 wk prior to the initiation of the study
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages in air-conditioned rooms
- Diet (e.g. ad libitum): Certified rodent diet 5002 (PMI Feeds, Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water from an automated watering system was available ad libitum
- Acclimation period: 2 weeks. All animals were assigned a temporary identification number at receipt and examined by the staff veterinarian during the acclimation period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12 during the acclimation and all nonexposure periods - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- other: nitrogen
- Details on inhalation exposure:
- Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Exposure levels were determined three times daily by gas chromatography. The highest concentration was approximately 75% of the lower explosive limit. Animals’ positions in the cages were rotated for each exposure to ensure uniform exposure of every animal.
Chamber Operation: Animals were housed individually in wire mesh, stainless steel cages within a 1000-L glass and stainless steel exposure chamber. Chamber temperature and humidity were monitored every half hour during exposure and maintained, to the extent possible, within the ranges of 20–24°C temperature and 40–60% relative humidity. Animals did not receive food or water during the exposure period. Exposure chambers were operated dynamically at a calibrated airflow rate of 200 L/min (lpm). Recordings of airflow and static pressure were made every half hour. All animals remained in the chamber for a minimum of 30 min at the end of exposure while the chamber was operated using clean air only. Chambers were exhausted through a system of coarse filter, HEPA filter, and charcoal filter.
Atmosphere Generation: LAND-2 was pumped directly from the 5-gal container, housed within a freezer constantly flushed with nitrogen, using a laboratory pump, equipped with a piston, that was insulated within a styrofoam container. An ice bag was placed on top of the piston to keep the piston chamber cold to inhibit volatilization of LAND-2 in the pump and delivery lines. LAND-2 was delivered onto the central glass helix of a countercurrent volatilization chamber (one generator per chamber). The glass helix was heated by an internal nichrome wire inserted in the center of the glass tube that supported the helix (external to the volatilization chamber) and was controlled by a variable autotransformer. House-line nitrogen delivered from a regulator with a backpressure gauge was divided with a stainless steel T into the generation flow system and a purge flow system. Purge nitrogen was delivered to the bottom of the tube containing the nichrome wire to continuously purge the area surrounding the wire, protecting it from oxidation. Nitrogen for the generation system was directed through a flowmeter to the ball-and-socket joint at the bottom of the volatilization chamber, flowed up the chamber, passed over the coil, and volatilized the test material. The LAND-2-laden nitrogen flowed through a T tube at the top of the volatilization chamber into the turret of a 1-m3 glass and stainless steel exposure chamber, where it mixed with room air to appropriate exposure concentrations as it was drawn into the chamber (flow rate of 200 lpm). Control animals were sham-exposed to nitrogen alone introduced into the turret and mixed with air in the chamber.
Exposure Chamber Monitoring: Samples for determination of analytical exposure levels and the major components of LAND-2 vapor were withdrawn by vacuum pump from the breathing zone in the exposure chambers three times per exposure for treated groups and once per exposure for controls. Samples were pulled through Teflon lines into the multipositional control module and directed to a Hewlett Packard 5890II gas chromatograph, equipped with a flame ionization detector for analysis by ASTM method D5134-92 (ASTM, 1992). Composition and stability of the test material were evaluated by characterizing neat LAND-2 and comparing the major components in the neat and generated atmospheres at the beginning and end of the study. Particle size distribution measurements of any background aerosol were performed once during each exposure for chambers and room air using a TSI Aerodynamic Particle Sizer. Samples were drawn for 20 s at a rate of 5 L/min. Mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD), and total mass concentration (TMC) were calculated. Nominal concentrations (mg/m3) were calculated from the loss of weight from the generation apparatus divided by the total air flow through the chamber during exposure. This value was converted to parts per million (ppm) using an average molecular weight for this mixture of 89.2. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Determined three times daily by gas chromatography.
Composition and Uniformity Chamber Gas Chromatographic Results (% Weight): n-Butane: 3.21; iso-Pentane: 34.343; 2,3-Dimethylbutane: 12.977, 2-Methylpentane: 4.096; 2,4-Dimethylpentane: 5.663; 2,3-Dimethylpentane: 2.680; 2,2,4-Trimethylpentane: 16.885; 2,3,4-Trimethylpentane: 3.578; 2,3,3 –Trimethylpentane: 4.505; 2,2,5-Trimethylhexane: 2.499. - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hours/day, 5 days/week
- Remarks:
- Doses / Concentrations:
0, 668, 2220, and 6646 ppm (0, 2.4, 8.1, and 24.3 g/m3)
Basis:
analytical conc. - No. of animals per sex per dose:
- 12
- Control animals:
- yes, sham-exposed
- Details on study design:
- Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures.
Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically. - Observations and examinations performed and frequency:
- Animals were evaluated twice daily for mortality and gross signs of toxicological or pharmacological effects. During each exposure, rats were observed as a group once for abnormal behavior. Detailed physical examinations were performed twice pretest and weekly during the study. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at wk 14 (terminal) and 18 (recovery).
Body Weights and Food Consumption. All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period.
Animals were transported from the room in which they were housed during non-exposure hours to the neurotoxicity laboratories. Behaviors were evaluated pretest and during wk 5, 9, 14, and 18 (recovery groups). Temperature, humidity, and illumination were measured and recorded to minimize variation in environmental conditions during evaluations. Noise levels were not recorded. Testing was staggered over 8 sessions within a 4-d period, each session consisting of approximately 2 rats/sex/treatment group.
Motor Activity. Locomotor activity was monitored as a function of the number of beam breaks in an activity box, using an automated photo- beam activity system. Sessions were 60 mm in length divided into twelve 5-mm intervals. Rats were evaluated at pretest, at 3 time points during the treatment period (wk 5, 9, and 1 4), and at the end of the recovery period. Treatment groups were counter balanced across test times. Three sets of analyses were performed. The first analysis was conducted using the pre-dose data with animals nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals. The second analysis used data from the 3 treatment time points, and animals were nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals nested within the 3 time periods. The third analysis was similar to the first, using the recovery data. All three analyses tested for treatment effects, sex differences, treatment group by sex interactions, and these effects crossed with periods (second analysis only) and intervals. Analyses were repeated using transformed rank data to achieve a normal distribution of the residuals. Residuals from the models were tested for normality by the Shapiro-Wilk W-test or the Kolomogorov D-test.
Functional Operational Battery. The battery was comprised of home- cage evaluations (posture, vocalizations, and palpebral closure), handling evaluations (reactivity to general stimuli, signs of autonomic function), open-field behavior (arousal level and gait, urination and defecation frequency, convulsions, tremor, abnormal behaviors, piloerection, and exophthalmos), and reflex assessments (response to visual and auditory stimuli, tail pinch, pupillary function). Animals were also evaluated for forelimb and hindlimb grip strength, landing foot splay, and air righting ability. For landing foot splay, a small dot of paint was applied to each hindpaw. The rat was dropped from a height of 2 ft above a flat surface and the distance between the marks left by the hindpaws was measured in centimeters. To evaluate air righting reflex ability, the rat was held upside down, dropped from a height of 2 ft above a container of bedding, and the landing position was observed. Treatment groups were counterbalanced across test times. The observer performing the evaluation did not know the identity of the animal’s dose group. - Sacrifice and pathology:
- All animals were sacrificed by intraperitoneal injection of sodium pentobarbital, and tissues were preserved in situ by transcardial perfusion with phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde/1 % glutaraldehyde in the same buffer. Animals were killed at termination of 13 wk of exposure (week 13 terminal sacrifices performed during week 14) or at the completion of the 4-wk recovery period (control and high-dose groups only). A complete macroscopic examination was performed on all animals and 12 organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus, and uterus. The length and width of the brain of each rat was measured.
Thirty-nine tissues were preserved from all animals in all dose groups. Tissues from all animals in the control and high-dose groups were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological examination. The kidneys of selected animals were also stained with Mallory-Heidenhain stain. In addition, tissues of the nervous system were fixed for all animals. Brain, spinal cord, ganglia, and spinal nerve roots were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin-eosin, Luxol fast blue, and Sevier-Munger stains. Peripheral nerve sections (sciatic, tibial, sural, and optic) were embedded in glycol methacrylate and stained with toluidine blue. Slides of nervous system tissues were examined from animals (6/sex/group) designated through random selection for neuropathology, in the control and high-dose groups sacrificed at the end of 13 weeks of exposure. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons, and medulla. - Statistics:
- Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett’s test at 1 % significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1 % significance, two- sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett’s test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn’s summed rank test was used to determine differences from controls.
