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

Endpoint summary

Description of key information

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

Repeated Dose Inhalation 90d – NOAEC ≥ 10400 mg/m³ for rats (similar to OECD TG 413)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 408: GLP

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

According to EPA guideline 82-1

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 238-295g (males); 180-236g (females)
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1990-12-17 To: 1991-07-13

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was mixed with corn oil to ensure a 10ml/kg dose volume at all dose levels.

Test material mixtures were administered by oral gavage at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as main test and allowed to recover for 28 days post-treatment.

VEHICLE
- Amount of vehicle (if gavage): 10ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test material in corn oil were analyzed by Gas Chromatography for concentration verification, stability and uniformity analysis. Concentration verification analysis showed the values to be within 5.6% of the target levels over a three month period. Samples of the 5% and 50% nominal concentration levels (500 and 5000 mg/kg/day, respectively) were kept under conditions of room temperature and refrigeration, prior to analyzing aliquots of these samples on days 0, 5 and 8. Sample aliquots were stable for up to 8 days under both conditions. To evaluate uniformity, triplicate aliquots of the 5% and 50% nominal concentration levels were analyzed. Mean values of triplicate aliquots were 5.25% ± 0.13 and 52.5% ± 0.51, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days/week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1 (Control)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Group 2 (Low Dose)

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Group 3 (Mid Dose)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 (High Dose)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group 5 (Satellite Group)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Test material mixtures were administered by oral gavage at three different doses at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as the main test and allowed to recover for 28 days post-treatment.

- Post-exposure recovery period in satellite groups: 28 days post-treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily monday-friday and once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, the day of dose initiation, and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
at study initiation and during the final week of the main study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals:all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following parameters were statistically analyzed for significant differences: mean hematology parameters, serum chemistry parameters, organ weights, organ to body weight ratios, body weights, mean food consumption. Comparisons were limited to within sex analysis. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test of ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differ significantly from control. In addition to ANOVA, a standard regression analysis for liner response in the dose groups and linear lack of fit were preformed.

For the nonparametric procedure the test of equality of means was performed using the Kruskal-Wallis test. If significant differences among the means was indicated, Dunn’s Summed Rank test was used to determine which treatment group differ significantly from control. In addition, Jonckheere’s test for monotonic trend in the dose response was performed.

The statistical t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. In addition, the t-test was used to compare the satellite group's and the control group's relative organ weights. The t-test was also used to compare the high dose and satellite groups to ensure similar results in order to accurately evaluate the recovery effects.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of animals in the control, low and mid-dose groups appeared normal. Very low sporadic incidences of scabs, alopecia, fur staining, dry/wet rales, dyspnea, dried red nasal discharge and hypoactivity were observed across all dose groups, but particularly in the high dose and satellite groups. The frequencies of these observations notably decreased over time during the satellite recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of fourteen unscheduled deaths were recorded across all dose groups for the duration of the study. With the exception of one 2500 mg/Kg female, for which the cause of death was not determined, all other unscheduled deaths were attributed to dosing trauma and/or incidental aspiration of test material based on post-mortem and histopathological findings. The animal deaths associated with the dosing procedures appeared to be related to physical characteristics of the test material and high dosage volume.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrements in mean body weights were observed for mid dose males from week 11 and high dose males from week 8 (p≤ 0.05 and p ≤0.01 significance level, respectively). Body weights for male rats in the satellite group were similar to those in the high dose groups, although there was a trend towards recovery following main study termination. Statistically significant body weight differences in treated female rats were small (≤10% difference) and restricted to mid and high dose groups at week 13. Mean body weights for females in the satellite group were similar to controls, suggesting that slight changes observed in the mid and high dose groups were not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of blood samples from rats at study termination showed a statistically significant, dose-dependent increase in platelet counts in all treated males and high dose females. In addition, red blood cell counts, hematocrit, hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin were statistically significantly decreased in mid dose males compared to controls. Although the cause of these decreases could not be ascertained, the lack of similar effects in the high dose males suggested these changes were not treatment related. With the exception of the increased platelet counts, all other effects were reversed in recovery group rats.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Examination of serum chemistry values revealed a statistically significant increase in blood urea nitrogen (BUN) and gamma glutamyl transferase (GGT) for high dose males and also mid dose males for BUN. Cholesterol levels were dose dependently increased relative to control in both males and females, with statistically significant increases occurring at the mid and high dose groups. Glucose values were significantly lower than the control values at the p ≤ 0.01 level for both males and females in the mid and high dose groups and for the male low dose group at the p ≤0.05 significance level. Statistically significant increases in alanine aminotransferase (ALT) levels of 2- and 2.4-fold were observed in mid and high dose males, respectively. In the females, the high dose group showed a slight but not statistically significant increase in GGT compared to controls. Also noted was a statistically significant increase in total bilirubin (TBIL) in the high dose groups for both sexes. With the exception of small decreases in chloride levels in mid and high dose females, no statistically significant changes were noted for serum levels of calcium, phosphate, sodium and potassium (data not shown). All changes reported as statistically significant at study termination showed evidence of recovery trends in satellite rats held for 28 days post last exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences in mean kidney weights, compared to vehicle control rats were observed for all treated male rats. Liver weights for all treated female rats and low and mid dose males were statistically significantly increased relative to respective vehicle controls. Although liver weights for high dose males showed an increasing trend relative to controls, they were not significantly different from control values. Mean adrenal weights were also significantly increased for high dose males, including mid and high dose females. With respect to changes in organ/body weight ratios, relative kidney weights were statistically significantly increased for all treated male rats. Similar changes were also observed for mid and high dose rat livers and adrenal glands for high dose males.
Relative testes weights for high dose males were statistically significantly increased (p ≤0.05); however the difference was small and may have been related to the differences in body weights. In females, statistically significant differences in relative liver and adrenal weights were observed for mid and high dose groups. Similar to male rats, relative kidney weights were also statistically significantly increased for all treated female rats. No changes were observed in relative ovary weights. All changes showed trends towards almost complete recovery in high dose rats held without treatment for 28 days post last exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic changes were observed in the kidneys of all treated male rats, livers of all treated male and female rats, and the stomach and/or anus of male and female rats in the mid and high dose groups. Microscopic evaluation of the stomach revealed a dose-dependent increase in the incidence and severity of thickening of the non-glandular mucosa due to hyperplasia and hyperkeratosis of the squamous epithelium. Edema and inflammatory cell infiltrations in the submucosa and focal necrosis of the superficial glandular mucosa were also noted, although at a lower incidence. These changes showed reversibility in the severity of the hyperplasia and hyperkeratosis of the mucosa.

