[No public or meaningful name is available]

Endpoint summary

Description of key information

Repeated dose dermal and inhalation toxicity studies in rats are available.
In a sub-chronic inhalation study no systemic effects were reported and a NOEC of 1710 mg/m3 was determined.
In a key sub-chronic dermal study, the systemic LOAEL was 1.00 mL/kg/day (estimated to be 835 mg/kg/day) based on moderate dermal irritation accompanied by decreased body weight (males only), increased food consumption, changes in differential leukocyte counts, decreased albumin (and albumin/globulin ratio), and increased incidence of lymphoid hyperplasia of the axilliary lymph node. The NOAEL was 0.1 mL/kg/day (estimated to be 83.5 mg/kg/day).

In addition, a recent study to OECD 411 and GLP was conducted on a member of the VHGO category. This showed low systemic toxicity (NOAEL 600 mg/kg/day), with higher doses not possible due to excessive local irritation. This study was conducted on a substance in the category low in PAH content (the constituent expected to drive toxicological hazard).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

1982-08-09 to 1983-01-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there is no GLP statement, but the study was generally conducted in accordance with OECD 413 guidelines.

Justification for type of information:

Read across justification included in 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)

Deviations:

yes

Remarks:

animals were exposed 4hrs/day, 2 days/week - total 26 exposures

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Massachussetts
- Age at study initiation: 18 to 21 weeks old
- Weight at study initiation: Approximately 500 grams for males and 260 to 290 grams for females
- Housing: Individaully in hanging, stainless steel, wire mesh cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 10 to 13 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 1982-08-09 To: 1983-01-24

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Ranged from 0.43 microns to 0.75 microns with a standard geometric deviation of 1.4 to 1.7

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: 1.5 m3 New York University style inhalation chambers
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: Vycor heater
- Temperature, humidity, pressure in air chamber: 20 to 24 degrees Celcius, 70% humidity, and air pressure not reported
- Air flow rate: Only specified as constant
- Air change rate: Not reported
- Method of particle size determination: Cascade impactation at various times during the experiment
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared backscatter probes and periodic filter samples taken for gravimetric determination, which were also analyzed by high performance liquid chromatography and gas chromatography
- Samples taken from breathing zone: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gravimetric data indicate that all concentrations were greater than the target concentrations by 40%, 17%, and 14% for the 250, 750, and 1500 mg/m3 target concentrations; however, the backscatter probes found the concentration of the 250 mg/m3 group to be lower than the target concentration. This was considered a result of the vapour concentration and aerosol concentrations were considered to be close enough to the target to be acceptable.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Twice a week

Remarks:

Doses / Concentrations:
0, 0.25, 0.75, or 1.50 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 0.35, 0.88, 1.71 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

24 for sham control and treatment groups and 12 for untreated controls

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: This was phase 3 of the testing protocol. Phase 1 and 2 were acute and short-term testing. Doses were based on the previous tests.
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period in satellite groups: 2 months
- Section schedule rationale (if not random): Not reported

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During exposure and on removal from the chamber
- Cage side observations were not reported.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At end of exposure and after 2 month recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked included red blood cell counts, white blood cell counts, and haematocrit

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Terminal sacrifice (immediately after exposure and after 2-month recovery)
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation, at various time points over the 13 week exposure period, and once a month during the 2-month recovery
- Dose groups that were examined: All groups
- Battery of functions tested: other: Breathing frequency and startle response

OTHER: The number of alveolar macrophages in pulmonary lavage fluid and pulmonary function were measured.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

Select organs were weighed (Table 2).

Statistics:

Analysis of variance

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present.

BODY WEIGHT AND WEIGHT GAIN: Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls (Table 1). Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively.

FOOD CONSUMPTION: Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

HAEMATOLOGY: There were no treatment-related effects on red blood cell counts, white blood cell counts, or haematocrit.

CLINICAL CHEMISTRY: Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported.

NEUROBEHAVIOUR: Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14th exposure, before the 26th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

ORGAN WEIGHTS: Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups (Table 2). Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups.

HISTOPATHOLOGY: Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals.

OTHER FINDINGS: The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected).Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

Dose descriptor:

NOAEC

Remarks:

Systemic effects

Effect level:

> 1.71 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: systemic effects

Dose descriptor:

NOAEC

Remarks:

Local effects

Effect level:

0.88 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: Lung weight

Critical effects observed:

not specified

 

Table 1. Body weight
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Initial	508.7±9.2	512.6±9.4	510.3±11.7	507.6±7.1
End of exposure	580.3±12.2	543.7±10.9 *	525.6±13.6 *	528.1±8.8 *
End of recovery	606.1±22.9	608.4±13.9	570.0±20.7	588.4±17.4
Females
Initial	284.6±3.5	287.7±4.0	266.0±3.7 *	267.4±3.7 *
End of exposure	331.5±6.1	301.3±5.1 *	279.4±4.2 *	283.5±4.3 *
End of recovery	336.5±9.9	336.3±12.1	311.2±9.4	321.4±9.7

Significantly different from the control * p<0.05

   

