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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-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).

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:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
Following an initial dose range-finding study, the test sample was applied either neat or diluted in acetone, under occlusion, to clipped rat skin 5 d/wk for 4 wk. Toxicological information collected during the study included details of on clinical signs, body weight effects, clinical chemistry, haematology, necropsy findings, organ weights and tissue histopathology.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Viscous hydrocarbon liquid
Details on test material:
- Name of test material (as cited in Sponsor documentation): F-115-01, FCCU clarified oil
- Dark blue-green to red-brown coloured liquid, slightly cracked or burnt odor
- Specific gravity: 1.06
- Flash Point (°F): 250

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Banton and Kingman, Fremont, CA USA.
- Age at study initiation: young adults
- Weight at study initiation: males (mean) 265-275 g, females (mean) 160-175 g
- Housing: individually housed in stainless steel, wire mesh-bottomed cages
- Diet (ad libitum): certified rodent diet
- Water (ad libitum): tap water
- Acclimation period: 7 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: 27 Nov 1989 To: 26-29 December 1989

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
The sample was applied (neat or diluted in acetone) to clipped dorsal skin (approx. 10% of body surface) once daily, 5 d/wk for 4 wk, and the test site covered with an occlusive dressing for 6 hr prior to wiping clean with gauze (no solvent). The study design included two control groups (sham control, acetone control).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hr occluded contact, 5 d/wk for 4 wk
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.00094, 0.00943 or 0.04717 mL/kg bw/d
Basis:
other: applied neat, nominal per unit body weight
Remarks:
Doses / Concentrations:
0, 1, 10 or 50 mg/kg bw/d
Basis:
other: applied neat, equivalent dose based on density of 1.06
Remarks:
Doses / Concentrations:
0, 0.001 (1%), 0.001 (10%), 0.010 (10%), 0.500 (10%)
Basis:
other: applied diluted in acetone (diluted to 1% or 10%), nominal per unit body weight
Remarks:
Doses / Concentrations:
0, 0.01, 0.10, 1, 10 or 50 mg/kg bw/d
Basis:
other: applied diluted in acetone, equivalent dose based on density of 1.06
No. of animals per sex per dose:
10 males, 10 females
Control animals:
yes, concurrent vehicle
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)
- Body weight
- Haematology
- Clinical chemistry
Sacrifice and pathology:
- Necropsy:
Animals were sacrificed (halothane anaesthesia) and blood collected for haematology and clinical chemistry analyses prior to exsanguination. Animals were then subject to a full necropsy, during which selected organs (adrenal, brain, kidney, liver, testes, ovaries) were weighed and representative samples collected from around 35 major organs and tissues (preserved in 10% neutral buffered formalin). Bone marrow smears were prepared and preserved.

- Histopathology:
Around 20 preserved tissues from control and high dose animals were processed for histological evaluation (H&E staining).
Statistics:
Bartlett’s test was applied to determine whether the control and test groups possessed equal variance. If variance was equal, data were analysed by one way ANOVA/Dunnett’s test with linear regression analysis used for line-fitting. If variance data was unequal, data were analysed by Kruskal-Wallis test/Dunn's Summed Rank test with Jonckheere’s test for trend analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
- In-life observations:
All animals survived until scheduled study end, with clinical signs restricted to not clearly dose related slight irritation of the test site (including barely perceptible or slight erythema, drying/flaking, slight eschar) in the low and intermediate dose groups (both treated neat and diluted with acetone) but absent from the majority of high dose animals. The overall irritancy of a given dose of F-115-01 appeared to be slightly greater after dilution with acetone than when applied neat.

- Body weight/body weight gain:
In animals treated with neat test substance, body weight was significantly decreased by around 10% in high dose females from study day 22, with a comparable reduction seen also in high dose males at study end. No effect on body weight was apparent in any group receiving F-115-01 in acetone.

- Haematology, clinical chemistry: neat F-115-01
Haematological analyses revealed significant (approx. 15%) decrements in red cell count, haematocrit and haemoglobin concentration in high dose males with platelet count significantly decreased (approx. 15%) in high dose females. Blood urea nitrogen was elevated 20-30% in high dose animals of both sexes, while serum cholesterol was significantly increased (+75%) in high dose females and potassium significantly increased (+10%) in females at 10 mg/kg bw/d and above.