Most rats in the high dose groups exhibited anal swelling with thickened skin and mucosa around the anus due to hyperplasia and hyperkeratosis. Areas of necrosis, neutrophilic inflammatory cell infiltrations and pustular formations in the superficial mucosa and epidermis of the anus and surrounding skin were also observed. All other microscopic changes were considered to have occurred spontaneously and to have been unrelated to treatment.

Microscopic examination of the kidneys of male rats showed changes that are typical of male rat-specific hydrocarbon nephropathy. Renal changes consisted of accumulations of hyaline droplets in the cytoplasm of the proximal convoluted tubules, dilatation and granular cast formations in the medullary tubules and increased basophilia of cortical tubules. Affected basophilic cortical tubules showed changes consistent with both degeneration and regeneration. The renal changes were observed only in male rats, and there were no differences in the incidence and/or severity of
the lesions across treatment groups. Microscopic examination of the kidneys in the satellite group male rats necropsied after the 28-day recovery period showed no evidence of hyaline droplets in the cortical tubules. However, there was a 50% incidence of dilated tubules with granular casts in the medulla and a 30% incidence of focal chronic nephritis in rats in the recovery group. There was no difference in the incidence of cortical basophilic tubules between the control and recovery group male rats, indicating that the renal changes were reversible with discontinuation of exposure to test material.

Treatment-related effects in the liver consisted of hepatocellular hypertrophy, predominantly in centrilobular areas. The incidence and severity of hepatocellular hypertrophy was dose-related, consistent with the increased liver weights seen in all treated rats irrespective of sex. The liver lesions were completely absent in recovery rats.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
One male and 1 female died in the control group, 2 females died in the 2500 mg/kg dose group, 4 females died in the 5000 mg/kg dose group, 2 males and 3 females died in the satellite group. With the exception of one 2500 mg/kg female, all of the other 13 listed spontaneous deaths appear to be a result of dosing trauma and/or aspiration of test material (due to physical characteristics of test material and the high dosage volume).

The majority of animals in the control, low and mid dose groups displayed no observable abnormal clinical signs. Observations included but are not limited to scabs, maloccluded incisors, alopecia and staining of fur, dry/wet rales, dyspnea, nasal discharge. The type and incidence of abnormal clinical signs were similar between the high dose and satellite groups with a dramatic increase in incidence when compared to mid dose group. Clinical signs most frequently noted included swollen anus, ano-genital staining, emaciation, and alopecia. During the satellite recovery period, the incidence of abnormal signs decreased over time with an increase in the number of animals exhibiting no observable abnormalities.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases from controls at the p<=0.05 level of significance were noted for mid dose males on days 77, 84, 91 and termination and for the high dose males on Day 42. A statistically significant decrease (p<=0.01) was noted for the high dose group males on Day 49 and continued through the end of the treatment period. Statistically significant decreases were noted for mid dose females (p<=0.05) on day 91 and for high dose females on days 77 and 91. At termination both mid and high dose females displayed a statistically significant decrease in body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings.

