Petroleum heavy fuel oil fraction, co-processed with renewable hydrocarbons of plant or animal origin

Repeated dose toxicity: dermal

Administrative data

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:

other: Guideline, GLP-compliant study. Adequate for assessment.

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. However, the primary objective of the testing required for REACH is the identification of hazard, for which the default exposure route under the regulation is oral as this is considered to maximise systemic exposure. To address the regulatory exposure route issue, Concawe will review the current data base for evidence of systemic toxicity after dermal exposure and will also conduct a number of oral OECD 422 studies on prioritized substances in each relevant petroleum category. The document attached provides a concise overview of the information to further support the dermal route of exposure and proposed additional work, as part of a larger testing strategy (the strategy document can be found in Annex 13).

Read across justification included in Section 13

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

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:

other: Guideline, GLP-compliant study. Adequate for assessment.

Justification for type of information:

Read across justification included in Section 13
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. However, the primary objective of the testing required for REACH is the identification of hazard, for which the default exposure route under the regulation is oral as this is considered to maximise systemic exposure. To address the regulatory exposure route issue, Concawe will review the current data base for evidence of systemic toxicity after dermal exposure and will also conduct a number of oral OECD 422 studies on prioritized substances in each relevant petroleum category. The document attached provides a concise overview of the information to further support the dermal route of exposure and proposed additional work, as part of a larger testing strategy (the strategy document can be found in Annex 13).

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Principles of method if other than guideline:

Following an initial dose range-finding study, the test sample was applied neat, under occlusion, to clipped rat skin 5 d/wk for up to 13 wk at dose levels of 0 (sham control), 1.06, 10.6, 53, 106 or 530 mg /kg bw/d. Toxicological information collected during the study included details of clinical signs (including irritation of the test site), body weight effects, clinical chemistry, haematology, necropsy findings, organ weights and tissue histopathology.

Apart from the use of occluded exposure, other aspects of this GLP-compliant study appeared consistent with those of a guideline investigation.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco Inc., Omaha, Nebraska, USA
- Age at study initiation: young adults
- Weight at study initiation: males (mean) 260-270 g, females (mean) 180-185 g
- Housing: individually housed in stainless steel, wire mesh-bottomed cages
- Diet (ad libitum): certified rodent diet
- Water (ad libitum): tap water
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 degrees
- Humidity (%): 40-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 1991 To: 20-24 May 1991

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

The sample was applied neat to clipped dorsal skin (approx. 10% of body surface) once daily, 5 d/wk for 13 wk, and the test site covered with an occlusive dressing for 6 hr prior to wiping clean with gauze (no solvent).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hr occluded contact, 5 d/wk for 13 wk

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 0.001, 0.010, 0.050, 0.100, 0.500 mL/Kg bw/d
Basis:
nominal per unit body weight

Remarks:

Doses / Concentrations:
0, 1.06, 10.6, 53, 106, 530 mg/kg bw/d
Basis:
other: equivalent dose based on density of 1.06

No. of animals per sex per dose:

20 males, 20 females

Control animals:

yes, sham-exposed

Details on study design:

Treatment levels were selected based on results from a dose-range finding study that assessed the irritation potential and toxicity of the test substance.

Observations and examinations performed and frequency:

- Clinical signs (including dermal irritation)
The animals were observed twice daily for viability and once daily (afternoon) for signs of toxicity. Dermal irritation at the site of application was evaluated daily (Draize criteria) just prior to the application of the test article and just prior to necropsy.

- Body weight and food consumption
Body weights were determined weekly during the study and also prior to necropsy. Food intake data were collected weekly commencing week 6 (reflecting lower body weight values for treated animals observed from around this time).

- Haematology
Erythrocyte count, total leucocyte count, differential leucocyte count, haemoglobin concentration, haematocrit, platelet count, mean corpuscular volume (MCV)

- Clinical chemistry
Sodium, potassium, chloride, calcium, phosphorous, blood urea nitrogen (BUN), glucose, creatinine, cholesterol, triglyceride, total protein, albumin, globulin (calculated), albumin/globulin ratio (calculated), alkaline phosphatise, aspartate aminotransferase (SGOT/AST), alanine aminotransferase (SGPT/ALT).

Sacrifice and pathology:

- Necropsy:
Animals were sacrificed (halothane anaesthesia) and blood collected for haematology and clinical chemistry analyses prior to exsanguination. Following gross necropsy (which included examination of external body surface, cranial cavity, thoracic and abdominal cavities) samples of selected organs were weighed and tissues collected and preserved (10% neutral buffered formalin) for later histopathological examination. Bone marrow smears were prepared and preserved.

The following organs were weighed after removal of fat and other contiguous tissue:
- adrenal glands (pair), brain, heart, kidneys (pair), liver, lungs (perfused after weighing), spleen, testes (pair), ovaries (pair), thymus. Paired tissues were weighed together to give a total (combined) weight.

The following tissues were sampled (all animals, all groups) and preserved in 10% neutral buffered formalin:
- accessory genital organs (prostate, seminal vesicles, epididymis), adrenal glands, aorta, brain (cerebrum, cerebellum, medulla pons), caecum, cervical lymph nodes, colon, duodenum, eyes, femur (with articular surface), heart, ileum, jejunum, kidneys, liver, lungs with trachea (perfused with fixative), mammary glands (female only), oesophagus, pancreas, peripheral nerve, pituitary, rectum, salivary glands, skeletal muscle (thigh), skin (treated site, untreated) spinal cord (cervical, mid thoracic, lumbar), spleen, sternum (with bone marrow), stomach, testes/ovaries, thyroid gland with parathyroid glands, thymus, urinary bladder, uterus, all gross lesions and masses.

