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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
Study period:
1984-11-19 to 1984-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it was conducted according to or similar to OECD Guideline 410 without exceptions.
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:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Oily liquid
Details on test material:
Test substance: API 83-12 (CAS No. 64742-53-6)
- Name of test material (as cited in study report): API 83-12


- Physical state: clear liquid
- Viscosity, SSU:
53.5 at 100°F
33.3 at 210°F
- API Gravity: 26.2
- Flash Point: 255 °F
- Distillation range (°F) ASTM D86 Equiv: 533-713 (10-95%)
Initial Boiling Point (°F): 464
Final Boiling Point (°F): 796
- Pour Point (°F): 60
- Aniline Point (°F): 148.3
- Colour ASTM: 0.5

- Composition of test material ASTM D-2007, Wt. %:
Saturates: 61.6
Aromatics 36.1
Polar compounds: 2.3

Sulfur, Wt%: 0.019

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Dutchland, Inc. (Denver, Pennsylvania)
- Age at study initiation: Not reported
- Weight at study initiation: Males: 2.2 - 3.1 Kg; Females: 2.6 - 3.0 Kg
- Fasting period before study: Not reported
- Housing: Individual in stainless steel cages with grid bottoms
- Diet (e.g. ad libitum): Purina Laboratory Rabbit Chow (ad libitum)
- Water (e.g. ad libitum): Ad libitum via automatic feeder system
- Acclimation period: 19 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 34 ± 12%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: From: 1984-11-01 To: 1984-12-21

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Dorsal trunk
- % coverage: 10%
- Type of wrap if used: Sheet of polyethylene material secured by hypoallergenic tape (Blenderm®)
- Time intervals for shavings or clippings: Prior to first application and as necessary thereafter


REMOVAL OF TEST SUBSTANCE
- Washing (if done): Wiped gently with a dry, clean absorbent guaze pad
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200, 1000, or 2000 mg/kg body weight
- Constant volume or concentration used: no, volume calculated based on weekly body weight


VEHICLE
- Justification for use and choice of vehicle (if other than water): TS administered without vehicle


USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
3 times/week for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 1000, or 2000
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on pilot study
- Rationale for animal assignment (if not random): Random
- Section schedule rationale (if not random):
Positive control:
Positive control not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Prior to TS administration and once daily thereafter


BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 of study, once weekly thereafter, and prior to termination


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


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: Yes
- Time schedule for examinations: Post termination
- Dose groups that were examined: 0, 200, 1000, and 2000 mg/kg


ORGAN WEIGHTS: yes
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (T-61®)
- Animals fasted: No data
- How many animals: 5/sex/dose
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: No data
- How many animals: 5/sex/dose
- Parameters checked in table [No.2] were examined.


URINALYSIS: Urine was collected for possible future evaluations. The urine was not used for urinalysis.
- Time schedule for collection of urine: Urine collected prior to dosing and at Termination
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data



NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes for control and high-dose animals (see table 3)
Statistics:
Body weights, clinical pathology, and absolute and relative organ weight data of the treated groups were statistically compared to the control group data of the same sex, using a two-tailed Student's t-test at the 5% probability level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed in control animals or at any dose level tested.

Soft feces was observed in 1 control (Days 24, 25), 1 mid-dose male (Day 8), and 2 high-dose males (Days 3, 4, and 12-15). 1 male rabbit dosed with API 83-12 at 2000 mg/kg also appeared thin from Days 15 to 29 and had a soiled anal area on Days 3,4, and 6 of the study period. One female treated at 1000 mg/kg was observed to have a limb injury on Day 12 and one high-dose (2000 mg/kg) female had a scab on its ear on Days 19 to 29 of the study period. Thinness was apparent in all female rabbits treated with API 83-12 at 2000 mg/kg body weight.

Control males and females did not exhibit any dermal irritation. Dermal administration of API 83-12 at 200 mg/kg caused minimal irritation in males (MIS = 0.1) and females (MIS = 0.4). Irritation was observed in males on 12 days and in females on 20 days of the 28-day study period. No edema was observed in either males or females dosed at 200 mg/kg. Dermal application of 1000 mg/kg API 83-12 resulted in mean irritation scores of 2.0 and 2.2 for males and females, respectively. Moderate irritation was observed in males on 14 of the 28 days and minimal to slight irritation was seen in females on 13 of the 28 days. Very slight edema accompanying well-defined erythema was also seen in both male and female rabbits dosed at 1000 mg/kg. Dermal application of API 83-12 at 2000 mg/kg resulted in mean irritation scores of 2.6 and 3.1 in males and females, respectively. Moderate irritation was observed 19 out of 28 days and 21 out of 28 days in males and females, respectively. Erythema and edema were consistently observed in all male and female animals dosed at 2000 mg/kg by Day 18 of the study period. Maximal erythema (4) was observed in two females on Day 20 and 1 female moderate edema on Day 17.