- Haematology, clinical chemistry: F-115-01 diluted in acetone
Red cell count, haematocrit and haemoglobin concentration were decreased by 10-15% (significant) in males receiving 10 mg/kg bw/d and above, with haematocrit and haemoglobin concentration decreased by around 15% (significant) in high dose females also. Platelet counts were decreased by around 25% (non-significant) in high dose females. Clinical chemistry analysis revealed that blood urea nitrogen was elevated significantly (25-35%) in high dose males and females, with serum cholesterol levels approximately doubled in high dose females only (males unaffected). SGPT activity was decreased significantly (10-30%) in females receiving 0.1-50 mg/Kg bw/d, however the toxicological relevance of this observation is unclear.

- Necropsy: neat F-115-01
Absolute thymus weights were significantly decreased by 25-35% in high dose males and females, with an approx. 20% reduction in relative thymus weight (significant in females only). Relative liver weights were significantly increased in high dose males (+25%) and in females given 10 mg/kg bw/d and above (elevated 20-25%).

- Necropsy: F-115-01 diluted in acetone
Absolute liver weight was significantly increased (20-25%) in high dose animals (both sexes) with a significant (approx. 45%) decrease in absolute thymus weight. Comparable alterations in relative liver weight (increased 30-35%) and relative thymus weight (decreased 40-45%) were also apparent for these animals, with relative liver weight increased 10-15% in both sexes receiving 10 mg/kg bw/d.

- Histopathology: neat F-115-01
No histopathological assessment was performed on tissues from animals treated with neat test sample.

- Histopathology: F-115-01 diluted in acetone
Histopathological evaluation of tissues from control (acetone) and high dose animals revealed trace to mild acanthosis and hyperkeratosis in skin from the test site (more evident in females than in males) but no treatment related changes in any other tissue (including those exhibiting weight changes).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity (applied neat)
Effect level:
10 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Decreased body weight, decreased haematological parameters, clinical chemistry effects, organ weight changes
Dose descriptor:
NOAEL
Remarks:
systemic toxicity (applied neat)
Effect level:
1 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Increased serum potassium, increased relative liver weight
Dose descriptor:
LOAEL
Remarks:
local effects (applied neat)
Effect level:
1 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Sporadic very slight erythema, eschar and dry skin
Dose descriptor:
NOAEL
Remarks:
systemic toxicity (applied in acetone)
Effect level:
1 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Decreased haematological parameters, increased relative liver weight
Dose descriptor:
NOAEL
Remarks:
systemic toxicity (applied in acetone)
Effect level:
1 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Increased relative liver weight
Dose descriptor:
LOAEL
Remarks:
local effects (applied in acetone)
Effect level:
0.01 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Sporadic very slight erythema, eschar and dry skin

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Selected results for the acetone treated groups (reflecting the lower NOAEL obtained for these animals); presented as mean [SD]:

Red blood cell count (million/mm3):
Male: 7.806 [0.343], 7.574 [0.540], 7.628 [0.356], 7.746 [0.595], 6.866 [0.518]**, 6.870 [0.522]**
Female: 7.294 [0.627], 7.230 [0.664], 7.052 [0.263], 6.552 [0.611], 6.228 [1.165]

Haematocrit (%):
Males: 43.02 [2.35], 41.52 [2.83], 42.14 [2.55], 41.58 [2.21], 37.46 [2.80]**, 37.36 [2.61]**
Females: 41.04 [3.59], 40.22 [3.97], 40.48 [2.48], 36.64 [8.32], 37.40 [3.25], 34.56 [6.58]*

Haemoglobin concentration (g/dL):
Males: 15.38 [0.57], 15.15 [0.38], 15.23 [0.54], 15.34 [0.57], 14.06 [1.10]**, 13.60 [0.89]**
Females: 14.97 [0.51], 15.20 [0.78], 14.68 [0.57], 14.74 [0.74], 14.08 [0.62], 12.51 [1.30]**

Blood urea nitrogen (mg/dL):
Males: 16.1 [2.1], 15.1 [1.4], 14.6 [1.3], 15.3 [2.0], 18.8 [3.5], 20.1 [3.5]**
Females: 14.8 [2.3],15.8 [2.1], 16.3 [2.9], 16.7 [2.7], 17.4 [1.4], 19.9 [4.8]*

Serum cholesterol (mg/dL):
Males: 37.7 [8.1], 45.3 [13.0], 38.2 [10.4], 35.7 [6.0], 43.0 [10.8], 42.0 [11.0]
Females: 43.6 [9.9], 46.1 [9.0], 48.4 [11.5], 49.5 [14.0], 64.1 [12.8]*, 85.5 [32.4]**

