Hydrocarbons, C9-C10, n-alkanes, isoalkanes, cyclics, <2% aromatics

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of toxicity were absent at all exposure levels when the animals were examined before and after each day's exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in male body weights related to exposure were observed. Female body weights appeared to be lower than control at the high exposure level at some time points during the course of the study (e.g. between week 8 and 10), without achieving significant difference and were within the
range of the control group at the end of study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Female food intakes showed significant reduction at the high exposure level at the second week of exposure, and there was a tendency for female rats at this exposure level to eat less than control rats throughout the study. Although lower food intake may be related to the apparent lower body weight gain, there were no clinical signs of toxicity. No differences in food intake were observed in male rats.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Some significant increases in water intake were seen in males exposed to the high concentration of test material at weeks 4 and 5, but returned to control levels in subsequent weeks until the end of the study. This change in water intake did not seem to have an impact on the study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small but statistically significant decrease (p≤0.05) in reticulocytes was observed in male rats in the high exposure group (other exposure levels were not assessed). Although this parameter is significantly lower (2.12 ± 0.9%) than controls (2.83 ± 1.0%), it was within the reference range of males rats at this age (< 6 months), with a mean of 2.0 ± 1.23% (Wolford et al., 1986), suggestive of a chance finding rather than being treatment related. There was no corresponding effect in female rats. All other red and white cell parameters were normal.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A significant decrease in alanine aminotransferase (ALT) values was observed in the low treatment group (p≤0.05) females, but not in males. Aside from the absence of any dose/response in the ALT levels in exposed females, the levels were within normal physiological range (Okamura et al., 2011) and did not appear to be toxicologically relevant. Compared to the control group, no significant differences were observed in other clinical parameters in males or females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood was present in the urine samples of two rats but this could be attributed to slight damage to the claws from the mesh floor of the urine collector. Glucose and protein were also present in the urine of many of the rats, but since the urine was collected over night between exposures and the animals had to be fed during the collection period, this was not unexpected.

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):

There was an increase in kidney and liver weights of the male rats from the high exposure group. When adjusted for terminal body weight, a statistical increase at all exposure levels was evident indicating that body weight gain during the study did not play a role in the organ weight increases but rather was a consequence of exposure to the test material. In females, terminal and adjusted liver weights were only increased at the high exposure level. These differences were not observed in terminal or adjusted female kidney weights at any exposure level.

These treatment-related and exposure level dependent increases in liver weights (7–16% over control) were not accompanied by histopathological changes or elevated levels of markers of liver damage, ALT and aspartate aminotransferase (AST), typical markers of liver damage. The observations suggest that the increase in liver weights was evidence of an adaptive response to increasing metabolic demand.

The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been
conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health.

A statistical increase in the adjusted heart weight of males in the low exposure group was also observed. As this was not observed in the higher exposure groups, and there was no concomitant clinical or histopathological change, this finding was not considered toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross examination at necropsy revealed an increased incidence of renal pallor and subcapsular granularity in the male rats exposed to the high concentration (6000 mg/m3). No other changes were found in either sex that could be attributed to the test material exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys
Kidneys of all male rats exposed to all concentrations of the test material contained multiple, hyaline, intracytoplasmic, inclusion-droplets in the epithelium of the proximal convoluted tubules and showed an increased incidence of local cortical, tubular basophilia. Hyaline droplets, were most frequently found in the proximal tubular epithelium of the outer cortex and varied in number and size from cell to cell.

Focally, epithelial cells appeared to be enlarged due to intense droplet aggregations in the cytoplasm. Free droplets or amorphous, hyaline material were seen occasionally in tubular lumina. Minor sloughing of tubular epithelium was evident focally but affected tubules showed no evidence of necrosis and no inflammatory response was visible in association with droplet formation or epithelial sloughing.

A series of special staining techniques were performed on a representative sample of kidneys from the high concentration and control males to determine the nature of the hyaline droplets. The droplets showed a positive reaction when stained for protein with eosin and Mallory's phloxin, and a negative reaction for the other staining techniques. Focal, tubular basophilia was seen more frequently in the renal cortices of treated males than in controls of the same sex. The basophilic foci were quantified on the basis of frequency of occurrence in paraffin sections stained with haematoxylin and eosin. Proximal tubules were most frequently affected. The main features of the lesions were pale-staining, filamentous cytoplasm, mild focal cytoplasmic swelling or vacuolation and mild to moderate thickening of tubular basement membranes. Larger foci showed minor interstitial fibrosis and lymphocyte/ macrophage infiltration. The involved cells did not appear necrotic; their nuclei were pale but of normal size and morphology. Hyaline droplets were rarely present in the cytoplasm of these basophilic foci. Neither the presence of hyaline inclusion droplets nor the incidence of total tubular basophilia exhibited a clear dose/response relationship; histological lesions in the kidneys of the low concentration males were similar in type and intensity to those of the high concentration group.

Hyaline droplets were not found in the kidneys of female rats at any exposure level. Nephrocalcinosis was a common observation in female rats in all groups including controls. Small foci of tubular basophilia were seen occasionally in both exposed and control females but the inflammatory or fibrotic changes found in some of the exposed male rats were rarely seen.

The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been
conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health.

Respiratory Tract
A low grade catarrhal inflammatory reaction was evident in the nasal cavities of a majority of the rats exposed to the medium concentration (3000 mg/m3). The lesions were confined to the olfactory epithelium and comprised mild mucosal and submucosal edema, focal congestion and diffuse low grade inflammatory cell infiltrates. Unilateral and bilateral lesions occurred. Some desquamation of olfactory epithelium was visible focally but there was no evidence of necrosis or significant degenerative changes. The respiratory epithelium was not affected and the animals exhibited no clinical signs of rhinitis. The nasal cavities of the low and high exposure groups were similar to the control group in all respects.