Table 2. Select Relative Organ Weights
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Liver After exposure 2 month post exposure	10.24±0.55 9.67±0.60	10.82±0.49 11.56±6.3 *	10.72±0.48 9.63±0.55	13.18±0.51 * 10.61±0.57
Lung (wet weight right middle lobe) After exposure 2 month post exposure	162±8 143±9	160±7 148±9	173±7 149±8	193±7 * 148±8
Lung (wet/dry ratio) After exposure 2 month post exposure	5.64±0.19 5.04±0.20	5.55±0.17 5.05±0.22	5.36±0.16 5.31±0.19	5.52±0.17 6.95±0.20 **
Females
Liver After exposure 2 month post exposure	10.49±0.72 11.63±0.65	11.12±0.89 11.38±0.67	10.83±0.47  11.17±0.78	11.98±0.92 * 11.41±0.74
Lung (wet weight right middle lobe) After exposure 2 month post exposure	157±10 149±9	161±12 157±9	171±14 144±11	185±13 * 161±11
Lung (wet/dry ratio) After exposure 2 month post exposure	5.53±0.25 4.99±0.23	5.35±0.31 4.91±0.23	5.15±0.0.33 5.01±0.27	5.31±0.32 7.67±0.26 **

Significantly different from the control * p<0.05; ** p< 0.0001

Conclusions:

The sub-chronic inhalation study of diesel fuel resulted in a conservative sub-chronic NOAEC of 750 mg/m3 determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 was established for systemic effects, based on no significant findings at this level.

Executive summary:

The sub-chronic inhalation toxicity of diesel fuel, including potential effects on neurological and pulmonary function, has been investigated in a well conducted, well reported study in which groups of male and female Sprague-Dawley rats were exposed whole body to 250, 750 or 1500 mg/m³aerosol (MMAD 0.43-0.75 microns) 4 hour per day, two days per week for 13 weeks (total of 26 exposures). The study also included a sham (chamber) control group and an untreated (animal room) control group, with animals in all groups aged 18-21 weeks at the start of the study. Body weight (all animals) and food consumption (6/sex/dose level) were recorded weekly. Breathing frequency was measured using a barometric method (groups of 12 rats/sex/dose level, temporarily housed in sealed chambers) prior to the first exposure (baseline), before the 14^th and 26^th exposures (i.e., study weeks 7 and 13), and after one or two months recovery. Startle reflex (assessed by measuring reaction time, peak time and peak height following exposure to five 10 msec pulses of 110 dB noise) was quantified in males only (8/dose) immediately after the first, 14^th and 26^th exposures (to evaluate acute/short term effects), and in both sexes (8/sex/dose) prior to the 14^th and 26^th exposures and after one and two months recovery (to assess chronic/cumulative changes). The type and number of free cells present in pulmonary lavage fluid and serum chemistry (alkaline phosphatase, aspartate aminotransferase, cholesterol, triglyceride, uric acid, urea nitrogen, glucose, bilirubin, creatinine, sodium, potassium) were determined in groups of animals (6/sex/dose) at study termination and following a two-month recovery period. Lung function tests (pulmonary resistance, nitrogen washout, carbon monoxide diffusion, functional reserve capacity, peak expiratory flow, total lung capacity, vital capacity, inspiratory capacity, functional residual capacity, residual volume, specific compliance) were performed on groups of anaesthetised animals (8/sex/dose level, fitted with a tracheal cannula) after 13 weeks exposure and also following a 2-month recovery period using whole body plethysmography. After completion of the lung function tests, animals were subject to necropsy (including gross examination and collection of organ weight data), followed by histological examination of around 15-20 tissues.

 

There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present. Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls. Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively. Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

 

Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14^th exposure, before the 26^th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14^th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

 

The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected). Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

 

Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups. Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups. Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported. Red cell and white cell counts were unaffected.

 

Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals. No treatment -related effects were noted in examination of the weight and histopathology of the testes.

 

These results demonstrate statistically significant alterations in a number of parameters (body weight, food consumption, startle reflex, certain lung function parameters) in rats following sub-chronic inhalation exposure to diesel aerosol, however the magnitude of these changes was small suggesting that they are of doubtful biological relevance. Statistically significant increases in relative liver weight and relative wet lung weight were observed in animals exposed to 1710 mg/m³ (actual concentration) diesel aerosol for 13 weeks, however there was no histopathological involvement, again making the relevance of these findings unclear.  It is noted that the use of whole body exposure probably resulted in ingestion of the test sample during grooming, and may account for the systemic findings that were observed. All of the changes present following 13 weeks exposure were reversed after a 2-month recovery period. A conservative sub-chronic NOAEC of 750 mg/m3 is determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 is established for systemic effects, based on no significant findings at this level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 710 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

1982-08-09 to 1983-01-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there is no GLP statement, but the study was generally conducted in accordance with OECD 413 guidelines.