- Histopathology:
The following preserved tissues were processed for microscopic examination after staining haematoxylin and eosin:
- adrenal glands, aorta, brain (cerebellum, cerebrum, medulla pons), cervical lymph nodes, oesophagus, gastrointestinal tract (stomach, duodenum, jejunum, ileum, colon, rectum), gross lesions, heart, kidneys, liver, lungs, pancreas, peripheral nerve, pituitary, prostate, salivary glands, skeletal muscle, skin (treated and untreated), spleen, sternum with bone marrow, testes/ovaries, thyroid (with parathyroid glands), thymus, trachea, urinary bladder, uterus.

Statistics:

Clinical pathology data, body weights, terminal organ weights, and organ to body weight ratios were analyzed using Bartlett's test. If variances were equal, data were analysed using one way ANOVA followed by Dunnett's test (to determine which treatment groups differ significantly from control) and linear regression. If variances differed, results were analysed using the Kruskal-Wallis test followed by Dunn's Summed Rank test with Jonckheere's test for trend to assess any dose response relationship present. Data for the sexes was analysed separately with ratios transformed (arc sine transformation, Cochran's transformation) to stabilise variances.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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

Details on results:

- In-life observations:
Nine animals (7 male, 2 female) from the 530 mg/kg bw/d group died from presumed treatment related causes, commencing on study day 59. Clinical findings in survivors, present primarily in the high dose group, included cold to the touch, pale colour, laboured breathing, ataxia and tremors. Tape irritation (associated with application/removal of the occlusive wrappings) was common in all groups, including controls. Intermittent irritation of the test site (slight erythema, slight eschar and slight dryness) was recorded in all treated groups, but was expressed inconsistently within the dose groups and appeared unrelated to applied dose.

- Body weight/body weight gain:
Body weight was significantly decreased in high dose males and females from study week 5-6 onwards, with the females recovering from week 10 but males still showing a significant decrease (-15%) during week 12 (the last time-point when the number of live high dose males was adequate for meaningful statistical analysis). Food intake was decreased significantly in high dose males on study weeks 7, 9, 11 and 12, and in high dose females between study weeks 7-9, with significant but not clearly dose related increases and decreases seen sporadically in the other treated groups also.

- Haematology:
Haematological analysis revealed generally significant, clearly dose related reductions of around 10-30% in RCB, HCT and HGB in males and females receiving doses of 53 mg/kg bw/d and above, with platelet counts significantly decreased by 20-40% in males at 10.6 mg/kg bw/d and above, and by 30-60% females at 106 mg/kg bw/d and above.

- Clinical chemistry
Clinical chemistry determinations demonstrated statistically significant increases of 50-130% in BUN in males at 53 mg/kg bw/d and above and females at 106 mg/kg bw/d and above, and a significant (60-100%) increase serum cholesterol in both sexes treated with 53 mg/kg bw/d and above. Mean serum creatinine was increased significantly by approx. 10-15% in high dose males and in females at 53 mg/kg bw/d and above. Mean SGOT exhibited a variable (30-110%) statistically significant increase in high dose females and males, with alkaline phosphatase activity increased by 40% or more in both sexes given 106 mg/kg bw/d and above (significant only in high dose females).

- Necropsy:
Treatment related findings at gross necropsy in animals that survived to scheduled necropsy included brown staining of the test site (no irritation reported), and a reduction in thymus size in high dose decedent animals and all other treated animals surviving to scheduled study termination. Absolute liver weight was significantly increased by 20-45% in both sexes at 53 mg/kg bw/d and above, with a significant dose related increase in relative liver weight (both sexes) at 10.6 mg/kg bw/d and above. Absolute lung weight was significantly increased by around 15% in males from the 53 and 106 mg/kg bw/d groups and by 10-15% in females given 53 mg/kg bw/d and above, with a significant dose related increase in relative lung weight in both sexes given 10.6 mg/kg bw/d and above. Absolute and relative thymus weights were significantly decreased by 40-60% in high dose animals of both sexes.

- Histopathology:
Histopathological examination of the test site revealed mild microscopic changes (including acanthosis, epidermal crusting and hyperkeratosis with occasional sub-acute inflammation and moderate ulceration) in both sexes at 530 mg/kg bw/d with increased hyperkeratosis in females at 106 mg/kg bw/d. Treatment related changes in the internal organs included alterations in liver (hepatic congestion, necrosis, vacuolar change) in both sexes at 53 mg/kg bw/d and above and thymus (atrophy, congestion) in males at 10.6 mg/kg bw/d and above, and in females at 53 mg/kg bw/d and above. Chronic inflammation of the thyroid (lymphocytic thyroiditis; 53 mg/kg bw/d and above) and cellular depletion of bone marrow (106 mg/kg bw/d and above) were also present.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1.06 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Decreased platelet count, increased relative liver weight (with histopathological change at higher treatments)

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1.06 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Increased relative liver weight

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

106 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Trace acanthosis and trace epidermal crusting at treatment site

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

53 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Hyperkeratosis at treatment site

Critical effects observed:

not specified

Selected body weight data (g; mean and [SD]) by dose level:

Study day 1:

- males: 271.6 [13.3], 268.1 [13.2], 267.0 [14.0], 263.8 [23.0], 268.5 [13.8], 264.6 [17.4]

- females: 184.1 [11.9], 183.6 [10.7], 185.1 [9.4], 184.6 [12.2], 181.4 [8.4], 181.7 [9.9]

Study day 25:

- males: 341.8 [19.5], 332.7 [17.8], 334.5 [23.5], 326.8 [22.6], 328.6 [22.3], 322.6 [27.7]

- females: 219.4 [16.1], 217.4 [12.8], 219.3 [11.5], 218.5 [14.7], 214.2 [8.9], 208.8 [14.4

Study day 32:

- males: 355.1 [21.3], 345.6 [16.0], 345.5 [27.2], 337.2 [24.9], 341.6 [23.5], 333.4 [31.4]

- females: 224.7 [15.1], 224.9 [12.4], 224.4 [12.2], 224.5 [14.8], 220.1 [10.3], 212.2 [15.9]*

Study day 39:

- males: 368.9 [21.9], 357.8 [18.8], 354.9 [29.4], 348.7 [28.2], 350.2 [27.9], 337.2 [35.2]*

- females 230.9 [16.6], 227.1 [13.6], 227.9 [12.6], 227.9 [16.6], 222.9 [12.6], 215.6 [18.5]*

Study day 88:

- males: 423.6 [24.1], 406.7 [22.8], 396.2 [32.9], 396.1 [40.8], 401.9 [37.1], 358.8 [43.0]**

- females: 253.3 [17.8], 249.1 [13.6], 249.3 [13.4], 253.5 [14.9], 245.5 [17.1], 239.1 [19.6]

Study day 95:

- males: 434.3 [26.4], 420.3 [27.3], 426.3 [27.0], 407.0 [19.5], 449.8 [42.2], 344.0 [73.5]

- females: 259.8 [20.1], 249.0 [11.4], 254.5 [17.1], 264.8 [19.1], 256.8 [5.9], 236.3 [18.5]

Selected haematological parameters (mean, [SD]) by dose level:

Red cell counts (million per mm3):

Males: 8.851 [0.444], 8.767 [0.436], 8.526 [0.480], 8.083 [0.783]*, 7.268 [1.369]**, 6.177 [1.852]**

Females: 8.135 [0.376], 8.268 [0.638], 8.092 [0.608], 7.804 [0.663], 7.366 [0.729]**, 6.371 [1.379]**

Haematocrit (%):

Males: 45.78 [2.23], 45.39 [3.95], 43.51 [2.21], 41.63 [2.85]**, 37.64 [5.40]**, 29.40 [8.83]**

Females: 43.32 [1.98], 42.89 [3.23], 41.95 [3.11], 40.20 [2.59]*, 37.81 [3.80]**, 32.49 [6.44]**

Haemoglobin concentration (g/dl):

Males: 16.41 [0.71], 16.06 [0.81], 15.70 [0.49], 14.95 [0.89]**, 13.76 [1.95]**, 11.86 [2.81]**

Females: 15.40 [0.52], 15.41 [0.89], 15.06 [0.83], 14.43 [0.73]**, 13.76 [1.04]**, 12.00 [2.18]**

Platelet count (thousand per mm3):

Males: 935.5 [203.3], 910.3 [92.7], 751.7 [259.8]**, 695.3 [259.2]**, 619.0 [289.5]**, 532.8 [216.4]**

Females: 824.0 [266.1], 794.2 [253.8], 869.2 [138.0], 711.4 [139.6], 581.3 [176.6]**, 373.3 [275.6]**

Selected clinical chemistry parameters (mean, [SD]) by dose level:

Blood urea nitrogen (mg/dL):

Males: 20.50 [2.21], 22.15 [2.30], 23.70 [5.96], 32.95 [13.66]**, 38.00 [13.88]**, 36.85 [15.63]**

Females: 23.05 [3.72], 23.65 [3.56], 24.75 [4.34], 26.70 [4.97], 30.10 [6.84]**, 30.17 [6.03]**

Cholesterol (mg/dL)

Males: 74.8 [11.7], 83.3 [30.7], 86.8 [25.7], 127.9 [22.1]**, 136.3 [46.8]**, 120.8 [33.8]**

Females: 82.1 [15.3], 88.4 [19.3], 104.8 [18.1], 131.9 [28.7]**, 153.2 [34.5]**, 166.4 [29.3]**

Creatinine (mg/dL):

Males: 0.81 [0.07], 0.81 [0.06], 0.80 [0.11], 0.88 [0.11], 0.88 [0.10], 0.92 [0.11]*

Females: 0.84 [0.07], 0.87 [0.07], 0.88 [0.08], 0.91 [0.08]*, 0.94 [0.09]**, 0.92 [0.09]*

SGOT (IU/L):

Males: 75.3 [13.4], 77.8 [15.6], 83.2 [15.6], 90.6 [36.0], 117.9 [123.1], 159.1 [155.1]*

Females: 86.9 [22.4], 94.6 [16.0]*, 90.9 [10.9], 89.8 [15.6], 96.8 [22.2], 111.7 [43.0]*

Alkaline phosphatase (IU/L):

Males: 97.8 [31.7], 119.2 [35.2], 124.3 [32.6], 136.3 [60.0], 141.8 [88.7], 140.6 [60.4]*