Most rats showed low-grade pulmonary lesions comprising minor perivascular lymphocyte and eosinophil infiltration, discrete aggregates of foamy macrophages and focal alveolar wall thickening due to macrophage, lymphocyte and neutrophil infiltration. The lungs of several animals showed focal granulomata. Such changes were not uncommon in control Wistar rats of the testing facility; the frequency and type of the lesions were similar in treated and control animals. No treatment-related changes were observed in the trachea or bronchi of animals.

Except for a range of minor spontaneous lesions found in some animals in all experimental groups that were within the historical range of the Wistar rat colony used in the testing facility, no other pathological changes were observed in other organ tissues of male or female rats that could be associated with exposure to the test material.

Histopathological findings: neoplastic:

not examined

Details on results:

Overall, the key treatment-related effects of exposure to the Naphthenic solvent, C9-C11, <2% aromatics, were restricted to male rat kidney damage and adaptive liver enlargement in males and females. These effects are commonly associated with exposures to structurally related straight chain or branched chain aliphatic hydrocarbons.

Key result

Dose descriptor:

NOAEC

Effect level:

6 000 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/m³ air

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

In the low and medium exposure groups the measured concentrations had a mean of 1500 (±16) mg/m³ and 2980 (±55) mg/m³ versus the target concentrations of 1500 and 3000 mg/m³ respectively. However there was more variability in the high exposure group in which the mean exposure was 5950 (±416) mg/m³, versus a target of 6000 mg/m³. When the concentration of this exposure level exceeded the target by more than 20% for longer than approximately 36 min (10% of a daily exposure) the approximate extent of the deviation was recorded. These extremes only occurred about once per week reaching about 7200 and 8400 mg/m³ for periods of 0.75–4.75 h.

Table 1. Mean Clinical Chemistry values after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m³

Parameters	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
Protein (g/l)	66.2 ± 3.0	64.1± 3.9	67.9± 2.9	67.2± 4.1	64.3± 4.4	65.3± 4.2	66.1± 2.9	65.6±3.8
Albumin (%)^a	53.2± 4.2	53.0± 4.9	52.4± 4.9	51.2± 4.0	57.7± 3.6	56.3± 3.2	55.9± 2.8	56.1± 4.5
Alpha 1 (%)^a	13.3± 2.0	12.1± 1.5	12.8± 1.6	12.5± 2.5	12.6± 1.1	12.3± 2.0	13.5± 1.5	12.7± 1.4
Alpha 2 (%)^a	9.4± 3.3	9.4± 1.4	9.4± 1.7	10.0± 1.8	7.3± 1.1	7.6± 1.0	7.5± 1.2	7.4± 1.9
Beta (%)^a	18.5± 2.9	19.6± 4.3	19.1± 3.1	20.1± 3.3	14.8± 1.8	15.4± 2.7	15.6± 2.8	16.5± 2.6
Gamma (%)^a	5.7± 3.4	5.8± 3.2	6.7± 3.5	6.3± 3.5	7.6± 2.0	8.3± 2.5	7.5± 1.7	7.4± 2.5
Urea (mm/l)	8.0± 0.8	8.2± 1.2	8.0± 0.8	8.3± 0.9	8.1± 1.3	8.1± 1.4	8.2± 1.5	8.4± 1.7
AP (IU)	71± 10	59± 16	69± 16	64± 18	43± 16	35± 12	33± 9	41± 13
ALT (IU)	22± 4.7	21± 6.7	21± 3.4	24± 14.2	23± 6.6	17± 3.6^*	19± 8.0	18± 3.7
AST (IU)	37± 9.7	35± 5.5	34± 6.3	35± 9.8	37± 8.3	38± 15.1	35± 8.9	32± 4.2
Na (mm/l)	145± 1.6	145± 2.7	145± 1.6	146± 1.6	143± 2.2	144± 2.2	144± 1.7	144± 1.5
K (mm/l)	5.3± 0.6	5.5± 0.8	5.5± 0.6	5.5± 0.8	5.4± 1.0	5.6± 0.8	5.2± 0.9	5.2± 0.9
CL (mm/l)	100± 2.6	99± 2.7	99± 1.9	99± 2.1	102± 2.3	102± 2.9	102± 1.9	102± 2.5
Glucose (mm/l)	4.0± 0.4	3.9± 0.4	3.8± 0.4	4.0± 0.4	4.7± 0.8	4.4± 0.8	4.1± 0.8	4.5± 0.6

* p≤0.05 significance of the difference between treatment and control means.

^a Protein electrophoresis fractions

Table 2. Mean Hematological values after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m3

Parameter	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
BBC (x10^6/cmm)	8.10± 0.5	7.89± 0.3	8.2± 0.5	7.94	7.30± 0.3	7.21± 0.4	7.46± 0.3	7.22± 0.5
Hb (g/100 ml)	15.3± 0.7	14.8± 0.5	15.3± 0.8	14.8± 0.5	14.6± 0.7	14.6± 0.9	15.0± 0.6	14.6± 0.7
HCT (%)	44± 1.9	43± 1.7	44± 2.1	43± 1.9	43± 1.6	42± 2.6	44± 1.9	42± 2.2
MCV (m^3)	53± 1.4	53± 1.9	53± 1.7	53± 1.4	57± 1.8	57± 1.9	57± 1.4	58± 1.9
MCH (pg)	18.5± 0.5	18.4± 0.6	18.3± 0.6	18.3± 0.7	19.6± 0.6	19.9± 0.5	19.8± 0.6	19.9± 0.7
MCHC (g/100ml)	34.4± 0.6	34.3± 0.7	34.4± 0.8	34.3± 0.7	34.1± 0.9	34.3± 0.8	34.3± 0.6	34.3± 0.6
PT (secs)	16.5± 1.0	17.0± 1.6	17.0± 1.4	16.9± 1.0	15.2± 0.7	15.1± 0.3	15.5± 1.8	15.1± 0.7
KCCT (secs)	27.7± 2.9	28.8± 5.4	28.8± 5.0	28.3± 4.2	25.0± 3.1	24.7± 3.0	25.1± 3.4	23.2± 4.0
Retics (%)	2.83± 1.0	-	-	2.12± 0.9^*	2.17± 1.2	-	-	2.44± 1.4
WBC (x10^3/mm)	4.7± 1.3	4.5± 1.2	4.7± 1.0	4.7± 1.2	3.6± 1.2	4.1± 1.5	3.8± 1.1	3.7± 1.0