HAEMATOLOGY
A statistically significant increase in platelets which was linearly related to dose in both the males and females was observed. In addition the male animals displayed a linear dose related increase in white blood cells. The mid dose male values were noted to differ significantly from those of controls for hematocrit and hemoglobin at the p<=0.01 level of significance and mean corpuscular volume and mean corpuscular hemoglobin at the p<=0.05 level of significance.

CLINICAL CHEMISTRY
Statistically significant increases in males (p<=0.01) for urea nitrogen and gamma glutamyl transpeptidase for the high dose males and also the mid dose males for urea nitrogen. An increase for cholesterol was noted for the mid and high dose groups of both sexes (p<=0.01). An increase in alanine aminotransferase was also noted for the mid and high dose males (p<=0.01). Glucose levels were significantly lower than the control values (p<=0.01) for both sexes in the mid and high dose and for the male low dose (P<=0.05). A statistically significant increase in bilirubin in the high dose of both sexes was observed. Other parameters showing statistically significant differences from controls included creatinine, chloride, tryglycerides.

ORGAN WEIGHTS
Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg. Testes weights were elevated in male rats at 5000 mg/kg. Both the male and female relative kidney weights for all treated groups were significantly different from the control value (p<=0.01).

GROSS PATHOLOGY
Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).

Key result

Dose descriptor:

other: BMDL

Effect level:

1 857 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Critical effects observed:

no

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 (×10^3/mm^3)	7.6±2.7	9.2±2.3	10.4±2.8	10.8±2.3	7.7±1.7
RBC (×10^6/mm^3)	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 (×10^3/mm^3)	770±40	877±45 *	1008±73 **	984±115 **	1050±96
Females	N=8	N=10	N=8	N=10	N=3
WBC (×10^3/mm^3)	5.4±2.5	4.4±1.0	4.2±0.8	6.8±1.6	5.1±1.9
RBC (×10^6/mm^3)	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 (×10^3/mm^3)	792±116	846±81	892±89	1023±110 **	1052±154

WBC – white blood cell

RBC – red blood cell

HGB – hemoglobin

HCT – hematocrit

MCV – mean cell volume

MCH – mean corpuscular hemoglobin

MCHC – mean corpuscular hemoglobin concentration,

PLT – platelet count

^a Measurements on day 125, 2 of 10 and 2 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table3. Mean Clinical Chemistry values after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

Parameter	Exposure dose (mg/kg/day)
	0 (control)	500	2500	5000	5000 (Recovery)^a
Males	N=8	N=10	N=10	N=10	N=8
GGT (lU/L)	1.80.18±0.83	1.60±0.97	2.90±0.99	4.90±1.91 **	0.88±0.83
Albumin (g/l)	4.2±0.1	4.2±0.1	4.1±0.2	4.1±0.1	3.9±0.2
Glucose (mg/dl)	128.6±11.3	113.8±10.5 *	94.9±12.5 **	93.8±12.6 **	117.5±13.8
Chol (mg/dl)	39.3±3.8	46.8±8.8	65.0±14.6 **	66.8±15.0 **	35.5±4.4
TBIL (mg/dl)	0.49±0.04	0.48±0.06	0.62±0.15	0.61±0.12 *	0.48±0.07
BUN (mg/dl)	9.8±1.5	10.4±1.3	13.3±3.4 **	14.5±2.4 **	18.8±2.5
ALT (IU/il)	37.5±3.9	42.5±8.0	75.0±18.3 **	90.7±26.0 **	31.8±6.2
AST IU/l)	94.8±18.4	88.0±15.2	91.7±18.3	106.0±10.0	119.4±19.4
Crea (mg/dl)	0.5±0.1	0.6±0.1	0.6±0.1	0.6±0.1	0.5±0.1
Females	N=9	N=10	N=8	N=10	N=3
GGT (lU/L)	1.0±0.87	0.60±0.70	1.38±0.92	1.90±1.60	2.33±1.15
Albumin (g/l)	4.6±0.3	4.8±0.3	5.0±0.2	4.8±0.4	4.3±0.1
Glucose (mg/dl)	107.2±6.4	104.0±13.6	89.5±9.7 **	81.4±6.8 **	118.0±18.7
Chol (mg/dl)	48.3±8.4	63.6±9.8	95.0±18.2 **	81.9±16.1 **	66.3±17.2
TBIL (mg/dl)	0.54±0.07	0.58±0.08	0.63±0.07	0.68±0.15 *	0.50±0.10
BUN (mg/dl)	12.8±2.6	12.2±1.9	11.6±1.8	13.2±1.9	17.7±0.6
ALT (IU/il)	60.7±48.2	38.0±39.5	51.5±22.4	69.3±16.0	26.7±5.5
AST IU/l)	113.9±49.0	93.6±38.3	97.3±13.6	115.9±13.9	88.7±23.7
Crea (mg/dl)	0.6±0.1	0.7±0.1 *	0.6±0.1	0.7±0.1	0.6±0.1