A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal orJonckheere’s test for monotonic trend in nonparametric cases. - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- All animals survived the treatment period and were sacrificed according to study design at the end of 13 wk or at 18 wk (recovery groups). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. No LAND-2-related ocular disease was observed. All groups showed similar mean body weights, body weight gains, and food consumption.
At wk 13 terminal sacrifice, there was a statistically significant decrease, relative to control values, in hemoglobin (5%), hematocrit (5%), and erythrocytes (7%) in blood of high dose males (data not shown). The hemoglobin was still decreased (4%) after the 28-d recovery period. However, because these differences were small and within the historical range for control animals in this laboratory, they are not considered toxicologically relevant. Clinical chemistry results showed a statistically significant decrease in aspartate aminotransferase (AST 32%) and alanine aminotransferase (ALT 46%) in females of the high-dose group (data not shown) compared to controls. However, several control female rats had elevated AST and ALT relative to the other nine female rats in the group and historical controls. Comparison of ALT and AST values from high-dose females with these elevated concurrent control values produced a statistical significance that is not toxicologically relevant. These results are not considered LAND-2 related.
NEUROBEHAVIORAL STUDIES
Motor Activity. Shapiro-Wilk analysis of data from the predose period indicated that the only statistically significant effects on response pattern occurred in the low-dose group due to inactivity among females for intervals 6—8, and increased activity of males during interval 10 (data not shown). Data from the treatment intervals and the recovery period were analyzed based on the Blom transformed data because the residuals from the model were not normally distributed by the Shapiro—Wilk statistic at the .01 level. There were statistically significant differences in the number (ct < .04) and relative pattern (cx. < .02) of beam breaks among the dose groups over the treatment testing periods. There were expected differences between sexes, and pattern differences across the 1 2 measuring intervals. In the recovery period in which only the room air controls and the high-dose animals were evaluated, there were no dose-group-related differences in response. Overall, dose-group differences did not occur in a dose-related pattern. Although statistically significant, the magnitudes of the differences were not large, and none of the treatment-group differences were larger than differences seen during the pre-dose period.
Functional Operational Battery. No differences were detected in the distance between foot splay for male or female rats in any dose group over any time interval evaluated. Grip strength of both fore- and hindlimbs in general increased from pretest through wk 14 for both sexes in all treatment groups. Values for control and high-dose recovery animals were lower at wk 18 than in previous treatment weeks but similar between the groups. There was no test-material-related effect on any endpoint evaluated within the functional operational battery of tests.
Pathology. At the wk 13 terminal sacrifice there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex, based on positive staining reaction by the Mallory-Heidenhain method, and increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific and are not considered relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at wk 13 sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Lung and brain weights were comparable to controls. Lungs were macroscopically and microscopically comparable to controls. Brain length and width measurements showed no test-material-related effects. There were no microscopic findings in the brain, spinal cord, or peripheral nerves that could be attributable to exposure to LAND-2. - Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 24 300 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no effects except adaptive response of liver weight to exposure equivalent to 6646 ppm
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
- Executive summary:
The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- 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
- 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.
Referenceopen allclose all
Under the conditions of this inhalation study in rats, the NOAEC was 8.4 mg/L.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 24 300 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- 1 key short-term substance specific study available, 2 key and 1 supporting sub-chronic studies available from structural analogues
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
- Critical effects observed:
- not specified
- Endpoint:
- sub-chronic toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Short-term repeated dose toxicity data is available for Octane. Additional short-term data is available for structural analogue Decane. Sub-chronic data is available for structural analogues Nonane, Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics and light alkylate naphtha distillate. 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:
Decane
In a key study (Sasol, 1995) groups of 10 male and 10 female Sprague Dawley rats were dosed with decane daily by gavage at exposure levels of 0, 25, 150, or 1000 mg/kg/day. Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation. Oral dosing of decane produced no evidence of any adverse effects on clinical observations, organ weights, gross pathology, neurobehavioral activity, clinical chemistry or hematology endpoints. Evidence of irritation of the nonglandular mucosa of the stomach was observed, but was considered an artifact of the dosing method and not attributed to the inherent toxicity of the test material. Based on these data, the no-observable- adverse effect level (NOAEL) for repeated dose toxicty was >=1000 mg/kg/day, the highest dose tested.