Females: 72.9 [28.9], 96.3 [30.6], 81.5 [24.2], 88.6 [27.4], 103.3 [56.8], 140.7 [70.7]**

Selected relative organ weights (mean, [SD]) by dose level:

Relative liver weight (g):

Males: 0.0256 [0.0017], 0.0272 [0.0035], 0.0285 [0.0025]*, 0.0318 [0.0036]**, 0.0345 [0.0037]**, 0.0350 [0.0029]**

Females: 0.0258 [0.0024], 0.0264 [0.0013], 0.0292 [0.0024]*, 0.0318 [0.0035]**, 0.0334 [0.0029]**, 0.0394 [0.0043]**

Relative lung weight (g/g bw):

Males: 0.0043 [0.0006], 0.0044 [0.0003], 0.0047 [0.0005], 0.0052 [0.0005]**, 0.0051 [0.0005]**, 0.0051 [0.0008]**

Females: 0.0056 [0.0004], 0.0057 [0.0004], 0.0061 [0.0004]*, 0.0063 [0.0004]**, 0.0066 [0.0004]**, 0.0069 [0.0008]**

Relative thymus weight (g/g bw):

Males: 0.0007 [0.0003], 0.0006 [0.0002], 0.0006 [0.0002], 0.0005 [0.0003], 0.0005 [0.0001], 0.0004 [0.0002]*

Females: 0.0008 [0.0002], 0.0009 [0.0001], 0.0008 [0.0003], 0.0007 [0.0003], 0.0006 [0.0002], 0.0004 [0.0002]**

Dose levels: 0 (sham), 1.06, 10.6, 53, 106 or 530 mg/kg bw/d

* = P<0.05; ** = P<0.01

Conclusions:

NOAEL for systemic toxicity = 1.06 mg/kg bw/d (both sexes)
NOAEL for local effects (skin irritation) = 106 mg/kg bw/d (males)
NOAEL for local effects (skin irritation) = 53 mg/kg bw/d (females)

Executive summary:

The sub-chronic dermal toxicity of a sample F-179 (0, 1.06, 10.6, 53, 106 or 530 mg/kg bw/d) was investigated in groups of 25 male and 25 female SD rats following repeated occluded dermal application to clipped skin, 6 hr/d, 5 d/wk for up to 13 weeks.

Nine animals (7 male, 2 female) from the 530 mg/kg bw/d group died from presumed treatment related causes, commencing on study day 59. Clinical findings in survivors, present primarily in the high dose group, included cold to the touch, pale colour, laboured breathing, ataxia and tremors. Intermittent irritation of the test site (slight erythema, slight eschar and slight dryness) was recorded in all treated groups, but was expressed inconsistently within the dose groups and appeared unrelated to applied dose. Body weight was significantly decreased in high dose males and females from study week 5-6 onwards, with the females recovering from week 10 but males still showing a significant decrease during week 12 (the last time-point when the number of live high dose males was adequate for meaningful statistical analysis). Food intake was decreased significantly in high dose males on study weeks 7, 9, 11 and 12, and in high dose females between study weeks 7-9, with significant but not clearly dose related increases and decreases seen sporadically in the other treated groups also.

Haematological analysis revealed generally significant, clearly dose-related reductions in red cell count, haematocrit and haemoglobin concentration in males and females receiving doses of 53 mg/kg bw/d and above, with platelet counts significantly decreased in males at 10.6 mg/kg bw/d and above, and females at 106 mg/kg bw/d and above. Clinical chemistry determinations demonstrated statistically significant increases in blood urea nitrogen in males at 53 mg/kg bw/d and above and females at 106 mg/kg bw/d and above, and a significant  increase serum cholesterol in both sexes treated with 53 mg/kg bw/d and above. Mean serum creatinine was increased significantly in high dose males and in females at 53 mg/kg bw/d and above. Mean SGOT exhibited a variable but statistically significant increase in high dose females and males, with alkaline phosphatase activity increased in both sexes given 106 mg/kg bw /d and above (significant only in high dose females).

Treatment-related findings at gross necropsy in animals that survived to scheduled necropsy included brown staining of the test site (no irritation reported), and a reduction in thymus size in high dose decedent animals and all other treated animals surviving to scheduled study termination. Absolute liver weight was significantly increased in both sexes at 53 mg/kg bw/d and above, with a significant dose related increase in relative liver weight (both sexes) at 10.6 mg/kg bw/d and above. Absolute lung weight was significantly increased in males from the 53 and 106 mg/kg bw/d groups and in females given 53 mg/kg bw/d and above, with a significant dose-related increase in relative lung weight in both sexes given 10.6 mg/kg bw/d and above. Absolute and relative thymus weights were significantly decreased in high dose animals of both sexes. Histopathological examination of the test site revealed mild microscopic changes (including acanthosis, epidermal crusting and hyperkeratosis with occasional sub-acute inflammation and moderate ulceration) in both sexes at 530 mg/kg bw/d with increased hyperkeratosis in females at 106 mg/kg bw/d. Treatment related changes in the internal organs included alterations in liver (hepatic congestion, necrosis, vacuolar change) in both sexes at  53 mg/kg bw/d and above and thymus (atrophy, congestion) in males at 10.6 mg/kg bw/d and above and in females at 53 mg/kg bw/d and above. Chronic inflammation of the thyroid (lymphocytic thyroiditis; 53 mg/kg bw/d and above) and cellular depletion of bone marrow (106 mg/kg bw/d and above) were also present.