Justification for type of information:

Read across justification included in 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)

Deviations:

yes

Remarks:

animals were exposed 4hrs/day, 2 days/week - total 26 exposures

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Massachussetts
- Age at study initiation: 18 to 21 weeks old
- Weight at study initiation: Approximately 500 grams for males and 260 to 290 grams for females
- Housing: Individaully in hanging, stainless steel, wire mesh cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 10 to 13 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 1982-08-09 To: 1983-01-24

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Ranged from 0.43 microns to 0.75 microns with a standard geometric deviation of 1.4 to 1.7

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: 1.5 m3 New York University style inhalation chambers
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: Vycor heater
- Temperature, humidity, pressure in air chamber: 20 to 24 degrees Celcius, 70% humidity, and air pressure not reported
- Air flow rate: Only specified as constant
- Air change rate: Not reported
- Method of particle size determination: Cascade impactation at various times during the experiment
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared backscatter probes and periodic filter samples taken for gravimetric determination, which were also analyzed by high performance liquid chromatography and gas chromatography
- Samples taken from breathing zone: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gravimetric data indicate that all concentrations were greater than the target concentrations by 40%, 17%, and 14% for the 250, 750, and 1500 mg/m3 target concentrations; however, the backscatter probes found the concentration of the 250 mg/m3 group to be lower than the target concentration. This was considered a result of the vapour concentration and aerosol concentrations were considered to be close enough to the target to be acceptable.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Twice a week

Remarks:

Doses / Concentrations:
0, 0.25, 0.75, or 1.50 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 0.35, 0.88, 1.71 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

24 for sham control and treatment groups and 12 for untreated controls

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: This was phase 3 of the testing protocol. Phase 1 and 2 were acute and short-term testing. Doses were based on the previous tests.
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period in satellite groups: 2 months
- Section schedule rationale (if not random): Not reported

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During exposure and on removal from the chamber
- Cage side observations were not reported.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At end of exposure and after 2 month recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked included red blood cell counts, white blood cell counts, and haematocrit

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Terminal sacrifice (immediately after exposure and after 2-month recovery)
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation, at various time points over the 13 week exposure period, and once a month during the 2-month recovery
- Dose groups that were examined: All groups
- Battery of functions tested: other: Breathing frequency and startle response

OTHER: The number of alveolar macrophages in pulmonary lavage fluid and pulmonary function were measured.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

Select organs were weighed (Table 2).

Statistics:

Analysis of variance

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present.

BODY WEIGHT AND WEIGHT GAIN: Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls (Table 1). Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively.

FOOD CONSUMPTION: Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

HAEMATOLOGY: There were no treatment-related effects on red blood cell counts, white blood cell counts, or haematocrit.

CLINICAL CHEMISTRY: Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported.

NEUROBEHAVIOUR: Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14th exposure, before the 26th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

ORGAN WEIGHTS: Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups (Table 2). Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups.

HISTOPATHOLOGY: Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals.

OTHER FINDINGS: The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected).Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

Dose descriptor:

NOAEC

Remarks:

Systemic effects

Effect level:

> 1.71 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: systemic effects

Dose descriptor:

NOAEC

Remarks:

Local effects

Effect level:

0.88 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: Lung weight

Critical effects observed:

not specified

 

Table 1. Body weight
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Initial	508.7±9.2	512.6±9.4	510.3±11.7	507.6±7.1
End of exposure	580.3±12.2	543.7±10.9 *	525.6±13.6 *	528.1±8.8 *
End of recovery	606.1±22.9	608.4±13.9	570.0±20.7	588.4±17.4
Females
Initial	284.6±3.5	287.7±4.0	266.0±3.7 *	267.4±3.7 *
End of exposure	331.5±6.1	301.3±5.1 *	279.4±4.2 *	283.5±4.3 *
End of recovery	336.5±9.9	336.3±12.1	311.2±9.4	321.4±9.7

Significantly different from the control * p<0.05

   

Table 2. Select Relative Organ Weights
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Liver After exposure 2 month post exposure	10.24±0.55 9.67±0.60	10.82±0.49 11.56±6.3 *	10.72±0.48 9.63±0.55	13.18±0.51 * 10.61±0.57
Lung (wet weight right middle lobe) After exposure 2 month post exposure	162±8 143±9	160±7 148±9	173±7 149±8	193±7 * 148±8
Lung (wet/dry ratio) After exposure 2 month post exposure	5.64±0.19 5.04±0.20	5.55±0.17 5.05±0.22	5.36±0.16 5.31±0.19	5.52±0.17 6.95±0.20 **
Females
Liver After exposure 2 month post exposure	10.49±0.72 11.63±0.65	11.12±0.89 11.38±0.67	10.83±0.47  11.17±0.78	11.98±0.92 * 11.41±0.74
Lung (wet weight right middle lobe) After exposure 2 month post exposure	157±10 149±9	161±12 157±9	171±14 144±11	185±13 * 161±11
Lung (wet/dry ratio) After exposure 2 month post exposure	5.53±0.25 4.99±0.23	5.35±0.31 4.91±0.23	5.15±0.0.33 5.01±0.27	5.31±0.32 7.67±0.26 **

Significantly different from the control * p<0.05; ** p< 0.0001

Conclusions:

The sub-chronic inhalation study of diesel fuel resulted in a conservative sub-chronic NOAEC of 750 mg/m3 determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 was established for systemic effects, based on no significant findings at this level.