 

Table 3. Mean and Adjusted Organ Weights (g) after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m³

Organ Weight (g)	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
Terminal body weight (g)	542± 40.3	532± 48.8	528± 44.9	530± 37.3	306± 18.3	301± 18.3	301± 17.8	299± 19.2
Brain	2.20± 0.10	2.21± 0.10	2.20± 0.08	2.24± 0.10	2.02± 0.08	2.01± 0.07	2.03± 0.05	2.02± 0.08
Heart	1.30± 0.13	1.41± 0.16	1.32± 0.11	1.39± 0.15	0.93± 0.08	0.91± 0.09	0.92± 0.6	0.90± 0.08
Liver	17.31± 2.08	18.21± 1.86	18.64± 2.02	20.11± 1.74^**	10.17± 0.61	11.07± 2.49	11.01± 0.85	11.92± 0.77^**
Spleen	0.96± 0.15	0.97± 0.07	0.96± 0.13	1.00± 0.10	0.72± 0.09	0.70± 0.10	0.71± 0.10	0.71± 0.10
Kidneys	3.22± 0.37	3.51± 0.37	3.51± 0.27	3.87± 0.32^**	2.03± 0.16	2.01± 0.13	2.04± 0.13	2.12± 0.16
Testes	3.45± 0.30	3.49± 0.29	3.49± 0.21	3.55± 0.23	-	-	-	-
Adjusted organ weights (g/100g body weight
Brain	0.41± 0.03	0.42± 0.04	0.42± 0.04	0.43± 0.03	0.66± 0.04	0.67± 0.04	0.68± 0.04	0.66± 0.05
Heart	0.24± 0.02	0.27± 0.03**	0.25± 0.02	0.26± 0.03	0.30± 0.03	0.30± 0.03	0.31± 0.03	0.30± 0.02
Liver	3.19± 0.21	3.43± 0.25^**	3.53± 0.22^**	3.80± 0.23^**	3.33± 0.17	3.67± 0.71	3.67± 0.30	3.99± 0.26^**
Spleen	0.18± 0.02	0.18± 0.02	0.18± 0.03	0.19± 0.02	0.23± 0.03	0.23± 0.03	0.24± 0.03	0.24± 0.03
Kidneys	0.59± 0.05	0.66± 0.05^**	0.67± 0.05^**	0.73± 0.06^**	0.67± 0.06	0.67± 0.04	0.68± 0.06	0.71± 0.06
Testes	0.64± 0.06	0.66± 0.06	0.67± 0.07	0.67± 0.05	-	-	-	-

Conclusions:

Based on the lack of adverse treatment-related effects observed, the NOAEC for hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics in this repeated dose inhalation toxicity study was determined to be 6000 mg/m3 in rats. 

Executive summary:

This data is being read across from the source study that tested Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics based on analogue read across.

In a key sub-chronic repeated dose inhalation toxicity study (Carrillo et al., 2018), the test material (hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics) was administered to groups of Wistar SPF albino rats (18/sex/concentration) at target concentrations of 1500, 3000, or 6000 mg/m³ 6 hours per day, 5 days per week, for a period of 13 weeks. Similar number of control animals were exposed to air only.

 

Animals were observed daily for general health and behavior and body weights, food consumption, and water intakes were recorded weekly. Individual urine samples were collected overnight from the control, low, and high exposure groups following their last exposure. Glucose, protein, ketones, bilirubin, pH, and blood pigments were estimated using semi-quantitative BM-test 8 (Boehringer). At the end of the experiment, blood was taken from all rats via cardiac puncture erythrocyte count (RBC), mean cell volume (MCV), hemoglobin (Hb), leucocyte count (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hematocrit (HTC), red cell fragilities, reticulocyte count, prothrombin time and kaolin-cephalin coagulation time were either measured or calculated. Differential white cell counts were also performed on stained blood films. For clinical chemistry analysis, estimations made on plasma included total protein, urea nitrogen, protein electrophoresis, alkaline phosphatase (AP), aspartate amino transferase (AST), alanine amino transferase (ALT), sodium, potassium, and chloride. In addition, estimations were made of glucose on blood samples taken from the tail vein. All animals surviving to the end of the experiment were euthanized by a lethal dose of intraperitoneal sodium pentobarbitone and subjected to a detailed post-mortem examination. Gross observations were undertaken externally and internally. The trachea was ligated before opening the thoracic cavity. After post-mortem examinations the following organs were weighed; brain, liver, kidneys, heart, spleen and testes. Tissues taken for historical examinations included main organs from following systems; digestive, reproductive, respiratory, nervous, renal, circulatory and endocrine. The organs of all animals exposed to the high and the medium concentration of the test material, plus the control animals, were examined histopathologically. The kidneys of the low concentration males and the nasal cavities of the low concentration males and females were also examined as effects were noted in the organs in animals from higher exposure groups. Other organs from low exposure group animals were not examined as pathological changes were not observed in organs from animals in higher exposure groups.