TBIL – total bilirubin

ALT – alanine amino transferase

AST – aspartate amino transferase

Chol – cholesterol

BUN – blood urea nitrogen

GGT – gamma glutamyl transferase

Crea – creatinine

^a Measurements on day 125, 2 of 10 and 3 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table 4. Mean Absolute and Relative Organ Weights after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

Parameter	Exposure dose (mg/kg/day)
	0 (control)	500	2500	5000	5000 (Recovery)^a
Males	N=8	N=10	N=10	N=10	N=8
Mean absolute (g)
Kidney	3.32±0.46	4.43±0.58 **	4.38±0.57 **	4.11±0.40 **	3.48±0.32
Liver	14.69±2.93	18.50±2.51 *	19.85±2.98 **	18.01±2.90	12.18±1.52
Adrenals	0.048±0.008	0.048±0.009	0.061±0.009	0.078±0.15 **	0.055±0.013
Testes	3.8519±0.3234	3.7200±0.3770	3.4243±0.7249	3.5136±0.3797	3.7671±0.2455
Mean relative (g)
Kidney	0.0063±0.0005	0.008±0.0011 **	0.01±0.0007 **	0.0102±0.0015 **	0.0078±0.0006
Liver	0..028±0.0001	0.033±0.001	0.045±0.004 **	0.044±0.004 **	0.027±0.001
Adrenals	0.0001±0.00001	0.00009±0.00001	0.00014±0.00001	0.0019±0.00003 **	0.00012±0.00002
Testes	0.0074±0.001	0.0067±0.0008	0.0078±0.0017	0.0087±0.0004 *	0.0084±0.0011
Females	N=9	N=10	N=8	N=10	N=3
Mean absolute (g)
Kidney	2.18±0.28	2.40±0.17	2.34±0.18	2.42±0.27	2.41±0.23
Liver	8.57±1.12	10.33±0.89 *	14.71±1.51 **	13.80±2.19 **	8.85±0.71
Adrenals	0.067±0.019	0.074±0.010	0.094±0.010 **	0.102±0.012 **	0.075±0.007
Testes	0.081±0.020	0.082±0.033	0.067±0.024	0.072±0.033	0.077±0.025
Mean relative (g)
Kidney	0.0072±0.0007	0.0084±0.0006 **	0.0089±0.0007 **	0.0091±0.0008 **	0.008±0.0002
Liver	0.028±0.002	0.036±0.002	0.056±0.005 **	0.052±0.008 **	0.029±0.001
Adrenals	0.00022±0.00006	0.00026±0.00004	0.00036±0.00004 **	0.00039±0.00006 **	0.00025±0.00004
Testes	0.00027±0.00007	0.00029±0.00012	0.00025±0.00008	0.00027±0.00012	0.00026±0.00009

^a Measurements on day 125, 2 of 10 and 3 of 6 rats excluded (due to death) in males and females, respectively

* P ≤0.05

** P≤0.01

Table 5. Incidence and Degree of Severity of Treatment-related Histopathological findings in the Kidney and Liver after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

 

Tissue/lesions	Male (mg/kg/day)					Female (mg/kg/day)
	0 (veh)	500	2500	5000	5000(Rec)^a	0 (veh)	500	2500	5000	5000(Rec)^a
Liver
No. examined	10	10	10	10	8	10	10	10	14	3
No. normal	3	4	4	2	2	4	4	1	2	1
Hypertrophy, hepatocellular, centrilobular
Minimal	0	3	4	1	0	0	3	5	6	0
Slight	0	0	1	3	0	0	0	4	4	0
Kidneys
No. examined	10	10	10	10	8	10	10	10	14	3
Normal	8	0	0	0	2	8	10	9	13	3
Basophilia, cortical tubules, multifocal
Minimal	1	3	3	2	2	0	0	0	1	0
Slight	0	2	3	6	0	0	0	0	0	0
Moderate	0	3	3	3	0	0	0	0	0	0
Dilated tubules/granular casts, medulla
Minimal	0	0	3	1	4	0	0	0	0	0
Slight	0	0	1	4	0	0	0	0	0	0
Moderate	0	3	3	3	0	0	0	0	0	0
Hyaline droplets, cortical tubules
	0	10	10	10	0	0	0	0	0	0

^a2 male and 3 female recovery rats died prior to the 90 day necropsy. 4 female recovery rats were also transferred to the female high dose group