Executive summary:

The sub-chronic inhalation toxicity of diesel fuel, including potential effects on neurological and pulmonary function, has been investigated in a well conducted, well reported study in which groups of male and female Sprague-Dawley rats were exposed whole body to 250, 750 or 1500 mg/m³aerosol (MMAD 0.43-0.75 microns) 4 hour per day, two days per week for 13 weeks (total of 26 exposures). The study also included a sham (chamber) control group and an untreated (animal room) control group, with animals in all groups aged 18-21 weeks at the start of the study. Body weight (all animals) and food consumption (6/sex/dose level) were recorded weekly. Breathing frequency was measured using a barometric method (groups of 12 rats/sex/dose level, temporarily housed in sealed chambers) prior to the first exposure (baseline), before the 14^th and 26^th exposures (i.e., study weeks 7 and 13), and after one or two months recovery. Startle reflex (assessed by measuring reaction time, peak time and peak height following exposure to five 10 msec pulses of 110 dB noise) was quantified in males only (8/dose) immediately after the first, 14^th and 26^th exposures (to evaluate acute/short term effects), and in both sexes (8/sex/dose) prior to the 14^th and 26^th exposures and after one and two months recovery (to assess chronic/cumulative changes). The type and number of free cells present in pulmonary lavage fluid and serum chemistry (alkaline phosphatase, aspartate aminotransferase, cholesterol, triglyceride, uric acid, urea nitrogen, glucose, bilirubin, creatinine, sodium, potassium) were determined in groups of animals (6/sex/dose) at study termination and following a two-month recovery period. Lung function tests (pulmonary resistance, nitrogen washout, carbon monoxide diffusion, functional reserve capacity, peak expiratory flow, total lung capacity, vital capacity, inspiratory capacity, functional residual capacity, residual volume, specific compliance) were performed on groups of anaesthetised animals (8/sex/dose level, fitted with a tracheal cannula) after 13 weeks exposure and also following a 2-month recovery period using whole body plethysmography. After completion of the lung function tests, animals were subject to necropsy (including gross examination and collection of organ weight data), followed by histological examination of around 15-20 tissues.

 

There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present. Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls. Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively. Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

 

Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14^th exposure, before the 26^th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14^th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

 

The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected). Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

 

Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups. Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups. Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported. Red cell and white cell counts were unaffected.

 

Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals. No treatment -related effects were noted in examination of the weight and histopathology of the testes.

 

These results demonstrate statistically significant alterations in a number of parameters (body weight, food consumption, startle reflex, certain lung function parameters) in rats following sub-chronic inhalation exposure to diesel aerosol, however the magnitude of these changes was small suggesting that they are of doubtful biological relevance. Statistically significant increases in relative liver weight and relative wet lung weight were observed in animals exposed to 1710 mg/m³ (actual concentration) diesel aerosol for 13 weeks, however there was no histopathological involvement, again making the relevance of these findings unclear.  It is noted that the use of whole body exposure probably resulted in ingestion of the test sample during grooming, and may account for the systemic findings that were observed. All of the changes present following 13 weeks exposure were reversed after a 2-month recovery period. A conservative sub-chronic NOAEC of 750 mg/m3 is determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 is established for systemic effects, based on no significant findings at this level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

750 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

1992-09-14 to 1993-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because it was GLP compliant and was generally conducted according to OECD 411 guidelines.

Justification for type of information:

Read across justification included in Section 13

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Remarks:

Doses / Concentrations:
0.01, 0.1, or 1.00 mL/kg/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

Twenty

Control animals:

yes, sham-exposed

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

0.1 other: mL/kg/day

Sex:

male/female

Basis for effect level:

other: body weight; food consumption; haematology; clinical chemistry; histopathology

Critical effects observed:

not specified

Conclusions:

The systemic LOAEL is 1.00 mL/kg/day (estimated to be 835 mg/kg/day) based on moderate dermal irritation accompanied by decreased body weight (males only), increased food consumption, changes in differential leukocyte counts, decreased albumin (and albumin/globulin ratio), and increased incidence of lymphoid hyperplasia of the auxilliary lymph node. The NOAEL is 0.1 mL/kg/day (estimated to be 83.5 mg/kg/day).

Executive summary:

In a 90-day dermal toxicity study, diesel fuel (Diesel 1) was applied to the shaved skin of twenty Sprague-Dawley rats/sex/dose at dose levels of 0, 0.01, 0.10, or 1.00 mL/kg bw/day, 6 hours/day for 5 days/week during a 90-day period.

 

There was moderate dermal irritation in the high-dose, slight dermal irritation in the mid-dose group, and very slight dermal irritation in the low-dose group. Body weight was decreased in high-dose males, but food consumption was increased in both sexes of the high-dose group. Changes in differential leukocyte counts, decreased albumin (and albumin/globulin ratio), and increased incidence of lymphoid hyperplasia of the auxilliary lymph node also occurred in the high-dose group and were considered to be secondary to the dermal irritation. The systemic LOAEL is 1.00 mL/kg/day (estimated to be 835 mg/kg/day) based on moderate dermal irritation accompanied by decreased body weight (males only), increased food consumption, changes in differential leukocyte counts, decreased albumin (and albumin/globulin ratio), and increased incidence of lymphoid hyperplasia of the auxilliary lymph node. No treatment-related effects were noted in examination of the weight of the testes and ovaries nor in the histopathology of the testis, ovary, prostate and uterus.