 

Exposure to the test material did not result in mortality or clinical evidence of treatment-related effects. There were no statistically significant differences in male or female body weights at study termination. However, a number of minor, spontaneous changes were observed but most of these changes were not considered to be toxicologically relevant in male/female rats. Changes in hematological parameters observed were limited to a statistically significant decrease in reticulocytes (%) in high exposure group males, but not in female rats. However, this was within the reference range data for rats at this age and with the lack of comparable changes in other red cell parameters, unlikely to be toxicologically meaningful. A significant decrease in alanine aminotransferase (ALT) values was observed in the low treatment group (p≤0.05) females, but not in males. Aside from the absence of any dose/response in the ALT levels in exposed females, the levels were within normal physiological range and did not appear to be toxicologically relevant. Compared to the control group, no significant differences were observed in other clinical parameters in males or females.Relative liver weights were increased in males at all exposure levels and in females at the high exposure level only. These treatment-related and exposure level dependent increases (7–16% over control) were not accompanied by histopathological changes or elevated levels of markers of liver damage, ALT and aspartate aminotransferase (AST), typical markers of liver damage. The observations suggest that the increase in liver weights was evidence of an adaptive response to increasing metabolic demand. Detailed histopathological examination of the kidneys revealed treatment-related renal hyaline droplets and tubular basophilia in males accompanied with increased relative kidney weights at all exposure levels. None of these changes was observed in females. The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health. A low grade catarrhal inflammatory reaction was evident in the nasal cavities of a majority of the rats exposed to the medium concentration (3000 mg/m³). The lesions were confined to the olfactory epithelium and comprised mild mucosal and submucosal edema, focal congestion and diffuse low grade inflammatory cell infiltrates. Unilateral and bilateral lesions occurred. Some desquamation of olfactory epithelium was visible focally but there was no evidence of necrosis or significant degenerative changes. The respiratory epithelium was not affected and the animals exhibited no clinical signs of rhinitis. The etiology of the olfactory lesions in the medium concentration group is uncertain. Such lesions are not uncommon in laboratory rats and the observed changes are consistent with low-grade infection. The absence of this finding in the high concentration group suggests that the olfactory changes were unlikely to be treatment-related.

 

Based on the lack of adverse treatment-related effects observed, the NOAEC for hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics in this repeated dose inhalation toxicity study was determined to be 6000 mg/m³ in rats. 

Data source

Reference

Reference Type:

publication

Title:

The sub-chronic toxicity of a naphthenic hydrocarbon solvent in rats

Author:

Carrillo, J.C., Adenuga, M.D., Momin, F., and McKee, R.H.

Year:

2018

Bibliographic source:

Regulatory Toxicology and Pharmacology 95 (2018): 323–332

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Remarks:

Publication does not specify

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

919-857-5

Molecular formula:

None available - not a single isomer - see remarks

IUPAC Name:

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

Details on test material:

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

Specific details on test material used for the study:

Name of substance: Hydrocarbons C9-C11, NIC,<2% aromatics (Source: Shell Kagaku, Tokyo)
The test material is a high naphthenic (∼70%), low aromatic (< 1%) hydrocarbon solvent in the white spirit boiling range (approximately 150 °C–200 °C). Although the abbreviation NIC indicates normal, iso and cyclo alkane constituents, the bulk of this material is cyclo-alkanes, also known as “Naphthenics”; predominantly centred at C10. The rest is made up of “paraffins” (normal and iso-alkanes) in varying proportions. Aromatics present at very low levels are mostly alkylated C9 ring structures.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

SPF albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall) breeding unit
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 10 - 13 weeks old
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: three of one sex per cage in hanging aluminum cages with stainless steel mesh bases
- Diet (e.g. ad libitum): Food (not specified) was provided ad libitum by means of a top loading food hopper; removed during exposure and the hoppers replenished daily thereafter
- Water (e.g. ad libitum): Plain drinking water from the public supply was available ad libitum in glass bottles
- Acclimation period: Not specified

DETAILS OF FOOD AND WATER QUALITY: Not specified in publication

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5°C - 25°C
- Humidity (%): 28 - 54%
- Air changes (per hr): Not specified (air flow rate: 0.4 and 0.55 m3/min)
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: Not specified

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Four aluminum chambers, with a volume of 1 m3 were 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. The total rate flow was
maintained between 1.8 and 2.0 m3 min−1. The individual flow rates through each chamber were balanced before the exposures began but were not checked further throughout the test because any significant changes would have been detected by the resulting changes in test material concentration.

During the test period laboratory temperature was recorded continuously. Chamber temperatures and relative humidity of the laboratory air were measured daily during the exposures.

TEST ATMOSPHERE
Test atmosphere was generated by completely evaporating the solvent into the streams of ventilating air entering the chambers using micro-metering pumps and vaporizers. The vaporizers consisted of electronically heated quartz tubes whose surface temperatures were adjusted during preliminary experiments to the minimum required for generation of the various atmospheres (high: 247°C; medium: 224°C; low; 163°C).

The chamber atmospheres were analyzed continuously by means of total hydrocarbon analyzers fitted with flame-ionization detectors. The calibration of each analyzer was checked at approximately weekly intervals. The control atmosphere was also monitored continuously throughout the exposure.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Air used as a vehicle but justification not specified in the publication.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber atmospheres were analyzed continuously by means of total hydrocarbon analyzers fitted with flame-ionization detectors. The calibration of each analyzer was checked at approximately weekly intervals. The control atmosphere was also monitored continuously throughout the exposure.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

Control (Air only)

Doses / concentrations 2

Dose / conc.:

1 500 mg/m³ air

Remarks:

Low Concentration

Doses / concentrations 3

Dose / conc.:

3 000 mg/m³ air

Remarks:

Medium Concentration

Doses / concentrations 4

Dose / conc.:

6 000 mg/m³ air

Remarks:

High Concentration

No. of animals per sex per dose:

18/sex/concentration

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 18 male and 18 female rats per exposure level were exposed to target concentrations of 6000 mg/m3 (high), 3000 mg/m3 (medium) or 1500 mg/m3 (low) for 6 h/day, 5 days/week for 13 weeks. Similar numbers of control animals were housed in an identical chamber but exposed to air only. The start and finish of the experiment were staggered in order that the optimum number of animals could be examined at necropsy after exposure. On each of 6 consecutive days, 3 male and 3 female rats per chamber were started on the experiment. Thirteen weeks later, 3 male and 3 female rats per chamber were removed from exposure for pathological examination on each of 6 consecutive days.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (recorded weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of experiment (18 hours after last exposure) by cardiac puncture
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: erythrocyte count (RBC), mean cell volume (MCV), hemoglobin (Hb), leucocyte count (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hematocrit (HTC), red cell fragilities, reticulocyte count, prothrombin time and kaolin-cephalin
coagulation time. Differential white cell counts were also performed on stained blood films.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of experiment
- Animals fasted: Not specified
- How many animals: all animals:
- Parameters checked: plasma: total protein, urea nitrogen, protein electrophoresis, alkaline phosphatase (AP), aspartate amino transferase (AST), alanine amino transferase (ALT), sodium, potassium and chloride. In addition, estimations were made of glucose on blood samples taken from the tail vein.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight following last exposure (control, low and high exposure groups)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: Glucose, protein, ketones, bilirubin, pH and blood pigments were estimated using semi-quantitative BM-test 8 (Boehringer).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals surviving to the end of the experiment were euthanized by a lethal dose of intraperitoneal sodium pentobarbitone and subjected to a detailed post-mortem examination. Gross observations were undertaken externally and internally. The trachea was ligated before opening the thoracic cavity. After post-mortem examinations the following organs were weighed; brain, liver, kidneys, heart, spleen and testes.

HISTOPATHOLOGY: Yes
Tissues taken for historical examinations included main organs from following systems; digestive, reproductive, respiratory, nervous, renal, circulatory and endocrine.

Tongue, femoral muscle, knee joint and femur were held in 4% neutral formalin and only processed for histological examination if indicated by clinical or other pathological findings. Also included were any other macroscopic lesions in any tissues.

Bone marrow smears were examined after staining with May-Grunwald/Giemsa. Sciatic nerves were examined after staining with haematoxylin/eosin, Glees-Marsland Silver Stain and Luxol Fast Blue/Cresyl Violet. To elucidate the nature of the renal changes, representative samples of top exposure level and control male kidneys were stained with Sudan IV, Sudan Black, Methyl Green Pyronin (MGP), Mallory's Phloxin, Heavy Eosin and Luxol Fast Blue/Cresyl Violet (LFB/CV) and treated by the Perl's Prussian Blue Reaction (PBR) and Periodic Acid Schiff (PAS) techniques.

The organs of all animals exposed to the high and the medium concentration of the test material, plus the control animals, were examined histopathologically. The kidneys of the low concentration males and the nasal cavities of the low concentration males and females were also examined as effects were noted in the organs in animals from higher exposure groups. Other organs from low exposure group animals were not examined as pathological changes were not observed in organs from animals in higher exposure groups.

Statistics:

Clinical and hematological parameters were compared across the exposure and control groups using analysis of variance (ANOVA). Organ weights were adjusted for terminal body weight to control for variations in total animal weight. Both unadjusted and adjusted organ weights were analyzed using ANOVA. Mean total body weight, food consumption, and water consumption were compared across groups within week of treatment and were also analyzed using ANOVA. For any differences between groups that were determined to be statistically significant, Tukey's honest significant difference (HSD) test was conducted to determine if statistically significant differences existed between the control group and treatment groups. All analyses were separated according to sex of animal to control for biological differences.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of toxicity were absent at all exposure levels when the animals were examined before and after each day's exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in male body weights related to exposure were observed. Female body weights appeared to be lower than control at the high exposure level at some time points during the course of the study (e.g. between week 8 and 10), without achieving significant difference and were within the
range of the control group at the end of study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Female food intakes showed significant reduction at the high exposure level at the second week of exposure, and there was a tendency for female rats at this exposure level to eat less than control rats throughout the study. Although lower food intake may be related to the apparent lower body weight gain, there were no clinical signs of toxicity. No differences in food intake were observed in male rats.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Some significant increases in water intake were seen in males exposed to the high concentration of test material at weeks 4 and 5, but returned to control levels in subsequent weeks until the end of the study. This change in water intake did not seem to have an impact on the study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small but statistically significant decrease (p≤0.05) in reticulocytes was observed in male rats in the high exposure group (other exposure levels were not assessed). Although this parameter is significantly lower (2.12 ± 0.9%) than controls (2.83 ± 1.0%), it was within the reference range of males rats at this age (< 6 months), with a mean of 2.0 ± 1.23% (Wolford et al., 1986), suggestive of a chance finding rather than being treatment related. There was no corresponding effect in female rats. All other red and white cell parameters were normal.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A significant decrease in alanine aminotransferase (ALT) values was observed in the low treatment group (p≤0.05) females, but not in males. Aside from the absence of any dose/response in the ALT levels in exposed females, the levels were within normal physiological range (Okamura et al., 2011) and did not appear to be toxicologically relevant. Compared to the control group, no significant differences were observed in other clinical parameters in males or females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood was present in the urine samples of two rats but this could be attributed to slight damage to the claws from the mesh floor of the urine collector. Glucose and protein were also present in the urine of many of the rats, but since the urine was collected over night between exposures and the animals had to be fed during the collection period, this was not unexpected.

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):

There was an increase in kidney and liver weights of the male rats from the high exposure group. When adjusted for terminal body weight, a statistical increase at all exposure levels was evident indicating that body weight gain during the study did not play a role in the organ weight increases but rather was a consequence of exposure to the test material. In females, terminal and adjusted liver weights were only increased at the high exposure level. These differences were not observed in terminal or adjusted female kidney weights at any exposure level.