Based on these overall findings the NOAEL for systemic toxicity in both sexes was 1.06 mg/kg bw/d (reflecting increased relative liver weight in both sexes, and the occurrence of liver histopathology and decreased platelet count in males only). The NOAEL for local effects (reflecting microscopic changes at the test site) was 106 mg/kg bw/d for males and 53 mg/kg bw/d for females.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Principles of method if other than guideline:

Following an initial dose range-finding study, the test sample was applied neat, under occlusion, to clipped rat skin 5 d/wk for up to 13 wk at dose levels of 0 (sham control), 1.06, 10.6, 53, 106 or 530 mg /kg bw/d. Toxicological information collected during the study included details of clinical signs (including irritation of the test site), body weight effects, clinical chemistry, haematology, necropsy findings, organ weights and tissue histopathology.

Apart from the use of occluded exposure, other aspects of this GLP-compliant study appeared consistent with those of a guideline investigation.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

64741-62-4

Cas Number:

64741-62-4

IUPAC Name:

64741-62-4

Test material form:

other: Viscous hydrocarbon liquid

Details on test material:

- Name of test material (as cited in Sponsor documentation): F-179, petrobase 100
- Dark blue-green to red-brown coloured liquid, slightly cracked or burnt odor
- Specific gravity 1.06
- Flash Point approx 250 °F

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco Inc., Omaha, Nebraska, USA
- Age at study initiation: young adults
- Weight at study initiation: males (mean) 260-270 g, females (mean) 180-185 g
- Housing: individually housed in stainless steel, wire mesh-bottomed cages
- Diet (ad libitum): certified rodent diet
- Water (ad libitum): tap water
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 degrees
- Humidity (%): 40-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 1991 To: 20-24 May 1991

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

The sample was applied neat to clipped dorsal skin (approx. 10% of body surface) once daily, 5 d/wk for 13 wk, and the test site covered with an occlusive dressing for 6 hr prior to wiping clean with gauze (no solvent).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hr occluded contact, 5 d/wk for 13 wk

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 0.001, 0.010, 0.050, 0.100, 0.500 mL/Kg bw/d
Basis:
nominal per unit body weight

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1.06, 10.6, 53, 106, 530 mg/kg bw/d
Basis:
other: equivalent dose based on density of 1.06

No. of animals per sex per dose:

20 males, 20 females

Control animals:

yes, sham-exposed

Details on study design:

Treatment levels were selected based on results from a dose-range finding study that assessed the irritation potential and toxicity of the test substance.

Examinations

Observations and examinations performed and frequency:

- Clinical signs (including dermal irritation)
The animals were observed twice daily for viability and once daily (afternoon) for signs of toxicity. Dermal irritation at the site of application was evaluated daily (Draize criteria) just prior to the application of the test article and just prior to necropsy.

- Body weight and food consumption
Body weights were determined weekly during the study and also prior to necropsy. Food intake data were collected weekly commencing week 6 (reflecting lower body weight values for treated animals observed from around this time).

- Haematology
Erythrocyte count, total leucocyte count, differential leucocyte count, haemoglobin concentration, haematocrit, platelet count, mean corpuscular volume (MCV)

- Clinical chemistry
Sodium, potassium, chloride, calcium, phosphorous, blood urea nitrogen (BUN), glucose, creatinine, cholesterol, triglyceride, total protein, albumin, globulin (calculated), albumin/globulin ratio (calculated), alkaline phosphatise, aspartate aminotransferase (SGOT/AST), alanine aminotransferase (SGPT/ALT).

Sacrifice and pathology:

- Necropsy:
Animals were sacrificed (halothane anaesthesia) and blood collected for haematology and clinical chemistry analyses prior to exsanguination. Following gross necropsy (which included examination of external body surface, cranial cavity, thoracic and abdominal cavities) samples of selected organs were weighed and tissues collected and preserved (10% neutral buffered formalin) for later histopathological examination. Bone marrow smears were prepared and preserved.

The following organs were weighed after removal of fat and other contiguous tissue:
- adrenal glands (pair), brain, heart, kidneys (pair), liver, lungs (perfused after weighing), spleen, testes (pair), ovaries (pair), thymus. Paired tissues were weighed together to give a total (combined) weight.

The following tissues were sampled (all animals, all groups) and preserved in 10% neutral buffered formalin:
- accessory genital organs (prostate, seminal vesicles, epididymis), adrenal glands, aorta, brain (cerebrum, cerebellum, medulla pons), caecum, cervical lymph nodes, colon, duodenum, eyes, femur (with articular surface), heart, ileum, jejunum, kidneys, liver, lungs with trachea (perfused with fixative), mammary glands (female only), oesophagus, pancreas, peripheral nerve, pituitary, rectum, salivary glands, skeletal muscle (thigh), skin (treated site, untreated) spinal cord (cervical, mid thoracic, lumbar), spleen, sternum (with bone marrow), stomach, testes/ovaries, thyroid gland with parathyroid glands, thymus, urinary bladder, uterus, all gross lesions and masses.