The NOAEL is 0.1 mL/kg/day (estimated to be 83.5 mg/kg/day).

 

This study received a Klimisch score of one and is classified as reliable without restrictions because it was GLP compliant and was generally conducted according to OECD 411 guidelines.

Reference 2

Endpoint:

sub-chronic toxicity: dermal

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:

guideline study

Justification for type of information:

Concawe believes that dermal is the most relevant exposure route, and is sufficiently robust, to identify any potential hazards from repeated exposures to petroleum products to be able to adequately manage the potentially associated risks. Please see attached document for further justification.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2250 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Ultra-Low Sulfur Diesel Fuel
CAS 68334-30-5
WIL sample ID no. 110129

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This species and strain of animal is recognized as appropriate for subchronic and chronic toxicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 45 days old upon receipt; at initiation 58 days
- Weight at study initiation: Individual body weights ranged from 209 g to 253 g for males and from 161 g to 188 g for females at randomization
- Housing: all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, available ad libitum except during the period of fasting prior to clinical pathology blood collection
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, available ad libitum
- Acclimation period: 13 days, during which time the animals were acclimated to wearing Elizabethanstyle collars on an incremental basis starting on study day -9 (1 hour), -8 (2 hours), -7 (4 hours), -6 (8 hours), -5 (24 hours)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71°F ± 5°F (22°C ± 3°C); Actual mean daily temperature ranged from 69.6°F to 72.3°F (20.9°C to 22.4°C)
- Humidity (%): 50% ± 20%; Actual mean daily relative humidity ranged from 46.0% to 70.3%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: To: 26th April 2011 to 7th August 2011

Type of coverage:

open

Vehicle:

other: mineral oil, USP lot no. ZH1000, exp. dates 13 May 2013 and 3 March 2012; lot no. 2AB0895, exp. date 16 May 2013; manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

Details on exposure:

TEST SITE
- Area of exposure: dorsal scapular area
- % coverage: 10%
- Type of wrap if used: no wrapping - Elizabethan collar worn to minimize ingestion of test substance
- Time intervals for shavings or clipplings: the day prior to initiation of dose administration and as often as necessary thereafter.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): patted with paper towel at the end of 6-hr exposure period; after each 5-day period residual test substance was removed (as much as possible without inducing irritation of the skin) from all animals using a warm water and mild soap solution (1% Ivory liquid soap in tap water) followed by a deionized water rinse and drying of the animals with a clean paper towel. Following each wash procedure, all animals were transferred to clean cages and the collars removed for a 2-day nondosing period
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.5 mL/kg (spread evenly using a glass rod)
- Concentration (if solution): 0, 66.7, 200, 400 mg/mL for control, 100, 300, 600 mg/kg/day groups respectively
- Constant volume or concentration used: no - Individual dosages and dose volumes were based on the most recently recorded body weights to provide the correct mg/kg/day dose

VEHICLE
mineral oil, USP lot no. ZH1000, exp. dates 13 May 2013 and 3 March 2012; lot no. 2AB0895, exp. date 16 May 2013; manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes - Elizabethan collars

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated GC/MS method was used for the determination of test substance concentration in suspension formulations. Two sets of duplicate samples from the top, middle, and bottom strata of the formulations prepared during study week 0, and from the middle stratum of those prepared in weeks 6 and12 at target test substance concentrations of 66.7, 200, and 400 mg/mL were analyzed to assess test substance homogeneity/concentration acceptability. Duplicate samples from the blank vehicle were also collected and analyzed. The analytical results met the applicable WIL Research SOP acceptance criteria for test substance homogeneity/concentration. No test article was detected in the analyzed vehicle administered to the control group.

Homogeneity, stability, and resuspension homogeneity of the test substance formulations were previously established at concentrations of 50 and 500 mg/mL following 8 days of room temperature storage during a preliminary study. Therefore, stability and resuspension homogeneity analyses were not conducted during the current study

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week for 6 hours per day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Remarks:

this was the highest tolerable dose due to dermal irritation observed in a 14 day rangefinder (moribund animals due to dermal irritation observed at 1000 mg/kg/day)

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

yes, sham-exposed

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design
- Fasting period before blood sampling for clinical biochemistry: during the period of overnight fasting prior to clinical pathology blood collection food, but not water, was withheld

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed on all animals prior to dose application and at approximately 1 to 2 hours following dose application. On nondosing days, the animals were observed once daily. Detailed physical examinations were conducted on all animals at least once during the pretest period, approximately weekly during the study, and on the days of the scheduled necropsy

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dermal observations were performed on all animals once during the pretest period and on all animals at the end of each 5-day dosing period at approximately 1 to 2 hours
following the removal of any residual test substance. The application sites were scored for erythema and edema

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly beginning during the pretest period and for the duration of the study. Body weights were collected prior to the
first dose of each 5-day dosing period (animals were weighed without collars). During study week 12, body weights were recorded twice weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Individual food consumption was recorded approximately weekly beginning during the pretest period and for the duration of the study. Food intake was calculated as g/animal/day for the corresponding body weight intervals