SGPT (IU/L):
Males: 43.9 [7.2], 40.3 [7.8], 36.3 [5.0]*, 38.4 [3.7], 33.2 [7.3]**, 31.5 [3.5]**
Females: 34.7 [4.7], 33.1 [5.2], 32.7 [4.8], 30.7 [5.1], 29.2 [5.4], 28.1 [6.5]

Relative liver weight (g/g bw):
Males: 0.0294 [0.0017], 0.0293 [0.0008], 0.0299 [0.0015], 0.0295 [0.0019], 0.0318 [0.0013]*, 0.0378 [0.0023]**
Females: 0.0323 [0.0021], 0.0319 [0.0022], 0.0338 [0.0020], 0.0343 [0.0023], 0.0367 [0.0031]*, 0.0437 [0.0051]**

Dose levels: 0, 0.01, 0.1, 1, 10 or 50 mg/kg bw/d
* = P<0.05; ** = P<0.01

Applicant's summary and conclusion

Conclusions:
NOAEL for systemic toxicity (neat) = 10 mg/Kg bw/d (males), 1 mg/Kg bw/d (females)
LOAEL for local effects (neat, skin irritation) = 1 mg/Kg bw/d (males and females)
NOAEL for systemic toxicity (in acetone) = 1 mg/Kg bw/d (males and females)
LOAEL for local effects (in acetone, skin irritation) = 0.01 mg/Kg bw/d (males and females)
Executive summary:

The sub-acute dermal toxicity of FCCU clarified oil (F-115-01) was investigated in male and female SD after application neat (0, 1, 10 or 50 mg/Kg bw/d) or diluted in acetone (0, 0.01, 0.1, 1.0, 10 or 50 mg/Kg bw/d). All animals survived until scheduled study end, with clinical signs restricted to not clearly dose-related slight irritation of the test site (including barely perceptible or slight erythema, drying/flaking, slight eschar) in the low and intermediate dose groups (both treated neat and diluted with acetone) but absent from the majority of high dose animals. The overall irritancy of a given dose of F-115-01 appeared to be slightly greater after dilution with acetone than when applied neat.
 

In animals treated with neat test substance, body weight was significantly decreased in high dose females from study day 22 and in high dose males at study end. Haematological analyses revealed significant decrements in RBC, HCT and HGB from high dose males with platelet count significantly decreased in high dose females. Blood urea nitrogen was elevated significantly in high dose animals of both sexes, while serum cholesterol was significantly increased in high dose females and potassium significantly increased in females at 10 mg/Kg bw/d and above. Organ weight measurements at necropsy revealed that absolute thymus weights were significantly decreased in high dose males and females, with relative thymus weight decreased also (significant in females only). In addition, relative liver weights were significantly increased in high dose males and in females given 10 mg/Kg bw/d and above, however no histopathological assessment was performed on tissues from these animals treated with neat test sample.
 

No effect of treatment on body weight was apparent in any group receiving F-115-01 in acetone, however significant decreases in RBC, HCT and HGB were present in males receiving 10 mg/Kg bw/d and above, with HCT and HGB decreased (significant) in high dose females also. Platelet counts were significantly decreased in high dose females. Clinical chemistry analysis revealed that BUN was elevated significantly in high dose males and females, with serum cholesterol levels approximately doubled in high dose females only (males unaffected). SGPT activity was decreased significantly in females receiving 0.1-50 mg/Kg bw/d, however the toxicological relevance of this observation is unclear. Findings at necropsy in high dose animals of both sexes treated with F-115-01 in acetone included a significant increase in absolute liver weight and a significant decrease in absolute thymus weight. Comparable alterations were apparent also in relative liver weight and relative thymus weight for these groups of animals, with relative liver weight increased in both sexes receiving 10 mg/kg bw/d. Histopathological evaluation of tissues from control and high dose (acetone-vehicle) animals revealed trace to mild acanthosis and hyperkeratosis in skin from the test site (more evident in females than in males) but no apparently treatment-related changes in any other tissue (including those exhibiting weight changes, as discussed above).
 

The NOAEL for systemic toxicity for the neat sample in males was 10 mg/Kg bw/day (based on decreased body weight, altered haematological and clinical parameters and organ weight effects) and 1 mg/Kg bw/d in females (reflecting changes in serum potassium and an increased relative liver weight). The LOAEL for local effects was 1 mg/Kg bw/d for both sexes (based on sporadic, very slight skin irritation).

The NOAEL for systemic toxicity for the sample applied in acetone was 1 mg/Kg bw/day in both sexes (based on decreased haematological parameters and/or increased relative liver weight) with a LOAEL for local effects of 0.01 mg/Kg bw/d for both sexes (based on sporadic, very slight skin irritation).