- Histopathology:
The following preserved tissues were processed for microscopic examination after staining haematoxylin and eosin:
- adrenal glands, aorta, brain (cerebellum, cerebrum, medulla pons), cervical lymph nodes, oesophagus, gastrointestinal tract (stomach, duodenum, jejunum, ileum, colon, rectum), gross lesions, heart, kidneys, liver, lungs, pancreas, peripheral nerve, pituitary, prostate, salivary glands, skeletal muscle, skin (treated and untreated), spleen, sternum with bone marrow, testes/ovaries, thyroid (with parathyroid glands), thymus, trachea, urinary bladder, uterus.

Statistics:

Clinical pathology data, body weights, terminal organ weights, and organ to body weight ratios were analyzed using Bartlett's test. If variances were equal, data were analysed using one way ANOVA followed by Dunnett's test (to determine which treatment groups differ significantly from control) and linear regression. If variances differed, results were analysed using the Kruskal-Wallis test followed by Dunn's Summed Rank test with Jonckheere's test for trend to assess any dose response relationship present. Data for the sexes was analysed separately with ratios transformed (arc sine transformation, Cochran's transformation) to stabilise variances.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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

Details on results:

- In-life observations:
Nine animals (7 male, 2 female) from the 530 mg/kg bw/d group died from presumed treatment related causes, commencing on study day 59. Clinical findings in survivors, present primarily in the high dose group, included cold to the touch, pale colour, laboured breathing, ataxia and tremors. Tape irritation (associated with application/removal of the occlusive wrappings) was common in all groups, including controls. Intermittent irritation of the test site (slight erythema, slight eschar and slight dryness) was recorded in all treated groups, but was expressed inconsistently within the dose groups and appeared unrelated to applied dose.

- Body weight/body weight gain:
Body weight was significantly decreased in high dose males and females from study week 5-6 onwards, with the females recovering from week 10 but males still showing a significant decrease (-15%) during week 12 (the last time-point when the number of live high dose males was adequate for meaningful statistical analysis). Food intake was decreased significantly in high dose males on study weeks 7, 9, 11 and 12, and in high dose females between study weeks 7-9, with significant but not clearly dose related increases and decreases seen sporadically in the other treated groups also.

- Haematology:
Haematological analysis revealed generally significant, clearly dose related reductions of around 10-30% in RCB, HCT and HGB in males and females receiving doses of 53 mg/kg bw/d and above, with platelet counts significantly decreased by 20-40% in males at 10.6 mg/kg bw/d and above, and by 30-60% females at 106 mg/kg bw/d and above.

- Clinical chemistry
Clinical chemistry determinations demonstrated statistically significant increases of 50-130% in BUN in males at 53 mg/kg bw/d and above and females at 106 mg/kg bw/d and above, and a significant (60-100%) increase serum cholesterol in both sexes treated with 53 mg/kg bw/d and above. Mean serum creatinine was increased significantly by approx. 10-15% in high dose males and in females at 53 mg/kg bw/d and above. Mean SGOT exhibited a variable (30-110%) statistically significant increase in high dose females and males, with alkaline phosphatase activity increased by 40% or more in both sexes given 106 mg/kg bw/d and above (significant only in high dose females).

- Necropsy:
Treatment related findings at gross necropsy in animals that survived to scheduled necropsy included brown staining of the test site (no irritation reported), and a reduction in thymus size in high dose decedent animals and all other treated animals surviving to scheduled study termination. Absolute liver weight was significantly increased by 20-45% in both sexes at 53 mg/kg bw/d and above, with a significant dose related increase in relative liver weight (both sexes) at 10.6 mg/kg bw/d and above. Absolute lung weight was significantly increased by around 15% in males from the 53 and 106 mg/kg bw/d groups and by 10-15% in females given 53 mg/kg bw/d and above, with a significant dose related increase in relative lung weight in both sexes given 10.6 mg/kg bw/d and above. Absolute and relative thymus weights were significantly decreased by 40-60% in high dose animals of both sexes.

- Histopathology:
Histopathological examination of the test site revealed mild microscopic changes (including acanthosis, epidermal crusting and hyperkeratosis with occasional sub-acute inflammation and moderate ulceration) in both sexes at 530 mg/kg bw/d with increased hyperkeratosis in females at 106 mg/kg bw/d. Treatment related changes in the internal organs included alterations in liver (hepatic congestion, necrosis, vacuolar change) in both sexes at 53 mg/kg bw/d and above and thymus (atrophy, congestion) in males at 10.6 mg/kg bw/d and above, and in females at 53 mg/kg bw/d and above. Chronic inflammation of the thyroid (lymphocytic thyroiditis; 53 mg/kg bw/d and above) and cellular depletion of bone marrow (106 mg/kg bw/d and above) were also present.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1.06 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Decreased platelet count, increased relative liver weight (with histopathological change at higher treatments)

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1.06 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Increased relative liver weight

3

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

106 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Trace acanthosis and trace epidermal crusting at treatment site

4

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

53 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Hyperkeratosis at treatment site

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Selected body weight data (g; mean and [SD]) by dose level:

Study day 1:

- males: 271.6 [13.3], 268.1 [13.2], 267.0 [14.0], 263.8 [23.0], 268.5 [13.8], 264.6 [17.4]

- females: 184.1 [11.9], 183.6 [10.7], 185.1 [9.4], 184.6 [12.2], 181.4 [8.4], 181.7 [9.9]

Study day 25:

- males: 341.8 [19.5], 332.7 [17.8], 334.5 [23.5], 326.8 [22.6], 328.6 [22.3], 322.6 [27.7]