BODY WEIGHT AND WEIGHT GAIN
Body weight gain was observed to be normal in males in the control, low-dose (200 mg/kg), and mid-dose (1000 mg/kg) groups. Body weight gain and mean body weights on days 8, 15, 22, and 29 for the high-dose (2000 mg/kg) males were observed to be lower (statistically significant) than that of the controls. Mean body weights and body weight gain was observed to be normal in control and low-dose (200 mg/kg) females while mean body weights for the mid-dose (1000 mg/kg) and high-dose (2000 mg/kg) females was found to be statistically lower than the corresponding controls.


HAEMATOLOGY
WBC counts in females dosed at 200 mg/kg were observed to be lower (statistically significant) than control animals. However, since this value was within the normal range for New Zealand White rabbits, the finding was considered incidental and not treatment-related. All other hematology parameters appeared to be normal in males and females in all dose groups.

CLINICAL CHEMISTRY
Clinical chemistry parameters appeared to be normal in males and females in all dose groups. One female in the mid-dose (1000 mg/kg) dose group exhibited abnormally high SGPT and SGOT levels but this was considered incidental, and not treatment-related.

ORGAN WEIGHTS
Mean terminal body weights of mid-dose (1000 mg/kg) females and high-dose (2000 mg/kg) males and females were observed to be lower (statistically significant) than the corresponding control animals. Absolute left and right testis weights and relative right testis weights for the high-dose (2000 mg/kg) dose males were also found to be lower (statistically significant) than the corresponsing controls. All other statistically significant differences in organ weights in both males and females were considered to be incidental and not treatment-related.

GROSS PATHOLOGY
Dry, scaly, rough, fissured, crusted, and/or thickened skin was observed in animals in the high-dose (2000 mg/kg) group. Two high-dose (2000 mg/kg) males were observed to have bilaterally small testes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Slight to moderate proliferative changes of the skin accompanied by increased granulopoeisis of the bone marrow were observed in all male and female rabbits dosed with 2000 mg/kg API 83-12. Testes of 3 of 5 high-dose (2000 mg/kg) males had bilateral diffuse tubular hypoplasia (atrophy) accompanied by aspermatogenesis and atrophy of accessory sex organs.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: The systemic toxicity NOAEL is 1,000 mg/kg, based on the lack of adverse systemic effects observed at this dose level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables


Table 1. Dermal irritation scores for male and female rabbits

Dose (mg/kg)

Day 2

Day 15

Day 29

Mean

Male

Female

Male

Female

Male

Female

Male

Female

0 (Control)

0.0

0.0

0.0

0.0

0.0

0.0

0

0

200

0.0

0.0

0.2

0.6

0.0

0.6

0.1

0.4

1000

0.0

0.2

2.0

2.2

3.0

3.0

2.0

2.2

2000

0.4

0.6

3.2

3.2

3.4

3.8

2.6

3.0

0 = Non-irritant

>0 but ≤1= Minimal

>1 but ≤2 = Slight

>2 but ≤5 = moderate

>5 = Severe



Table 2. Mean body weight gain for male and female rabbits

Dose (mg/kg)

Males (kg)

Females (Kg)

0 (Control)

0.5 (0.2)

0.3 (0.1)

200

0.3 (0.1)

0.4 (0.1)

1000

0.3 (0.2)

0.0*(0.2)

2000

0.1*(0.2)

-0.2*(0.1)

*  statistically significantly lower than contol (p0.05)

Standard deviation (SD) represented in parenthesis

Table 3. Absolute and Relative organ weights in male and female rabbits

Dose group (mg/kg)

Terminal body weight (Kg)

Right Testis

Left Testis

Male

Female

Absolute (g)

Relative (%)

Absolute (g)

Relative (%)

0 (Control)

3.3 (0.3)

3.2 (0.2)

2.86 (0.72)

0.09 (0.02)

2.74 (0.79)

0.08 (0.02)

200

3.0 (0.3)

3.3 (0.2)

