Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Details of age and body weight at study initiation, acclimation not reported; particle size distribution not reported; no satellite (reversibility) groups included
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
The sub-chronic toxicity in rats of isoparaffinic solvents.
Author:
Carrillo JC, Adenuga MD, McKee RH, Roth RN, Steup D and Simpson BJ.
Year:
2013
Bibliographic source:
Regul Toxicol Pharmacol. 67(3):446-455.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Details of age and body weight at study initiation, acclimation not reported; particle size distribution not reported; no satellite (reversibility) groups included
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Renewable hydrocarbons C9-C15, n-alkanes, isoalkanes, <0.005% aromatics
- Analytical purity: >98 %
- Composition of test material, percentage of components:
Composition of the C10–C12 isoparaffinic solvent by hydrocarbon type
C8 Paraffins: <1 %
C9 Paraffins: 1 %
C9 Naphthenes: <1 %
C10 Paraffins: 15 %
C10 Naphthenes: 1 %
C11 Paraffins: 39 %
C12 Paraffins: 44 %
Total Paraffins: 99 %
Total naphthenes: 1 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: Rats were housed in groups of three by sex, in hanging aluminum cages fitted with steel mesh bases.
- Food was not available during the exposure periods, but water was freely available at all times.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4-26.1 °C
- Humidity: 37-74 %

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Aluminum exposure chambers (1 m3) were used; these were ventilated by air drawn from the laboratory through dust filters.
- Twelve cages arranged in two layers in each exposure chamber. The animals remained in the exposure chambers throughout the study.
- Temperature, humidity, pressure in air chamber: During the study, the laboratory temperature varied between 19.4 and 26.1 °C, the relative humidity ranged between 37 and 74 %, and the barometric pressure ranged between 753 and 768 mmHg.
- Air flow rate: Air flow rate was monitored continuously throughout the experiment using an electro-anemometer mounted in the duct and slight adjustments made when necessary to maintain a constant exhaust rate. Individual flow rates through each chamber were balanced and adjusted to 0.50 m3 per minute before exposures began, but were not checked during the exposure.
- Treatment of exhaust air: The exhaust ducts from each chamber entered a common exhaust duct through which the air was drawn by fan at a rate of 2.0 ± 0.03 m3 per minute.

TEST ATMOSPHERE
- Test atmospheres were generated by complete volatilization of test material into the stream of ventilating air. The vaporizers were electrically heated quartz tubes whose surface temperatures were adjusted during preliminary experiments to the minimal required for complete vaporization of the test material. A total hydrocarbon analyzer fitted with a flame-ionization detector (Beckman 109A) was used to sequentially analyze the test atmospheres.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h each day, 5 days each week for thirteen weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
2600, 5200 and 10400 mg/m3 (2529, 5200 and 10186 mg/m3 measured)
Basis:

No. of animals per sex per dose:
Groups of 18 rats of each sex were exposed to fully vaporized test material at target concentrations of 2600, 5200 and 10400 mg/m3.
Control animals:
yes
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed daily for general health and behavior.

BODY WEIGHT, FOOD AND WATER CONSUMPTION: Yes
- Time schedule for examinations: Body weights, food intakes and water intakes were recorded weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- After 10 weeks exposure, blood samples taken from the tail vein were analyzed for glucose concentration.
- At the study termination, blood was taken from all surviving rats by cardiac puncture, and the following hematological parameters determined: erythrocyte count (RBC), mean cell volume (MCV), hemoglobin concentration (Hb), leukocyte count (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), prothrombin time and kaolin-cephalin coagulation time.
- Clinical chemical determinations on plasma comprised: total protein, urea nitrogen, alkaline phosphatase, alanine amino transferase (ALT), aspartate amino transferase (AST), sodium, potassium, chloride and albumin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; At the end of the study all animals were administered a lethal dose of sodium pentobarbitone and underwent a detailed postmortem examination.

HISTOPATHOLOGY: Yes; Brain, heart, kidneys, liver, spleen and testes were weighed and a wide range of tissues were removed for fixation and subsequent histopathological examination. The tissues of all high and medium dose group animals, and controls were examined microscopically. Additionally the kidneys from the low dose group animals were examined microscopically.
Other examinations:
None
Statistics:
- Body and organ weights were analyzed by covariance analysis using the initial body weight as the covariate. Means were adjusted for initial body weight if a significant covariance relationship existed. Where no 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 weights were reported as adjusted if a significant covariance relationship existed. Clinical chemical and hematological data were examined using analysis of variance. The analysis allowed for the fact that the animals were multi-housed.
- The significance of any difference between control and treated groups was tested using Williams’ t test (Williams, 1971,1972). If a monotonic dose response could not be assumed, Dunnet’s t test (Dunnet, 1964) was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
- The general health of the rats was good throughout the study. There were no clinical signs of toxicity in either the low or medium dose groups. Both males and females in the high dose group were slightly lethargic when examined one hour after exposure had ceased, but had recovered the following day prior to the next exposure to test material.