Table 6. Incidence and Degree of Severity of Treatment-related Gastritis and Peri-anal Irritation after a 90 day oral gavage study of C10-C13 dearomatised hydrocarbon solvent

 

Tissue/lesions	Male (mg/kg/day)					Female (mg/kg/day)
	0 (veh)	500	2500	5000	5000(Rec)^a	0 (veh)	500	2500	5000	5000(Rec)^a
Stomach
No. examined	10	10	10	10	8	10	10	10	14	3
No. normal	10	9	3	1	5	10	9	2	3	2
Edema/inflammation, sub mucosa
Slight	0	0	1	0	0	0	0	0	0	0
Moderate	0	0	1	1	0	0	0	1	0	0
Hyperplasia/hyperkeratoasis, non-glandular mucosa
Minimal	0	0	4	0	2	0	0	6	0	0
Slight	0	0	1	3	1	0	0	2	2	0
Moderate	0	0	1	6	0	0	0	0	9	0
Marked	0	0	1	0	0	0	0	0	0	0
Anus
No. examined	0	0	0	8	0	0	0	0	14	0
No. normal	0	0	0	0	0	0	0	0	0	0
Hyperplasia/hyperkeratosis
Slight	0	0	0	1	0	0	0	0	2	0
Moderate	0	0	0	4	0	0	0	0	14	0
Infiltration, neutrophilic/pustules
Minimal	0	0	0	2	0	0	0	0	2	0
Slight	0	0	0	2	0	0	0	0	9	0
Moderate	0	0	0	3	0	0	0	0	3	0
Marked	0	0	0	1	0	0	0	0	0	0
Necrosis, mucosa
Slight	0	0	0	1	0	0	0	0	0	0
Moderate	0	0	0	1	0	0	0	0	0	0
Marked	0	0	0	1	0	0	0	0	0	0

^a2 male and 3 female recovery rats died prior to the 90 day necropsy. 4 female recovery rats were also transferred to the female high dose group

Table 7. Benchmark Dose Estimation of the Point of Departure for ALT Responses in Male Rats.

Model	BMD (mg/kg)	BMDL (mg/kg)	GOF^ap-value	AIC	Scaled residual for dose group
Default BMR (1SD)
Exponential M4	271.174	163.321	0.2286	233.7707	-1.084
BMR – (adverse effect as 100% above concurrent control mean)
Polynomial	2588.94	1857.37	0.2902	233.439874	0.577

^aGoodness of fit (p-value). The chosen model is considered as an acceptable model fit to the data when the p-value is greater than 0.1.

Conclusions:

Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg.

Executive summary:

MRD-89-582 was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/kg, 7 days a week for 13 weeks to assess the subchronic toxicity.  An additional group of animals, dosed at 5000 mg/kg/day, was held for 4 weeks to assess reversibility.  No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/kg dose groups.  Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day.  Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg.  Testes weights were elevated in male rats at 5000 mg/kg.  Kidney effects occurred in males at all dose levels, and are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver.  Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups.  These findings are believed to have been a compensatory response and not an indication of toxicity.  Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans.  Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance.  These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity.  All treatment-related effects were reversible within the 4-week recovery period. Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided

for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The ‘justification for the read across’ is provided in the ‘Attached justification’ section below.

Species:

rat

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1 key read across sub-chronic toxicity study from a structural analogue available for assessment. BMDL level determined and presented in the additional information section.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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: supporting information

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

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.

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

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

Dose / conc.:

0 mg/m³ air

Remarks:

Control (Air only)

Dose / conc.:

1 500 mg/m³ air

Remarks:

Low Concentration

Dose / conc.:

3 000 mg/m³ air

Remarks:

Medium Concentration

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.

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.

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:

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. 

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 413.

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Species:

rat

Strain:

other: albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory Breding Unit
- Age at study initiation: 10-13 weeks
- Housing: three of one sex per cage
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum

During the period of the test the laboratory temperature varied between 19.4°C and 26.1°C and the relative humidity between 37% and 74%.
Barometric pressure was within the range 753 to 768 mm Hg

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Details on inhalation exposure:

The atmospheres were generated by completely evaporating the solvent into the streams of ventilating air entering the chambers using micrometering pumps and vaporizers. The vaporizers consisted of electrically heated quartz tubes whose surface temperatures were adjusted during preliminary experiments to the minimal for complete evaporation of the solvent.

Each chamber was constructed of aluminum, with a volume of 1 m3 and was ventilated by air drawn from the laboratory through dust filters. The exhaust ducts from each chamber entered a common exhaust duct through which the air was drawn by a fan situated on the roof of the laboratory.