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to randomization (study week -1) and prior to the scheduled necropsy (study week 12)
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected from all animals at the scheduled necropsy (study week 12)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table 1 [in any other information on materials and methods] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected from all animals at the scheduled necropsy (study week 12)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table 1 [in any other information on materials and methods] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were euthanized by isoflurane inhalation. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The following organs were weighed from all animals at the scheduled necropsy:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary
Prostate
Spleen
Testes
Thymus
Thyroid with parathyroids*
Uterus
Paired organs were weighed together. Designated organs (*) were weighed after fixation.
Organ to final body weight and organ to brain weight ratios were calculated

HISTOPATHOLOGY: Yes (see table)
Microscopic examination was performed on all tissues listed in table 2 [in any other information on materials and methods] from all animals. After fixation, protocol-specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase [GGT]), and organ weight (absolute and relative) data, for the vehicle control group and all test substance-treated groups were subjected to a one-way analysis of variance to determine intergroup differences. If significant intergroup variance was found, Dunnett's test was used to compare the test
substance-treated groups to the vehicle control group.
The vehicle control group data were evaluated using the Student’s t-Test and compared to the sham control group at a 95% confidence level (p < 0.05).

GGT values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. GGT data were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test was used to compare the test substance-treated groups to the vehicle control group. In addition, the sham control group and vehicle control group were compared using a separate Kruskal-Wallis nonparametric ANOVA test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects noted during the dermal observations. Residual test substance within the dose site was noted in the 100, 300, and 600 mg/kg/day group males and females.
Dermal findings of very slight, slight, and/or moderate erythema, very slight and/or slight edema, and/or scabbing within dose site were noted in the 100, 300, and/or 600 mg/kg/day group males and females, as well as in the vehicle control group. Therefore, these dermal observations were considered most likely vehicle-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights were unaffected by test substance administration.

Statistically significant differences in mean body weight changes were noted in the 600 mg/kg/day group females between the two weighings conducted in study week 12 when compared to the vehicle control group. Differences in body weight gain were probably due to biological variability and were not considered related to test substance administration due to lack of a dose-response trend. In addition, a statistically significant mean body weight loss was noted in the vehicle control group females during the same period when compared to the sham control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was unaffected by test substance administration.

Statistically significantly higher mean food consumption was noted in the 300 mg/kg/day group females (study week 10 to 11) and statistically significantly lower mean food consumption was noted in the 100 mg/kg/day group females (during double observation in study week 12’); however, due to the magnitude of change and the lack of a dose-response trend, these values were not considered test substance-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in hematology and coagulation parameters. There were no statistically significant differences when the vehicle control and test substance-treated groups were compared.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in serum chemistry parameters.

A statistically significant difference was observed when the vehicle and test substance-exposed groups were compared. The albumin to globulin (A/G) ratio in the 600 mg/kg/day group males was higher than the vehicle control group but was not considered test substance-related as the value was similar to the sham control male group value.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No observations that were considered to be associated with application of the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic findings.

Findings in the skin unrelated to test substance exposure included mononuclear infiltrates in the treated, untreated, and inguinal skin. These infiltrates consisted of lymphocytes and plasma cells in the superficial dermis and rarely surrounding follicles in all groups. Inguinal skin from test substance-exposed males and females had a slightly higher incidence of mononuclear infiltrates compared to the sham control group. Untreated skin in the 300 and 600 mg/kg/day group males and females had a slightly higher incidence of mononuclear infiltrates when compared to the sham and vehicle control groups. These findings were not considered test substance-related as they were noted in skin that was not exposed to the test substance and there was no microscopic evidence of systemic exposure. One 600 mg/kg/day group male had moderate chronic-active inflammation (characterized by lymphocytes, plasma cells, neutrophils and macrophages accompanied by fibroplasia). in the inguinal skin. Inflammatory cells were observed surrounding occasionally degenerate hair follicles and extended focally into the adjacent panniculus muscle. This inflammatory lesion was not considered test substance-related as this area was not exposed to test substance and was more consistent with a background lesion.

Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test article. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects noted at top dose tested

Key result

Critical effects observed:

no

Conclusions:

There were no treatment-related effects at 600 mg/kg/day, establishing the no-observed-effect level (NOEL) the highest dosage level evaluated.

Executive summary:

Based on the results of this study, dermal application of ultra-low sulfur diesel fuel over an area of approximately 10% of the shaved body surface area to Crl:CD(SD) rats for approximately 6 hours per day, 5 days per week, for a minimum 90-day treatment period at dosage levels of 100, 300, and 600 mg/kg/day was well tolerated at all dosage levels.

There were no treatment-related effects at 600 mg/kg/day, establishing the no-observed-effect level (NOEL) the highest dosage level evaluated.