BODY WEIGHT AND WEIGHT GAIN
- Body weights of the males in the high dose group were slightly lower (2.7–5.6 %) than the corresponding controls (P ≤ 0.1) during the first 6 weeks of exposure. Thereafter, although the weights were still below control values (approx. 2–3 %), the differences were not statistically significant.
- At various times, female body weights in all dose groups were also lower than the controls (P ≤ 005), but the differences were less than for the males (2.3–4.7 %) and did not appear to be dose-related. As with the males, at study termination the average body weights of all female dosed groups were not significantly different than control values.

FOOD CONSUMPTION
- During the first five weeks of the study, food consumption in the high dose males was less than the controls (up to an approximately 18 % reduction), but thereafter, there were no differences with control values.
- Reduced food consumption was seen sporadically among females in the high dose, but was not significantly different from control values during the final weeks of the study.

WATER CONSUMPTION
- Weekly water intakes were greater than controls in the high dose males (9–46 %) throughout the study. Apart from a few sporadic differences, the water intakes for all other treated groups were similar to those of the corresponding controls.

HAEMATOLOGY
- With the exception of a slight but statistically significant decrease in percentage of neutrophils and leukocytes in the female high dose group, none of the other hematological parameters in females was affected with treatment. However, a slight reduction in hematological parameters was observed in all male exposed groups including small but statistically significant reductions (approximately 3–5 %) in hemoglobin concentrations, packed cell volumes and red cell counts. Although there was only a 1% increase in mean corpuscular hemoglobin concentration in the high dose males, it was nevertheless statistically significant (P ≤ 0.01).
- Total white cell count was reduced in the high dose males and the percentage of neutrophils was increased (47 %) and the percentage of leucocytes was decreased slightly (11 %). The percentage of eosinophils was approximately doubled in both the mid- and high dose group males, and was significantly different from the control value.

CLINICAL CHEMISTRY
- Blood glucose concentrations of treated groups after 10 weeks of exposure did not differ from those of the controls.
- After 13 weeks exposure to test material, the serum protein concentrations in the high dose males were unaffected but were increased by approximately 7 % in the high dose females. The serum albumin concentrations were elevated in both high dose males (4 %) and females (12 %).
- Alkaline phosphatase was increased (16 %) in the high dose male group, but not in any other treatment group of either sex. Decreases in ALT (23–30 %) and AST (20–32 %) were seen at all exposure levels in the females, but levels of these enzymes in males were not significantly different from control values.
- Potassium levels were increased by 8 % in the high dose males and chloride levels were increased by 2 % in both the medium and high dose group males.
- Blood urea nitrogen was unaffected by treatment.

ORGAN WEIGHTS
- Neither brain weights nor spleen weights (absolute) in either sex, nor testis weights in males were affected by exposure to C10–C12 isoparaffinic solvent. There was a slight (6 %) increase in heart weight (absolute) for high dose males only.
- Kidney weights (absolute) were significantly increased in the low (21.5 %), medium (25 %) and high (34.5 %) male dose groups. Additionally, increases in liver weights (absolute) of 10, 14 and 32 % were seen in the low, medium and high male dose groups, respectively. In females, the liver weights (absolute) were increased in both the medium (10 %) and high (37 %) dose groups, while the kidney weights (absolute) were increased only in the high (14 %) dose group. The organ weights adjusted for terminal body weight (relative organ weights) showed the same pattern of changes and in addition, showed an increase (8 %) in spleen weight for the high dose group males.

HISTOPATHOLOGY
- Treatment-related tubular degeneration and hyaline inclusion droplets in the tubular epithelium were found in the kidneys of male rats exposed to all concentrations of C10–C12 isoparaffinic solvent. The findings in the male kidneys consisted of 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 proximal tubular cells of male rats also showed eosinophilic droplets, but there did not appear to be a relationship between these droplets and the intensity of the other renal lesions. Treatment-related changes were absent from the kidneys of females exposed to C10–C12 isoparaffinic solvent. While mineralization was commonly seen, mainly at the cortico-medullary junction, the incidence was similar in treated and control animals.
- Many of the rats, males and females, showed low-grade pulmonary lesions comprising a minimal increase in alveolar wall thickening and chronic infiltration in the lungs. Such changes were not uncommon in Wistar rats of this age at the testing facility, and the frequency and severity of the lesions were similar in treated and control animals.
- The increased liver weights were not associated with any histological findings, and there were no notable histological findings in any of the other organs examined.
- A papilloma of the renal pelvis in one male, and a renal mesenchymoma in a female were observed, both of which were in the control groups.
- No other treatment-related pathological findings were observed.

