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Repeated dose toxicity: oral

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Administrative data

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.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
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 source
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:

This data is being read across from the source study that tested Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics based on analogue read across.

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991
Reference Type:
publication
Title:
The sub-chronic oral toxicity of dearomatized hydrocarbon solvents in Sprague–Dawley rats
Author:
David Adenuga, Juan-Carlos Carrillo, Richard H. Mckee
Year:
2014
Bibliographic source:
Regul Toxicol Pharmacol . 2014 Dec;70(3):659-72.
Report date:
2014

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics
EC Number:
918-481-9
IUPAC Name:
Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics
Details on test material:
- Name of test material (as cited in study report): MRD-89-582
- Physical state: colorless liquid
- Analytical purity: assumed 100% pure
- Lot/batch No.: Batch II
- Expiration date of the lot/batch:

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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).

Effect levels

open allclose all
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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.