- females: 219.4 [16.1], 217.4 [12.8], 219.3 [11.5], 218.5 [14.7], 214.2 [8.9], 208.8 [14.4

Study day 32:

- males: 355.1 [21.3], 345.6 [16.0], 345.5 [27.2], 337.2 [24.9], 341.6 [23.5], 333.4 [31.4]

- females: 224.7 [15.1], 224.9 [12.4], 224.4 [12.2], 224.5 [14.8], 220.1 [10.3], 212.2 [15.9]*

Study day 39:

- males: 368.9 [21.9], 357.8 [18.8], 354.9 [29.4], 348.7 [28.2], 350.2 [27.9], 337.2 [35.2]*

- females 230.9 [16.6], 227.1 [13.6], 227.9 [12.6], 227.9 [16.6], 222.9 [12.6], 215.6 [18.5]*

Study day 88:

- males: 423.6 [24.1], 406.7 [22.8], 396.2 [32.9], 396.1 [40.8], 401.9 [37.1], 358.8 [43.0]**

- females: 253.3 [17.8], 249.1 [13.6], 249.3 [13.4], 253.5 [14.9], 245.5 [17.1], 239.1 [19.6]

Study day 95:

- males: 434.3 [26.4], 420.3 [27.3], 426.3 [27.0], 407.0 [19.5], 449.8 [42.2], 344.0 [73.5]

- females: 259.8 [20.1], 249.0 [11.4], 254.5 [17.1], 264.8 [19.1], 256.8 [5.9], 236.3 [18.5]

Selected haematological parameters (mean, [SD]) by dose level:

Red cell counts (million per mm3):

Males: 8.851 [0.444], 8.767 [0.436], 8.526 [0.480], 8.083 [0.783]*, 7.268 [1.369]**, 6.177 [1.852]**

Females: 8.135 [0.376], 8.268 [0.638], 8.092 [0.608], 7.804 [0.663], 7.366 [0.729]**, 6.371 [1.379]**

Haematocrit (%):

Males: 45.78 [2.23], 45.39 [3.95], 43.51 [2.21], 41.63 [2.85]**, 37.64 [5.40]**, 29.40 [8.83]**

Females: 43.32 [1.98], 42.89 [3.23], 41.95 [3.11], 40.20 [2.59]*, 37.81 [3.80]**, 32.49 [6.44]**

Haemoglobin concentration (g/dl):

Males: 16.41 [0.71], 16.06 [0.81], 15.70 [0.49], 14.95 [0.89]**, 13.76 [1.95]**, 11.86 [2.81]**

Females: 15.40 [0.52], 15.41 [0.89], 15.06 [0.83], 14.43 [0.73]**, 13.76 [1.04]**, 12.00 [2.18]**

Platelet count (thousand per mm3):

Males: 935.5 [203.3], 910.3 [92.7], 751.7 [259.8]**, 695.3 [259.2]**, 619.0 [289.5]**, 532.8 [216.4]**

Females: 824.0 [266.1], 794.2 [253.8], 869.2 [138.0], 711.4 [139.6], 581.3 [176.6]**, 373.3 [275.6]**

Selected clinical chemistry parameters (mean, [SD]) by dose level:

Blood urea nitrogen (mg/dL):

Males: 20.50 [2.21], 22.15 [2.30], 23.70 [5.96], 32.95 [13.66]**, 38.00 [13.88]**, 36.85 [15.63]**

Females: 23.05 [3.72], 23.65 [3.56], 24.75 [4.34], 26.70 [4.97], 30.10 [6.84]**, 30.17 [6.03]**

Cholesterol (mg/dL)

Males: 74.8 [11.7], 83.3 [30.7], 86.8 [25.7], 127.9 [22.1]**, 136.3 [46.8]**, 120.8 [33.8]**

Females: 82.1 [15.3], 88.4 [19.3], 104.8 [18.1], 131.9 [28.7]**, 153.2 [34.5]**, 166.4 [29.3]**

Creatinine (mg/dL):

Males: 0.81 [0.07], 0.81 [0.06], 0.80 [0.11], 0.88 [0.11], 0.88 [0.10], 0.92 [0.11]*

Females: 0.84 [0.07], 0.87 [0.07], 0.88 [0.08], 0.91 [0.08]*, 0.94 [0.09]**, 0.92 [0.09]*

SGOT (IU/L):

Males: 75.3 [13.4], 77.8 [15.6], 83.2 [15.6], 90.6 [36.0], 117.9 [123.1], 159.1 [155.1]*

Females: 86.9 [22.4], 94.6 [16.0]*, 90.9 [10.9], 89.8 [15.6], 96.8 [22.2], 111.7 [43.0]*

Alkaline phosphatase (IU/L):

Males: 97.8 [31.7], 119.2 [35.2], 124.3 [32.6], 136.3 [60.0], 141.8 [88.7], 140.6 [60.4]*

Females: 72.9 [28.9], 96.3 [30.6], 81.5 [24.2], 88.6 [27.4], 103.3 [56.8], 140.7 [70.7]**

Selected relative organ weights (mean, [SD]) by dose level:

Relative liver weight (g):

Males: 0.0256 [0.0017], 0.0272 [0.0035], 0.0285 [0.0025]*, 0.0318 [0.0036]**, 0.0345 [0.0037]**, 0.0350 [0.0029]**