These treatment-related and exposure level dependent increases in liver weights (7–16% over control) were not accompanied by histopathological changes or elevated levels of markers of liver damage, ALT and aspartate aminotransferase (AST), typical markers of liver damage. The observations suggest that the increase in liver weights was evidence of an adaptive response to increasing metabolic demand.

The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been
conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health.

A statistical increase in the adjusted heart weight of males in the low exposure group was also observed. As this was not observed in the higher exposure groups, and there was no concomitant clinical or histopathological change, this finding was not considered toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross examination at necropsy revealed an increased incidence of renal pallor and subcapsular granularity in the male rats exposed to the high concentration (6000 mg/m3). No other changes were found in either sex that could be attributed to the test material exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys
Kidneys of all male rats exposed to all concentrations of the test material contained multiple, hyaline, intracytoplasmic, inclusion-droplets in the epithelium of the proximal convoluted tubules and showed an increased incidence of local cortical, tubular basophilia. Hyaline droplets, were most frequently found in the proximal tubular epithelium of the outer cortex and varied in number and size from cell to cell.

Focally, epithelial cells appeared to be enlarged due to intense droplet aggregations in the cytoplasm. Free droplets or amorphous, hyaline material were seen occasionally in tubular lumina. Minor sloughing of tubular epithelium was evident focally but affected tubules showed no evidence of necrosis and no inflammatory response was visible in association with droplet formation or epithelial sloughing.

A series of special staining techniques were performed on a representative sample of kidneys from the high concentration and control males to determine the nature of the hyaline droplets. The droplets showed a positive reaction when stained for protein with eosin and Mallory's phloxin, and a negative reaction for the other staining techniques. Focal, tubular basophilia was seen more frequently in the renal cortices of treated males than in controls of the same sex. The basophilic foci were quantified on the basis of frequency of occurrence in paraffin sections stained with haematoxylin and eosin. Proximal tubules were most frequently affected. The main features of the lesions were pale-staining, filamentous cytoplasm, mild focal cytoplasmic swelling or vacuolation and mild to moderate thickening of tubular basement membranes. Larger foci showed minor interstitial fibrosis and lymphocyte/ macrophage infiltration. The involved cells did not appear necrotic; their nuclei were pale but of normal size and morphology. Hyaline droplets were rarely present in the cytoplasm of these basophilic foci. Neither the presence of hyaline inclusion droplets nor the incidence of total tubular basophilia exhibited a clear dose/response relationship; histological lesions in the kidneys of the low concentration males were similar in type and intensity to those of the high concentration group.

Hyaline droplets were not found in the kidneys of female rats at any exposure level. Nephrocalcinosis was a common observation in female rats in all groups including controls. Small foci of tubular basophilia were seen occasionally in both exposed and control females but the inflammatory or fibrotic changes found in some of the exposed male rats were rarely seen.

The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been
conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health.

Respiratory Tract
A low grade catarrhal inflammatory reaction was evident in the nasal cavities of a majority of the rats exposed to the medium concentration (3000 mg/m3). The lesions were confined to the olfactory epithelium and comprised mild mucosal and submucosal edema, focal congestion and diffuse low grade inflammatory cell infiltrates. Unilateral and bilateral lesions occurred. Some desquamation of olfactory epithelium was visible focally but there was no evidence of necrosis or significant degenerative changes. The respiratory epithelium was not affected and the animals exhibited no clinical signs of rhinitis. The nasal cavities of the low and high exposure groups were similar to the control group in all respects.

Most rats showed low-grade pulmonary lesions comprising minor perivascular lymphocyte and eosinophil infiltration, discrete aggregates of foamy macrophages and focal alveolar wall thickening due to macrophage, lymphocyte and neutrophil infiltration. The lungs of several animals showed focal granulomata. Such changes were not uncommon in control Wistar rats of the testing facility; the frequency and type of the lesions were similar in treated and control animals. No treatment-related changes were observed in the trachea or bronchi of animals.

Except for a range of minor spontaneous lesions found in some animals in all experimental groups that were within the historical range of the Wistar rat colony used in the testing facility, no other pathological changes were observed in other organ tissues of male or female rats that could be associated with exposure to the test material.

Histopathological findings: neoplastic:

not examined

Details on results:

Overall, the key treatment-related effects of exposure to the Naphthenic solvent, C9-C11, <2% aromatics, were restricted to male rat kidney damage and adaptive liver enlargement in males and females. These effects are commonly associated with exposures to structurally related straight chain or branched chain aliphatic hydrocarbons.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

6 000 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/m³ air

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

In the low and medium exposure groups the measured concentrations had a mean of 1500 (±16) mg/m³ and 2980 (±55) mg/m³ versus the target concentrations of 1500 and 3000 mg/m³ respectively. However there was more variability in the high exposure group in which the mean exposure was 5950 (±416) mg/m³, versus a target of 6000 mg/m³. When the concentration of this exposure level exceeded the target by more than 20% for longer than approximately 36 min (10% of a daily exposure) the approximate extent of the deviation was recorded. These extremes only occurred about once per week reaching about 7200 and 8400 mg/m³ for periods of 0.75–4.75 h.