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.
Additionally, an OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) test is proposed for structural analogue, Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics.This endpoint will be updated subsequent to ECHA's approval of the testing proposal and availability of data upon completion of the study.
Inhalation:
Octane
In a key study (CEFIC, 2000), 8 male rats/dose were exposed via inhalation to 0, 1400, 4200 or 14000 mg/m³ Octane for 8 hours/day for 3 consecutive days. Animals were observed for clinical signs of toxicity and tested daily for effects on motor activity, functional observation measures and learned performance of a visual discrimination task. No significant treatment-related effects were observed up to the highest dose tested. Therefore, the NOAEC was determined to be >14000 mg/ m³.
Nonane
In a key study conducted similar to OECD 413 (Carpenter et al., 1978), groups of male rats were exposed by whole body inhalation to 0, 360, 590, or 1600 ppm n-nonane for 6 hours/day, 5 days/week, for 13 weeks. Clinical signs included salivation, mild loss of coordination and fine tremors throughout the first 4 days of exposure in the high dose group. Salivation and lacrimation were observed during the remaining exposure periods. Mean body weights or mean body weight changes of rats at 1600 ppm were statistically significantly lower than controls throughout the study. Histopathological evaluation conducted on weeks 4, 8, and 13 revealed only common sporadic lesions that were not considered to be treatment-related. No effects were noted in the 360 or 590 ppm dose groups throughout the study. The NOAEC for this study was determined to be 8400 mg/m³ corresponding to 1600 ppm.
Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics
In a supporting study (ExxonMobil, 1978), the test material (Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics) was administered by inhalation to Sprague-Dawley rats for 6 hours/day, 5 days/week for 12 weeks at nominal vapor concentrations of 300 ppm and 900 ppm to assess subchronic inhalation toxicity. Ten animals per sex per group were examined at 4 weeks, 8 weeks and all survivors were sacrificed and examined at 12 weeks. Male body weight gain was significantly decreased at 900 ppm. There were no treatment-related effects in any of the hematology and serum chemistry values. Liver and kidney weights were increased in male rats at 900 ppm, and adrenal weights were increased in female rats at 900 ppm. The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy. Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats. These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes. These kidney effects are specific to male rats and are not considered to be of biological relevance to humans. Histopathological examination did not reveal any abnormalities that were considered treatment related. As there were no pathologic changes, changes in organ weight to body weight ratios were judged to have been compensatory rather than toxic effects. Based on these results, the No Observed Adverse Effect Concentration (NOAEC) was greater than or equal to 900 ppm (>=5220 mg/m3).
Light alkylate naphtha distillate
A key 13-week inhalation toxicity study (Schreiner et al., 1998), was conducted using wholly vaporized light alkylate naphtha distillate (LAND-2) generated in nitrogen. Male and female rats were exposed by inhalation in whole-body exposure cages 6 hours/day, 5 days/week for 13 weeks at analytical concentrations of 0, 668, 2220, and 6646 ppm. All animals survived the treatment period and were sacrificed according to study design at the end of week 13 or 18 (recovery group). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. At week 13, there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex as well as an increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific. Therefore, these effects are not considered to be relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at week 13 after sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Thus, the NOAEC for systemic toxicity was 24,300 mg/m³ corresponding to 6646 ppm.
Other routes:
Octane
Enzyme activity and potential for hepatotoxicity have been studied (Khanet al., 1980) in female rats using octane by daily intraperitoneal injection of 1 mL (corresponding to ca. 703 mg/kg bw) for 2 or 7 days. Reduction in specific activity of the enzymes, benzo(a)pyrene hydroxylase, benzphetamine-N-demethylase, p-nitroanisole-o-demethylase 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.
In another study (Khan and Pandya, 1980) examining enzyme activity and potential for hepatotoxicity, female rats received a daily intraperitoneal injection of 1 mL (corresponding to ca. 703 mg/kg bw) for 2 or 7 days. 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.
In another study examining enzyme activity and potential for hepatotoxicity, administration of octane (intraperitoneal injection of 1 mL corresponding to ca. 703 mg/kg bw) 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 substance specific and read across data, Octane 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).
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