The total air flow rate through the main duct exhausting all four chambers was recorded continuously throughout the test by means of an electro—anemometer mounted in the duct. Slight adjustments were made as required to compensate for the effects of wind at the efflux point. The total flow rate was maintained at 2.0 + 0.03 m3 ∙min- 1. The individual flow rates through each chamber were balanced before the exposures began but were not checked further throughout the test since any significant changes would have been detected by the resulting changes in toxicant concentration. The flow rates were adjusted to 0.50 m3 ∙min- 1.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmospheres were analyzed sequentially by means of a total hydrocarbon analyzer fitted with a flame-ionisation detector (Beckman 109A). The analyzer was calibrated during the test by means of known concentrations of SHELLSOL TD, prepared in a Teflon FEP gas sampling bag.

The recorder traces from the analyser were examined daily and a ‘daily mean concentration’ value was estimated by visual inspection. The daily mean concentrations for each of the test atmospheres were then ‘pooled’ to give weekly mean concentrations. The overall means of the weekly mean concentrations are given below:
Nominal concentration Observed concentration
(mg/m3) (mg/m3) (ppm)
10400* 10186 SD 327 1444
5200 5200 SD 207 737
2600 2529 SD 116 359
*83% saturated.

The desired concentrations of solvent in the test atmospheres were reached within 10 mm of the start of each exposure period. They then stayed remarkably constant throughout the 6 h exposure period.

Duration of treatment / exposure:

Six hours/day

Frequency of treatment:

five days/week for 13 weeks

Remarks:

Doses / Concentrations:
0, 2600, 5200, 10400 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

6 animals/sex/dose (total of 12 animals/dose)

Control animals:

yes, sham-exposed

Details on study design:

The start and finish of the experiment was staggered in order that the optimum number of animals could be examined histopathologically after exposure. On each of four consecutive days, four male and four female rats per chamber were started on the experiment. The remaining two males and two females were started the next day. Thirteen weeks later, four male and four female rats per chamber were removed from the experiment for pathological examination on each of four consecutive days. The remaining two males and two females were removed the next day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations: daily

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined weekly: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly


OPHTHALMOSCOPIC EXAMINATION: No



HAEMATOLOGY: Yes
- Time schedule for collection of blood: 18h after the last 13 week exposure
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 18h after the last 13 week exposure
- How many animals: all



URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No



OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes for all animals exposed to the high and medium concentrations, plus the control animals. Kidneys of low concentration males were also examined.

Other examinations:

Organ weights
After post-mortem examinations the following organs were weighed:
Brain
Liver
Heart
Spleen
Kidneys
Testes

Histopatholgy. Tissues taken for histological examination were:

Mammary gland (posterior site with skin)
Mesenteric lymph node
Pancreas
Stomach
Intestine at 5 levels
Caecum
Spleen
Liver (middle, left and triangular lobes)
Adrenals
Kidneys
Ovaries or testes
Uterus or prostate
Seminal vesicles
Urinary bladder
Thyroid (with oesophagus and trachea)
Trachea (mid course and bifurcation)
Heart
Lungs
Nasal cavity
Thymus
Eye and lacrimal glands
Salivary gland (submaxillary)
Brain
Spinal cord (thoracic)
Pituitary
Tongue
Sciatic nerves
Muscle (femoral)
Knee joint and femur
Plus any other macroscopic lesion in any tissues.
The samples marked were held in 4% neutral formalin and only processed for histological examination if indicated by clinical or other pathological findings.

Statistics:

Body and organ weights were analysed by covariance analysis using initial body weight as the covariate. Reported means were adjusted for initial body weight if a significant covariance relationship existed: where no significant covariance relationship was found, unadjusted means were reported.

Organ weights were further examined by covariance analysis using the terminal body weight as the covariate. The organ weight means are reported as adjusted for terminal body weight if a significant covariance relationship existed. Although not a true covariance analysis (because the terminal body weights are dependent upon treatment), the analysis does provide an aid to the interpretation of organ weights when there are differences in terminal body weights. The analysis attempts to predict what the organ weights would have been, had all the animals had the same terminal body weight.
Clinical, chemical and haematological parameters were examined using analysis of variance.

The analysis allowed for the fact that animals were multihoused. Differences in response can be affected by cage environment as well as by treatment but this effect is minimal in a study of this duration.
The significance of any difference between treated and control group means was tested using the Williams t test (1971, 1972). However, if a monotonic dose response could not be assumed Dunnett’s test (1964) was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

No deaths were recorded and clinical signs of toxicity were absent in the low and medium exposure groups; the high exposure groups were slightly lethargic when examined up to one hour after cessation of exposure. Body weight gain was slightly reduced in all female groups and in high exposure males. Water intake was increased in the high exposure males only.