Table 1. Mean Clinical Chemistry values after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m³

Parameters	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
Protein (g/l)	66.2 ± 3.0	64.1± 3.9	67.9± 2.9	67.2± 4.1	64.3± 4.4	65.3± 4.2	66.1± 2.9	65.6±3.8
Albumin (%)^a	53.2± 4.2	53.0± 4.9	52.4± 4.9	51.2± 4.0	57.7± 3.6	56.3± 3.2	55.9± 2.8	56.1± 4.5
Alpha 1 (%)^a	13.3± 2.0	12.1± 1.5	12.8± 1.6	12.5± 2.5	12.6± 1.1	12.3± 2.0	13.5± 1.5	12.7± 1.4
Alpha 2 (%)^a	9.4± 3.3	9.4± 1.4	9.4± 1.7	10.0± 1.8	7.3± 1.1	7.6± 1.0	7.5± 1.2	7.4± 1.9
Beta (%)^a	18.5± 2.9	19.6± 4.3	19.1± 3.1	20.1± 3.3	14.8± 1.8	15.4± 2.7	15.6± 2.8	16.5± 2.6
Gamma (%)^a	5.7± 3.4	5.8± 3.2	6.7± 3.5	6.3± 3.5	7.6± 2.0	8.3± 2.5	7.5± 1.7	7.4± 2.5
Urea (mm/l)	8.0± 0.8	8.2± 1.2	8.0± 0.8	8.3± 0.9	8.1± 1.3	8.1± 1.4	8.2± 1.5	8.4± 1.7
AP (IU)	71± 10	59± 16	69± 16	64± 18	43± 16	35± 12	33± 9	41± 13
ALT (IU)	22± 4.7	21± 6.7	21± 3.4	24± 14.2	23± 6.6	17± 3.6^*	19± 8.0	18± 3.7
AST (IU)	37± 9.7	35± 5.5	34± 6.3	35± 9.8	37± 8.3	38± 15.1	35± 8.9	32± 4.2
Na (mm/l)	145± 1.6	145± 2.7	145± 1.6	146± 1.6	143± 2.2	144± 2.2	144± 1.7	144± 1.5
K (mm/l)	5.3± 0.6	5.5± 0.8	5.5± 0.6	5.5± 0.8	5.4± 1.0	5.6± 0.8	5.2± 0.9	5.2± 0.9
CL (mm/l)	100± 2.6	99± 2.7	99± 1.9	99± 2.1	102± 2.3	102± 2.9	102± 1.9	102± 2.5
Glucose (mm/l)	4.0± 0.4	3.9± 0.4	3.8± 0.4	4.0± 0.4	4.7± 0.8	4.4± 0.8	4.1± 0.8	4.5± 0.6

* p≤0.05 significance of the difference between treatment and control means.

^a Protein electrophoresis fractions

Table 2. Mean Hematological values after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m3

Parameter	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
BBC (x10^6/cmm)	8.10± 0.5	7.89± 0.3	8.2± 0.5	7.94	7.30± 0.3	7.21± 0.4	7.46± 0.3	7.22± 0.5
Hb (g/100 ml)	15.3± 0.7	14.8± 0.5	15.3± 0.8	14.8± 0.5	14.6± 0.7	14.6± 0.9	15.0± 0.6	14.6± 0.7
HCT (%)	44± 1.9	43± 1.7	44± 2.1	43± 1.9	43± 1.6	42± 2.6	44± 1.9	42± 2.2
MCV (m^3)	53± 1.4	53± 1.9	53± 1.7	53± 1.4	57± 1.8	57± 1.9	57± 1.4	58± 1.9
MCH (pg)	18.5± 0.5	18.4± 0.6	18.3± 0.6	18.3± 0.7	19.6± 0.6	19.9± 0.5	19.8± 0.6	19.9± 0.7
MCHC (g/100ml)	34.4± 0.6	34.3± 0.7	34.4± 0.8	34.3± 0.7	34.1± 0.9	34.3± 0.8	34.3± 0.6	34.3± 0.6
PT (secs)	16.5± 1.0	17.0± 1.6	17.0± 1.4	16.9± 1.0	15.2± 0.7	15.1± 0.3	15.5± 1.8	15.1± 0.7
KCCT (secs)	27.7± 2.9	28.8± 5.4	28.8± 5.0	28.3± 4.2	25.0± 3.1	24.7± 3.0	25.1± 3.4	23.2± 4.0
Retics (%)	2.83± 1.0	-	-	2.12± 0.9^*	2.17± 1.2	-	-	2.44± 1.4
WBC (x10^3/mm)	4.7± 1.3	4.5± 1.2	4.7± 1.0	4.7± 1.2	3.6± 1.2	4.1± 1.5	3.8± 1.1	3.7± 1.0

 

Table 3. Mean and Adjusted Organ Weights (g) after 13 weeks inhalation of Naphthenic solvent, C9-C11, <2 % aromatics at 0, 1500, 3000 or 6000 mg/m³

Organ Weight (g)	Nominal Concentrations mg/m^3
	Male				Female
	Control	1500	3000	6000	Control	1500	3000	6000
Terminal body weight (g)	542± 40.3	532± 48.8	528± 44.9	530± 37.3	306± 18.3	301± 18.3	301± 17.8	299± 19.2
Brain	2.20± 0.10	2.21± 0.10	2.20± 0.08	2.24± 0.10	2.02± 0.08	2.01± 0.07	2.03± 0.05	2.02± 0.08
Heart	1.30± 0.13	1.41± 0.16	1.32± 0.11	1.39± 0.15	0.93± 0.08	0.91± 0.09	0.92± 0.6	0.90± 0.08
Liver	17.31± 2.08	18.21± 1.86	18.64± 2.02	20.11± 1.74^**	10.17± 0.61	11.07± 2.49	11.01± 0.85	11.92± 0.77^**
Spleen	0.96± 0.15	0.97± 0.07	0.96± 0.13	1.00± 0.10	0.72± 0.09	0.70± 0.10	0.71± 0.10	0.71± 0.10
Kidneys	3.22± 0.37	3.51± 0.37	3.51± 0.27	3.87± 0.32^**	2.03± 0.16	2.01± 0.13	2.04± 0.13	2.12± 0.16
Testes	3.45± 0.30	3.49± 0.29	3.49± 0.21	3.55± 0.23	-	-	-	-
Adjusted organ weights (g/100g body weight
Brain	0.41± 0.03	0.42± 0.04	0.42± 0.04	0.43± 0.03	0.66± 0.04	0.67± 0.04	0.68± 0.04	0.66± 0.05
Heart	0.24± 0.02	0.27± 0.03**	0.25± 0.02	0.26± 0.03	0.30± 0.03	0.30± 0.03	0.31± 0.03	0.30± 0.02
Liver	3.19± 0.21	3.43± 0.25^**	3.53± 0.22^**	3.80± 0.23^**	3.33± 0.17	3.67± 0.71	3.67± 0.30	3.99± 0.26^**
Spleen	0.18± 0.02	0.18± 0.02	0.18± 0.03	0.19± 0.02	0.23± 0.03	0.23± 0.03	0.24± 0.03	0.24± 0.03
Kidneys	0.59± 0.05	0.66± 0.05^**	0.67± 0.05^**	0.73± 0.06^**	0.67± 0.06	0.67± 0.04	0.68± 0.06	0.71± 0.06
Testes	0.64± 0.06	0.66± 0.06	0.67± 0.07	0.67± 0.05	-	-	-	-