Reference 3

Endpoint:

sub-chronic toxicity: dermal

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:

guideline study

Justification for type of information:

Concawe believes that dermal is the most relevant exposure route, and is sufficiently robust, to identify any potential hazards from repeated exposures to petroleum products to be able to adequately manage the potentially associated risks. Please see attached document for further justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2250 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Ultra-Low Sulfur Diesel Fuel
CAS 68334-30-5
WIL sample ID no. 110129

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This species and strain of animal is recognized as appropriate for subchronic and chronic toxicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 45 days old upon receipt; at initiation 58 days
- Weight at study initiation: Individual body weights ranged from 209 g to 253 g for males and from 161 g to 188 g for females at randomization
- Housing: all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, available ad libitum except during the period of fasting prior to clinical pathology blood collection
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, available ad libitum
- Acclimation period: 13 days, during which time the animals were acclimated to wearing Elizabethanstyle collars on an incremental basis starting on study day -9 (1 hour), -8 (2 hours), -7 (4 hours), -6 (8 hours), -5 (24 hours)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71°F ± 5°F (22°C ± 3°C); Actual mean daily temperature ranged from 69.6°F to 72.3°F (20.9°C to 22.4°C)
- Humidity (%): 50% ± 20%; Actual mean daily relative humidity ranged from 46.0% to 70.3%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: To: 26th April 2011 to 7th August 2011

Type of coverage:

open

Vehicle:

other: mineral oil, USP lot no. ZH1000, exp. dates 13 May 2013 and 3 March 2012; lot no. 2AB0895, exp. date 16 May 2013; manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

Details on exposure:

TEST SITE
- Area of exposure: dorsal scapular area
- % coverage: 10%
- Type of wrap if used: no wrapping - Elizabethan collar worn to minimize ingestion of test substance
- Time intervals for shavings or clipplings: the day prior to initiation of dose administration and as often as necessary thereafter.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): patted with paper towel at the end of 6-hr exposure period; after each 5-day period residual test substance was removed (as much as possible without inducing irritation of the skin) from all animals using a warm water and mild soap solution (1% Ivory liquid soap in tap water) followed by a deionized water rinse and drying of the animals with a clean paper towel. Following each wash procedure, all animals were transferred to clean cages and the collars removed for a 2-day nondosing period
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.5 mL/kg (spread evenly using a glass rod)
- Concentration (if solution): 0, 66.7, 200, 400 mg/mL for control, 100, 300, 600 mg/kg/day groups respectively
- Constant volume or concentration used: no - Individual dosages and dose volumes were based on the most recently recorded body weights to provide the correct mg/kg/day dose

VEHICLE
mineral oil, USP lot no. ZH1000, exp. dates 13 May 2013 and 3 March 2012; lot no. 2AB0895, exp. date 16 May 2013; manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes - Elizabethan collars

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated GC/MS method was used for the determination of test substance concentration in suspension formulations. Two sets of duplicate samples from the top, middle, and bottom strata of the formulations prepared during study week 0, and from the middle stratum of those prepared in weeks 6 and12 at target test substance concentrations of 66.7, 200, and 400 mg/mL were analyzed to assess test substance homogeneity/concentration acceptability. Duplicate samples from the blank vehicle were also collected and analyzed. The analytical results met the applicable WIL Research SOP acceptance criteria for test substance homogeneity/concentration. No test article was detected in the analyzed vehicle administered to the control group.

Homogeneity, stability, and resuspension homogeneity of the test substance formulations were previously established at concentrations of 50 and 500 mg/mL following 8 days of room temperature storage during a preliminary study. Therefore, stability and resuspension homogeneity analyses were not conducted during the current study

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week for 6 hours per day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Remarks:

this was the highest tolerable dose due to dermal irritation observed in a 14 day rangefinder (moribund animals due to dermal irritation observed at 1000 mg/kg/day)

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

yes, sham-exposed

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design
- Fasting period before blood sampling for clinical biochemistry: during the period of overnight fasting prior to clinical pathology blood collection food, but not water, was withheld

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed on all animals prior to dose application and at approximately 1 to 2 hours following dose application. On nondosing days, the animals were observed once daily. Detailed physical examinations were conducted on all animals at least once during the pretest period, approximately weekly during the study, and on the days of the scheduled necropsy

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dermal observations were performed on all animals once during the pretest period and on all animals at the end of each 5-day dosing period at approximately 1 to 2 hours
following the removal of any residual test substance. The application sites were scored for erythema and edema

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly beginning during the pretest period and for the duration of the study. Body weights were collected prior to the
first dose of each 5-day dosing period (animals were weighed without collars). During study week 12, body weights were recorded twice weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Individual food consumption was recorded approximately weekly beginning during the pretest period and for the duration of the study. Food intake was calculated as g/animal/day for the corresponding body weight intervals

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to randomization (study week -1) and prior to the scheduled necropsy (study week 12)
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected from all animals at the scheduled necropsy (study week 12)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table 1 [in any other information on materials and methods] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected from all animals at the scheduled necropsy (study week 12)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table 1 [in any other information on materials and methods] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were euthanized by isoflurane inhalation. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The following organs were weighed from all animals at the scheduled necropsy:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary
Prostate
Spleen
Testes
Thymus
Thyroid with parathyroids*
Uterus
Paired organs were weighed together. Designated organs (*) were weighed after fixation.
Organ to final body weight and organ to brain weight ratios were calculated

HISTOPATHOLOGY: Yes (see table)
Microscopic examination was performed on all tissues listed in table 2 [in any other information on materials and methods] from all animals. After fixation, protocol-specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase [GGT]), and organ weight (absolute and relative) data, for the vehicle control group and all test substance-treated groups were subjected to a one-way analysis of variance to determine intergroup differences. If significant intergroup variance was found, Dunnett's test was used to compare the test
substance-treated groups to the vehicle control group.
The vehicle control group data were evaluated using the Student’s t-Test and compared to the sham control group at a 95% confidence level (p < 0.05).