Female aspartate amino transferase and alanine amino transferase were decreased in all female groups exposed to SHELLSOL-TD. No pathological changes were detected which could explain the observed decreases in these enzymes. In view of this lack of supporting evidence and the fact that the control values for these two parameters were high when compared with historical controls in the laboratory, these changes were not considered toxicologically significant.

Male alkaline phosphatase, potassium, chloride and albumin were increased at the high exposure level. These were considered to represent biological variation in the rat and were not considered treatment-related.

Male kidney weights were increased at all exposure levels. Hyaline intracytoplasmic inclusions and an increased incidence of tubular degeneration and/or dilatation were seen in the cortical tubules of all exposed males. These are a common effect observed in repeated-dose animal studies with hydrocarbon solvents. These kidney changes have been identified to result from an alpha2u-globulin-mediated process that because of its sex and species specificity, is not regarded as relevant to humans.

A low grade anemia was evident in all males exposed to SHELLSOL TD, characterized by slight reductions in haemoglobin, packed cell volume and total erythrocyte counts. Splenic weight was increased in the high concentration males. These changes were not seen in females and were not considered dose-related and therefore considered not toxicologically relevant.

Male and female liver weights were increased at the high and medium exposures, and male liver weights at the low exposures also. No lesions were identified histologically in the livers of treated animals that could account for the increased weight. This change was considered a physiological response to exposure rather than a toxic response and as such is not of toxicological significance.

Key result

Dose descriptor:

NOAEC

Effect level:

> 10 400 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment-related mortality or significant adverse clinical effects occurred.

Critical effects observed:

not specified

Conclusions:

The NOAEC for SHELLSOL TD is 10186 mg/m3 (actual) (1444 ppm) under the test conditions of this study.

Executive summary:

SHELLSOL TC was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 10400 mg/m³, 5200 mg/m³, and 2600 mg/m³ to assess inhalation toxicity.  No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed.  Liver and kidney weights were increased in male rats at all exposure levels, male heart weights were increased at the highest exposure level and liver and kidney weights were increased in female rats at 10400 mg/m3.  In addition, the male rats exposed to SHELLSOL TC at all concentrations showed tubular degeneration and hyaline inclusion-droplets in the epithelium.  There was also scattered degeneration of the proximal renal tubules which showed cytoplasmic pallor and shrinkage. Occasionally the degenerate tubules were surrounded by a lymphocyte infiltrate. Many tubules also showed dilatation of the cortico-medullary junction, the dilated tubule being filled with a flocculent eosinophilic material. The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.  Histopathological examination did not reveal any abnormalities that were considered treatment related.  As there were no pathologic changes, changes in organ weights mentioned above were judged to have been compensatory rather than toxic effects.  Based on these results, the No Observed Adverse Effect Concentration (NOAEC) was greater than or equal to 10400 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

10 400 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

2 key and two supporting read across sub-chronic toxicity studies from structural analogues available for assessment.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no repeated dose toxicity data for Hydrocarbons, C9-C10, n-alkanes, isoalkanes, cyclics <2% aromatics. However, short-term data is available for structural analogues Hydrocarbons, C10-C12, isoalkanes, <2% aromatics and Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics. Sub-chronic data is available for structural analogues Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics,

Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C10-C12, isoalkanes, <2% aromatics, and Hydrocarbons, C10 -C13, n-alkanes, isoalkanes, cyclics, <2% aromatics.

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

Oral:

 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key repeated dose oral toxicity study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/Kg, 7 days a week for 13 weeks to assess the subchronic toxicity. An additional group of animals, dosed at 5000 mg/Kg/day, was held for 4 weeks to assess reversibility. No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/Kg dose groups. Liver weights were elevated in male and female rats at 2500 and 5000 mg/Kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/Kg. Testes weights were elevated in male rats at 5000 mg/Kg. Kidney effects occurred in males at all dose levels, and are indicative of alpha-2u-globulin nephropathy. Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats. These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes. These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.

 

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver.  Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups. These findings are believed to have been a compensatory response and not an indication of toxicity. Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans. Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance. These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity. All treatment-related effects were reversible within the 4-week recovery period. Based on a significant increase in ALT levels in the 2500 and 5000 mg/kg/day treatment groups in male rats, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 500 mg/Kg/day.

 

This NOAEL value is dependent on doses selected in the study and may not represent a true biological threshold. In order to circumvent the problem of dose-selection bias, benchmark analysis was used to determine a benchmark dose for this study, using individual ALT dose–response values in male rats as the critical effect. Since the minimal level of change in the endpoint (increased serum ALT) that would be considered biologically significant was known (2–4-fold increase compared to concurrent control values), this value was used as the Benchmark response in the derivation of a BMDL, although the BMDL value using the EPA default BMR of 1SD from the mean was provided

for comparison. The use of the 1SD default for the BMR resulted in an overly conservative BMDL value, 3-fold lower than would have been predicted using the NOAEL/ LOAEL approach. When the BMR was more accurately defined in terms of a 2-fold minimum level of change over the control mean, the estimated BMDL value was 1857 mg/Kg/day.