Effect levels

Dose descriptor:
NOAEC
Effect level:
> 10 000 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related mortality or signifcant adverse clinical effects occured.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.2/2: Clinical chemistry – results

 

Parameter

Exposure concentration (ppm)

SD of single observation

0 (control)

359

737

1444

Males

Protein (g/l)

66.4

66.6

66.5

67.9

2.29

Urea (mm/l)

9.2

9.2

9.2

9.1

0.67

APa (IU)

86

87

92

100.0b

18.0

ALT (IU)

29

30

34

36

12.4

AST (IU)

49

46

46

47

11.9

Na (mm/l)

143

143

143

144

1.6

K (mm/l)

4.8

4.9

5.1

5.2c

0.46

Cl (mm/l)

101

102

103.0c

103.0c

1.2

Albumin (g/l)

39.4

38.9

39.1

41.3c

1.53

Glucosed (mm/l)

5.8

5.9

5.8

5.8

0.68

Females

Protein (g/l)

69.3

70.0

71.1

74.5c

3.17

Urea (mm/l)

9.0

8.5

8.4

8.6

1.11

APa (IU)

56

55

50

54

15.2

ALT (IU)

27

21.0b

21.0b

19.0c

7.2

AST (IU)

54

43.0c

43.0c

37.0c

8.2

Na (mm/l)

143

143

144

144

1.7

K (mm/l)

4.7

4.5

4.2

4.7

0.91

Cl (mm/l)

106

106

106

106

1.6

Albumin (g/l)

42.5

43.8

44.2

47.8c

2.7

Glucosed (mm/l)

5.3

5.4

5.5

5.7

0.65

 

a Abbreviations: AP – alkaline phosphatase, ALT – alanine amino transferase, AST – aspartate amino transferase, Na – sodium, Cl – chlorine.

bP ≤ 0.05

cP ≤ 0.01

dGlucose determinations were made after 10 weeks exposure.

 

 Table 7.5.2/3: Hematology results - males

 

Parameter

Exposure concentration (ppm)

SD of single observation

0 (control)

359

737

1444

Hba(g/100 ml)

15.0

14.6b

14.3c

14.4c

0.45

PCV (%)

41

40.0c

39.0c

39.0c

1.3

RBC (x 106/cm3)

7.79

757b

7.46c

7.46c

0.301

WBC (x 106/cm3)

4.5

4.6

4.0

3.60c

0.68

MCV (µ3)

53

53

53

52

1.4

MCH (pg)

19.3

19.3

19.2

19.3

0.51

MCHC (g/100 ml)

36.1

36.4

36.2

36.6b

0.41

PT (s)

15.3

15.6

15.9

16.3

1.71

KCCT (s)

24.3

25.0

26.5

26.8b

3.31

 

a Abbreviations: Hb – hemoglobin, PCV – packed cell volume, RBC – red blood cell, WBC – white blood cell, MCV – mean cell volume, MCH – mean corpuscular hemoglobin,

pg – pictogram, MCHC – mean corpuscular hemoglobin concentration, PT – prothrombin time, KCCT – kaolin-cephalin coagulation time.

bP ≤ 0.05

cP ≤ 0.01

 

Table 7.5.2/4: Liver and kidney weights of rats

 

Parameter

Exposure concentration (ppm)

SD of single observation

0 (control)

359

737

1444

Males

Absolute organ weight

Liver

15.4

17.0a

17.6b

20.4b

1.760

Kidney

2.78

3.38b

3.48b

3.74b

0.289

Heart

1.17

1.24

1.23

1.24a

0.089

Organ weight adjusted for terminal body weight

Liver

15.40

16.59b

17.40b

21.00b

1.258

Kidney

2.77

3.33b

3.45a

3.83b

0.285

Heart

1.17

1.22

1.22

1.27b

0.081

Females

Absolute organ weight

Liver

9.06

9.19

10.11b

12.61b

0.953

Kidney

1.80

1.85

1.89

2.05a

0.155

Heart

0.84

0.83

0.85

0.82

0.100

Organ weight adjusted for terminal body weight

Liver

8.92

9.29

10.09b

12.67b

0.840

Kidney

1.78

1.87

1.880

2.06b

0.154

Heart

0.83

0.84

0.85

0.82

0.100

 