GGT values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. GGT data were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test was used to compare the test substance-treated groups to the vehicle control group. In addition, the sham control group and vehicle control group were compared using a separate Kruskal-Wallis nonparametric ANOVA test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects noted during the dermal observations. Residual test substance within the dose site was noted in the 100, 300, and 600 mg/kg/day group males and females.
Dermal findings of very slight, slight, and/or moderate erythema, very slight and/or slight edema, and/or scabbing within dose site were noted in the 100, 300, and/or 600 mg/kg/day group males and females, as well as in the vehicle control group. Therefore, these dermal observations were considered most likely vehicle-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights were unaffected by test substance administration.

Statistically significant differences in mean body weight changes were noted in the 600 mg/kg/day group females between the two weighings conducted in study week 12 when compared to the vehicle control group. Differences in body weight gain were probably due to biological variability and were not considered related to test substance administration due to lack of a dose-response trend. In addition, a statistically significant mean body weight loss was noted in the vehicle control group females during the same period when compared to the sham control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was unaffected by test substance administration.

Statistically significantly higher mean food consumption was noted in the 300 mg/kg/day group females (study week 10 to 11) and statistically significantly lower mean food consumption was noted in the 100 mg/kg/day group females (during double observation in study week 12’); however, due to the magnitude of change and the lack of a dose-response trend, these values were not considered test substance-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in hematology and coagulation parameters. There were no statistically significant differences when the vehicle control and test substance-treated groups were compared.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in serum chemistry parameters.

A statistically significant difference was observed when the vehicle and test substance-exposed groups were compared. The albumin to globulin (A/G) ratio in the 600 mg/kg/day group males was higher than the vehicle control group but was not considered test substance-related as the value was similar to the sham control male group value.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No observations that were considered to be associated with application of the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic findings.

Findings in the skin unrelated to test substance exposure included mononuclear infiltrates in the treated, untreated, and inguinal skin. These infiltrates consisted of lymphocytes and plasma cells in the superficial dermis and rarely surrounding follicles in all groups. Inguinal skin from test substance-exposed males and females had a slightly higher incidence of mononuclear infiltrates compared to the sham control group. Untreated skin in the 300 and 600 mg/kg/day group males and females had a slightly higher incidence of mononuclear infiltrates when compared to the sham and vehicle control groups. These findings were not considered test substance-related as they were noted in skin that was not exposed to the test substance and there was no microscopic evidence of systemic exposure. One 600 mg/kg/day group male had moderate chronic-active inflammation (characterized by lymphocytes, plasma cells, neutrophils and macrophages accompanied by fibroplasia). in the inguinal skin. Inflammatory cells were observed surrounding occasionally degenerate hair follicles and extended focally into the adjacent panniculus muscle. This inflammatory lesion was not considered test substance-related as this area was not exposed to test substance and was more consistent with a background lesion.

Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test article. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects noted at top dose tested

Key result

Critical effects observed:

no

Conclusions:

There were no treatment-related effects at 600 mg/kg/day, establishing the no-observed-effect level (NOEL) the highest dosage level evaluated.

Executive summary:

Based on the results of this study, dermal application of ultra-low sulfur diesel fuel over an area of approximately 10% of the shaved body surface area to Crl:CD(SD) rats for approximately 6 hours per day, 5 days per week, for a minimum 90-day treatment period at dosage levels of 100, 300, and 600 mg/kg/day was well tolerated at all dosage levels.

There were no treatment-related effects at 600 mg/kg/day, establishing the no-observed-effect level (NOEL) the highest dosage level evaluated.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

83.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

Repeated inhalation exposure of rats for 90 days did not cause any systemic effects and a NOEC of 1710 mg/m3 was reported.

Three repeat dose dermal studies in rats were identified. In one of the key sub-chronic studies (ARCO 1994), a LOAEL of 1.0 ml/kg/day (estimated to be 835 mg/kg/day) was reported, based on moderate dermal irritation accompanied by decreased body weight (males only), increased food consumption, changes in differential leukocyte counts, decreased albumin (and albumin/globulin ratio), and increased incidence of lymphoid hyperplasia of the axilliary lymph node. The NOAEL was 0.1 mL/kg/day (estimated to be 83.5 mg/kg/day).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
well conducted and reported study

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
well conducted and reported study. Increased wet lung weight in top dose group but no histopathological change.

Repeated dose toxicity: dermal - systemic effects (target organ) cardiovascular / hematological: lymph nodes

Justification for classification or non-classification

Based on the findings in a sub-chronic dermal study in rats, a reported LOEL of 875 mg/kg and a NOAEL of 87.5 mg/kg, it is considered that the material meets the criteria for classification as STOT-RE, Category 2.