 

Inhalation:

 

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

In a supporting study (ExxonMobil, 1978), the test material (Hydrocarbons, C9-C11, isoalkanes, cyclics, <2% aromatics) was administered by inhalation to Sprague-Dawley rats for 6 hours/day, 5 days/week for 12 weeks at nominal vapor concentrations of 300 ppm and 900 ppm to assess subchronic inhalation toxicity.  Ten animals per sex per group were examined at 4 weeks, 8 weeks and all survivors were sacrificed and examined at 12 weeks. Male body weight gain was significantly decreased at 900 ppm.  There were no treatment-related effects in any of the hematology and serum chemistry values.  Liver and kidney weights were increased in male rats at 900 ppm, and adrenal weights were increased in female rats at 900 ppm.  The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.  Histopathological examination did not reveal any abnormalities that were considered treatment related.  As there were no pathologic changes, changes in organ weight to body weight ratios were judged to have been compensatory rather than toxic effects.  Based on these results, the No Observed Adverse Effect Level (NOAEL) was greater than or equal to 900 ppm (>=5220 mg/m³).

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

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/m36 hours per day, 5 days per week, for a period of 13 weeks. Similar number of control animals were exposed to air only. 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. 

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

In a key study (Shell, 1980), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by inhalation to albino rats for 6 hours/day, 5 days/week for 13 weeks at nominal vapor concentrations of 10400 mg/m³, 5200 mg/m³, and 2600 mg/m³to assess inhalation toxicity.  No mortality or treatment-related effects in any of the hematology and serum chemistry values were observed.  Liver and kidney weights were increased in male rats at all exposure levels, male heart weights were increased at the highest exposure level and liver and kidney weights were increased in female rats at 10400 mg/m3.  In addition, the male rats exposed to the test material at all concentrations showed tubular degeneration and hyaline inclusion-droplets in the epithelium.  There was also scattered degeneration of the proximal renal tubules which showed cytoplasmic pallor and shrinkage. Occasionally the degenerate tubules were surrounded by a lymphocyte infiltrate. Many tubules also showed dilatation of the cortico-medullary junction, the dilated tubule being filled with a flocculent eosinophilic material. The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.  Histopathological examination did not reveal any abnormalities that were considered treatment related.  As there were no pathologic changes, changes in organ weights mentioned above were judged to have been compensatory rather than toxic effects.  Based on these results, the No Observed Adverse Effect Concentration (NOAEC) was greater than or equal to 10400 mg/m³.

In a supporting study (Chevron, 1969), four rhesus monkeys were exposed to 4.2 mg/L of the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) for 6 hours/day, for 3 days a week for 4 weeks. A total of 13 treatments were completed. There were no noted changes in behavior, clinical chemistry, hematological, or histopathological parameters. The NOAEC in primates was determined to be > 4.2 mg/L (> 4200 mg/m³).

In a supporting study (ExxonMobil, 1978) the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by inhalation to rats at vapor concentrations of 300 or 900 ppm for 6 hours/day, 5 days/week for 12 weeks.  No treatment-related effects on mortality were observed and there were no significant alterations in hematology or clinical chemistry parameters.  Body weights were decreased and kidney weights were elevated in male rats at 300 and 900 ppm.  Relative mean liver weights were elevated in males at 900 ppm, but no changes were noted in histopathology.  Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) is greater than 900 ppm (> 5220 mg/m³). 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a supporting study (Chevron, 1969), test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics) vapor was introduced into a stainless steel and glass exposure chamber (volume‑85. cubic feet) at a rate of 4.25 cubic feet per minute. The final concentration of test material in the chamber was calculated to be 4.2 mg/L (654 ppm). An equilibrium period of 15 minutes was allowed prior to introduction of the test animals into the chamber. The exposure lasted for six hours per day, three days per week (Monday, Wednesday, and Friday), for a total of 13 exposures. Observations throughout the one month exposure period were conducted for behavioral patterns, body weight, food consumption, hematology, clinical chemistry, urine analysis, gross pathology, and microscopic pathology. All four rhesus monkeys survived the study. Observations for behavioral patterns, body weight, food consumption, clinical chemistry, urine analysis, gross pathology, and microscopic pathology were unremarkable. A slight lymphocytopenia and a slight neutrophilia were noted in the differential leucocyte count at the mid point of the study and after the last exposure.

Justification for classification or non-classification

Based on the available read across data, Hydrocarbons, C9-C10, n-alkanes, isoalkanes, cyclics, <2% aromatics does not meet the criteria for classification for repeated dose toxicity (STOT-RE) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).