aP ≤ 0.05

bP ≤ 0.01

Applicant's summary and conclusion

Conclusions:
No Observed Adverse Effect Concentration (NOAEC) was the highest level used in this study, >10,000 mg/m3. As the effects seen in this study were consistent with those reported in 4 other studies of isoparaffinic solvents with differing carbon numbers and other low aromatic C9–C14 aliphatic solvents (Amoruso et al., 2008), it seems reasonable to consider the effects as generic and to use the results of the study of this C10–C12 isoparaffinic solvent as representative of other low aromatic C9–C14 aliphatic solvents, and, further, to use the NOAEC for purposes of assessing risk and/or developing risk management measures including occupational exposure limits for solvents of this type.
Executive summary:

In a repeated dose toxicity study conducted similarly to the OECD Guideline 413, groups of 18 Wistar rats of each sex were exposed to fully vaporized test material at target concentrations of 2600, 5200 and 10400 mg/m3 (2529, 5200 and 10186 mg/m3 measured) for 6 hours per day, 5 days per week for 13 weeks. Control animals received air only. During the study, clinical condition, body weight, food and water consumption, haematology clinical chemistry, organ weight and histopathology investigations were undertaken.

There were no clinical signs of toxicity in either the low or medium dose groups. Both males and females in the high dose group were slightly lethargic, but had recovered the following day prior to the next exposure to test material.

 

Body weights of the males in the high dose group were slightly lower than the corresponding controls (P ≤ 0.1) during the first 6 weeks of exposure. Body weights of the females in all dose groups were also lower than the controls (P ≤ 005), but the differences were less than for the males (2.3–4.7 %) and did not appear to be dose-related. At study termination the average body weights of all dose groups were not significantly different than control values.

 

During the first five weeks of the study, food consumption in the high dose males was less than the controls, but thereafter, there were no differences with control values. Reduced food consumption was seen sporadically among females in the high dose, but was not significantly different from control values during the final weeks of the study. Weekly water intakes were greater than controls in the high dose males throughout the study. Apart from a few sporadic differences, the water intakes for all other treated groups were similar to those of the corresponding controls.

 

No treatment-related changes detected in haematological parameters of females, except slight but statistically significant decrease in percentage of neutrophils and leukocytes in the female high dose group. However, a slight reduction in hematological parameters was observed in all male exposed groups including small but statistically significant reductions in hemoglobin concentrations, packed cell volumes and red cell counts. Total white cell count was reduced in the high dose males and the percentage of neutrophils was increased (47 %) and the percentage of leucocytes was decreased slightly (11 %). The percentage of eosinophils was approximately doubled in both the mid- and high dose group males, and was significantly different from the control value.

 

Blood glucose concentrations of treated groups after 10 weeks of exposure did not differ from those of the controls. After 13 weeks exposure to test material, the serum protein concentrations in the high dose males were unaffected but were increased in the high dose females. Elevated serum albumin concentrations in both sexes; increased alkaline phosphatase & potassium in males at high dose groups; whereas chloride levels were increased in both the medium and high dose group males. Decreases in ALT and AST were seen at all exposure levels in the females, but levels of these enzymes in males were not significantly different from control values. Blood urea nitrogen was unaffected by treatment.

 

Kidney and liver weights (absolute) were significantly increased in the low, medium and high male dose groups. In females, the liver weights (absolute) were increased in both the medium and high dose groups, while the kidney weights (absolute) were increased only in the high dose group. The organ weights adjusted for terminal body weight (relative organ weights) showed the same pattern of changes and in addition, showed an increase in spleen weight for the high dose group males.

 

Treatment-related tubular degeneration and hyaline inclusion droplets in the tubular epithelium were found in the kidneys of male rats in all dose groups; whereas no treatment-related changes were observed in females. The increased liver weights were not associated with any histological findings, and there were no notable histological findings in any of the other organs examined. No other treatment-related pathological findings were observed.

 

The male rat kidney changes were due to an α2u globulin process and not relevant for human health or risk assessment. The liver enlargement without pathologic changes or elevations in liver enzyme markers was considered to be an adaptive response. The reason for the reductions in haematological parameters that were observed in males only is not clear, but it is suggested that these were either due to normal variation or a secondary consequence of the nephropathy.

 

The overall No Observed Adverse Effect Concentration (NOAEC) was greater than10000 mg/m3 in rats.