2.57 (0.57)

0.09 (0.02)

2.57 (0.66)

0.09 (0.02)

1000

3.1 (0.2)

2.7*(0.2)

2.32 (0.52)

0.08 (0.02)

2.38 (0.50)

0.08 (0.02)

2000

2.7*(0.2)

2.5*(0.2)

1.23*(0.83)

0.05*(0.03)

1.21*(0.79)

0.05 (0.03)

* Statistically significantly lower than control (p≤0.05)

  Standard deviation (SD) represented in parenthesis


Applicant's summary and conclusion

Conclusions:
The systemic toxicity NOAEL for this 28-day dermal toxicity study in the rabbit is 1,000 mg/kg, based on the lack of adverse systemic effects observed at this dose level.
Executive summary:

In a 28-Day repeat dose dermal toxicity study, five New Zealand White rabbits/sex/dose were topically administered hydrotreated light naphthenic oil six hours/day, three times a week for a period of 28-days at concentrations of 0, 200, 1000, or 2000 mg/kg body weight.

All animals were observed twice daily for mortality and signs of clinical toxicity and dermal irritation was scored daily (according to the Draize system). Body weights were measured and recorded for each rabbit at the end of the quarantine period, at weekly intervals during the study, and prior to termination.

No mortality was observed in control animals or at any dose level tested. Soft feces was observed in some male and female rabbits in the control, mid-dose (1000 mg/kg) and high-dose (2000 mg/kg) dose groups. All female rabbits dosed at 2000 mg/kg hydrotreated light naphthenic oil appeared thin during the study period. Control males and females did not exhibit any dermal irritation while minimal irritation was observed in males (MIS = 0.1) and females (MIS = 0.4) dosed at 200 mg/kg hydrotreated light naphthenic oil. Slight to moderate irritation accompanied by very slight edema and well-defined erythema was observed in males (MIS = 2.0) and females (MIS = 2.2) dosed at 1000 mg/kg. Moderate irritation with consistent erythema and edema was seen in males (MIS = 2.6) and females (MIS = 3.1) dosed at 2000 mg/kg hydrotreated light naphthenic oil. A couple of females in the high-dose (2000 mg/kg) group also exhibited maximal erythema (MIS = 4.0) on day 20 of the study period. Body weight and body weight gain appeared to be normal in males in the control, low-dose (200 mg/kg), and mid-dose (1000 mg/kg) dose group. Mean body weights and body weight gain were lower (statistically significant) than control in the high-dose (2000 mg/kg) males and in the mid-dose (1000 mg/kg) and high-dose (2000 mg/kg) females. Most of the hematology parameters were found to be normal for males and females in all dose groups. WBC counts in the low-dose (200 mg/kg) females were lower than those observed in control animals but were considered incidental and not treatment-related. Clinical chemistry parameters appeared to be normal in males and females in all dose groups. One female in the mid-dose (1000 mg/kg) dose group exhibited abnormally high SGPT and SGOT levels but this was considered incidental, and not treatment-related. Mean terminal body weights of mid-dose (1000 mg/kg) females and high-dose (2000 mg/kg) males and females were observed to be lower (statistically significant) than the corresponding control animals. Absolute left and right testis weights and relative right testis weights for the high-dose (2000 mg/kg) dose males were also found to be lower (statistically significant) than the corresponsing controls. All other statistically significant differences in organ weights in both males and females were considered to be incidental and not treatment-related. Post-termination gross morphology examinations revealed dry, scaly, rough, fissured, crusted, and/or thickened skin in animals in the high-dose (2000 mg/kg) group. Two high-dose (2000 mg/kg) males were also observed to have bilaterally small testes. Histopathological examinations revealed slight to moderate proliferative changes of the skin accompanied by increased granulopoeisis of the bone marrow in all male and female rabbits dosed with 2000 mg/kg API 83-12. Testes of 3 of 5 high-dose (2000 mg/kg) males were observed to have bilateral diffuse tubular hypoplasia (atrophy) accompanied by aspermatogenesis and atrophy of accessory sex organs. Systemic effects may be a secondary effect due to effects at primary site of application.

The systemic toxicity NOAEL for this 28-day dermal toxicity study in the rabbit is 1,000 mg/kg, based on the lack of adverse systemic effects observed at this dose level. Systematic effects may be a secondary effect due to effects at primary site of application.

This study is classified as reliable without restriction because it was conducted according to or similar to OECD Guideline 410 without exceptions.