Females: 0.0258 [0.0024], 0.0264 [0.0013], 0.0292 [0.0024]*, 0.0318 [0.0035]**, 0.0334 [0.0029]**, 0.0394 [0.0043]**

Relative lung weight (g/g bw):

Males: 0.0043 [0.0006], 0.0044 [0.0003], 0.0047 [0.0005], 0.0052 [0.0005]**, 0.0051 [0.0005]**, 0.0051 [0.0008]**

Females: 0.0056 [0.0004], 0.0057 [0.0004], 0.0061 [0.0004]*, 0.0063 [0.0004]**, 0.0066 [0.0004]**, 0.0069 [0.0008]**

Relative thymus weight (g/g bw):

Males: 0.0007 [0.0003], 0.0006 [0.0002], 0.0006 [0.0002], 0.0005 [0.0003], 0.0005 [0.0001], 0.0004 [0.0002]*

Females: 0.0008 [0.0002], 0.0009 [0.0001], 0.0008 [0.0003], 0.0007 [0.0003], 0.0006 [0.0002], 0.0004 [0.0002]**

Dose levels: 0 (sham), 1.06, 10.6, 53, 106 or 530 mg/kg bw/d

* = P<0.05; ** = P<0.01

Applicant's summary and conclusion

Conclusions:

NOAEL for systemic toxicity = 1.06 mg/kg bw/d (both sexes)
NOAEL for local effects (skin irritation) = 106 mg/kg bw/d (males)
NOAEL for local effects (skin irritation) = 53 mg/kg bw/d (females)

Executive summary:

The sub-chronic dermal toxicity of a sample F-179 (0, 1.06, 10.6, 53, 106 or 530 mg/kg bw/d) was investigated in groups of 25 male and 25 female SD rats following repeated occluded dermal application to clipped skin, 6 hr/d, 5 d/wk for up to 13 weeks.

Nine animals (7 male, 2 female) from the 530 mg/kg bw/d group died from presumed treatment related causes, commencing on study day 59. Clinical findings in survivors, present primarily in the high dose group, included cold to the touch, pale colour, laboured breathing, ataxia and tremors. Intermittent irritation of the test site (slight erythema, slight eschar and slight dryness) was recorded in all treated groups, but was expressed inconsistently within the dose groups and appeared unrelated to applied dose. Body weight was significantly decreased in high dose males and females from study week 5-6 onwards, with the females recovering from week 10 but males still showing a significant decrease during week 12 (the last time-point when the number of live high dose males was adequate for meaningful statistical analysis). Food intake was decreased significantly in high dose males on study weeks 7, 9, 11 and 12, and in high dose females between study weeks 7-9, with significant but not clearly dose related increases and decreases seen sporadically in the other treated groups also.

Haematological analysis revealed generally significant, clearly dose-related reductions in red cell count, haematocrit and haemoglobin concentration in males and females receiving doses of 53 mg/kg bw/d and above, with platelet counts significantly decreased in males at 10.6 mg/kg bw/d and above, and females at 106 mg/kg bw/d and above. Clinical chemistry determinations demonstrated statistically significant increases in blood urea nitrogen in males at 53 mg/kg bw/d and above and females at 106 mg/kg bw/d and above, and a significant  increase serum cholesterol in both sexes treated with 53 mg/kg bw/d and above. Mean serum creatinine was increased significantly in high dose males and in females at 53 mg/kg bw/d and above. Mean SGOT exhibited a variable but statistically significant increase in high dose females and males, with alkaline phosphatase activity increased in both sexes given 106 mg/kg bw /d and above (significant only in high dose females).

Treatment-related findings at gross necropsy in animals that survived to scheduled necropsy included brown staining of the test site (no irritation reported), and a reduction in thymus size in high dose decedent animals and all other treated animals surviving to scheduled study termination. Absolute liver weight was significantly increased in both sexes at 53 mg/kg bw/d and above, with a significant dose related increase in relative liver weight (both sexes) at 10.6 mg/kg bw/d and above. Absolute lung weight was significantly increased in males from the 53 and 106 mg/kg bw/d groups and in females given 53 mg/kg bw/d and above, with a significant dose-related increase in relative lung weight in both sexes given 10.6 mg/kg bw/d and above. Absolute and relative thymus weights were significantly decreased in high dose animals of both sexes. Histopathological examination of the test site revealed mild microscopic changes (including acanthosis, epidermal crusting and hyperkeratosis with occasional sub-acute inflammation and moderate ulceration) in both sexes at 530 mg/kg bw/d with increased hyperkeratosis in females at 106 mg/kg bw/d. Treatment related changes in the internal organs included alterations in liver (hepatic congestion, necrosis, vacuolar change) in both sexes at  53 mg/kg bw/d and above and thymus (atrophy, congestion) in males at 10.6 mg/kg bw/d and above and in females at 53 mg/kg bw/d and above. Chronic inflammation of the thyroid (lymphocytic thyroiditis; 53 mg/kg bw/d and above) and cellular depletion of bone marrow (106 mg/kg bw/d and above) were also present.

Based on these overall findings the NOAEL for systemic toxicity in both sexes was 1.06 mg/kg bw/d (reflecting increased relative liver weight in both sexes, and the occurrence of liver histopathology and decreased platelet count in males only). The NOAEL for local effects (reflecting microscopic changes at the test site) was 106 mg/kg bw/d for males and 53 mg/kg bw/d for females.