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Administrative data

Description of key information

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

Repeated Dose Inhalation 90d – NOAEC ≥ 10400 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

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
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 (×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

** P0.01

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

** P0.01

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

** P0.01

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.

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
Type of information:
experimental study
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.

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:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
2 key and 1 supporting read across studies from structural analogues available for assessment. BMDL level determined and presented in the additional information section below.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
10 400 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 Key and 3 supporting sub-chronic read across 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, C12-C16, isoalkanes, cyclics, <2% aromatics. However, data is available for structural analogues, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% atomatics; Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics and Hydrocarbons, C9-C11, isoalkanes, cyclics, <2%. This data is read across to base on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Oral

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

A 90-day sub-chronic study (ExxonMobil, 1990) was conducted in rats to assess the toxicity of Isopar M (Hydrocarbons, C10-C12, isoalkanes, <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. No treatment-related mortalities or clinical effects were observed. Animals in all dose group exhibited an overall mean weight gain. Minimal changes were noted in the hematology and serum chemistry values, however, all were considered to be either within normal biological variation or not adverse effects. The mean absolute and relative liver weights for both the 500 and 1000 mg/Kg dose groups (both sexes) were significantly greater than the corresponding control values; however, these changes were found to be reversible following the 28 day recovery period. Slight increases in mean kidney weights were also noted in the 500 and 1000 mg/Kg female dose groups, however, these changes were not considered to be adverse effects. All histopathological findings were minimal and no treatment related adverse effects were noted. Based on the data recorded in this study, the NOAEL for Hydrocarbons, C10-C12, isoalkanes, <2% aromatics is greater than 1000 mg/Kg.

 

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

In another key 90-day sub-chronic study (ExxonMobil, 1991) conducted in rats the test material (Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics) 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 hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics is >1000mg/Kg.

Additionally, OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) tests are proposed for structural analogues, Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics and Hydrocarbons, C14-C19, isoalkanes, cyclics, <2% aromatics. This data is read across to base on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13. This endpoint will be updated subsequent to ECHA's approval of the testing proposals and availability of data upon completion of the studies.

Inhalation

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).

 

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

In a key 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/m3to 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 greater than or equal to 10400 mg/m3.

 

In a supporting study (Chevron, 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).

 

In a supporting study (ExxonMobil, 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) is greater than 900 ppm (> 5220 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 (volume85. 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 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/m3and 3000 mg/m3, and 6000 mg/m3to 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.

Justification for classification or non-classification

Based on the available read across data, Hydrocarbons, C12-C16, isoalkanes, cyclics, <2% does not meet the criteria to be classified as a repeated dose toxicant under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).