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed, the NOAEC for hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics in this repeated dose inhalation toxicity study was determined to be 6000 mg/m3 in rats. 

Executive summary:

In a key sub-chronic repeated dose inhalation toxicity study (Carrillo et al., 2018), the test material (hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics) was administered to groups of Wistar SPF albino rats (18/sex/concentration) at target concentrations of 1500, 3000, or 6000 mg/m³ 6 hours per day, 5 days per week, for a period of 13 weeks. Similar number of control animals were exposed to air only.

 

Animals were observed daily for general health and behavior and body weights, food consumption, and water intakes were recorded weekly. Individual urine samples were collected overnight from the control, low, and high exposure groups following their last exposure. Glucose, protein, ketones, bilirubin, pH, and blood pigments were estimated using semi-quantitative BM-test 8 (Boehringer). At the end of the experiment, blood was taken from all rats via cardiac puncture erythrocyte count (RBC), mean cell volume (MCV), hemoglobin (Hb), leucocyte count (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hematocrit (HTC), red cell fragilities, reticulocyte count, prothrombin time and kaolin-cephalin coagulation time were either measured or calculated. Differential white cell counts were also performed on stained blood films. For clinical chemistry analysis, estimations made on plasma included total protein, urea nitrogen, protein electrophoresis, alkaline phosphatase (AP), aspartate amino transferase (AST), alanine amino transferase (ALT), sodium, potassium, and chloride. In addition, estimations were made of glucose on blood samples taken from the tail vein. All animals surviving to the end of the experiment were euthanized by a lethal dose of intraperitoneal sodium pentobarbitone and subjected to a detailed post-mortem examination. Gross observations were undertaken externally and internally. The trachea was ligated before opening the thoracic cavity. After post-mortem examinations the following organs were weighed; brain, liver, kidneys, heart, spleen and testes. Tissues taken for historical examinations included main organs from following systems; digestive, reproductive, respiratory, nervous, renal, circulatory and endocrine. The organs of all animals exposed to the high and the medium concentration of the test material, plus the control animals, were examined histopathologically. The kidneys of the low concentration males and the nasal cavities of the low concentration males and females were also examined as effects were noted in the organs in animals from higher exposure groups. Other organs from low exposure group animals were not examined as pathological changes were not observed in organs from animals in higher exposure groups.

 

Exposure to the test material did not result in mortality or clinical evidence of treatment-related effects. There were no statistically significant differences in male or female body weights at study termination. However, a number of minor, spontaneous changes were observed but most of these changes were not considered to be toxicologically relevant in male/female rats. Changes in hematological parameters observed were limited to a statistically significant decrease in reticulocytes (%) in high exposure group males, but not in female rats. However, this was within the reference range data for rats at this age and with the lack of comparable changes in other red cell parameters, unlikely to be toxicologically meaningful. A significant decrease in alanine aminotransferase (ALT) values was observed in the low treatment group (p≤0.05) females, but not in males. Aside from the absence of any dose/response in the ALT levels in exposed females, the levels were within normal physiological range and did not appear to be toxicologically relevant. Compared to the control group, no significant differences were observed in other clinical parameters in males or females.Relative liver weights were increased in males at all exposure levels and in females at the high exposure level only. These treatment-related and exposure level dependent increases (7–16% over control) were not accompanied by histopathological changes or elevated levels of markers of liver damage, ALT and aspartate aminotransferase (AST), typical markers of liver damage. The observations suggest that the increase in liver weights was evidence of an adaptive response to increasing metabolic demand. Detailed histopathological examination of the kidneys revealed treatment-related renal hyaline droplets and tubular basophilia in males accompanied with increased relative kidney weights at all exposure levels. None of these changes was observed in females. The male rat specificity of the kidney effects is consistent with findings with other light hydrocarbons of similar carbon ranges. These findings have been conclusively linked to a α2u-globulin-mediated process, which, although associated with renal tumors in male rats, has no relevance to human health. A low grade catarrhal inflammatory reaction was evident in the nasal cavities of a majority of the rats exposed to the medium concentration (3000 mg/m³). The lesions were confined to the olfactory epithelium and comprised mild mucosal and submucosal edema, focal congestion and diffuse low grade inflammatory cell infiltrates. Unilateral and bilateral lesions occurred. Some desquamation of olfactory epithelium was visible focally but there was no evidence of necrosis or significant degenerative changes. The respiratory epithelium was not affected and the animals exhibited no clinical signs of rhinitis. The etiology of the olfactory lesions in the medium concentration group is uncertain. Such lesions are not uncommon in laboratory rats and the observed changes are consistent with low-grade infection. The absence of this finding in the high concentration group suggests that the olfactory changes were unlikely to be treatment-related.

 

Based on the lack of adverse treatment-related effects observed, the NOAEC for hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics in this repeated dose inhalation toxicity study was determined to be 6000 mg/m³ in rats.