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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
chronic toxicity: dermal
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987-03-20 to 1989-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction. The study closely adhered to OCED guideline 453.
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:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
only single dose level used
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Read Across to Other Lubricant Base Oils
-Test Substance: MRD-87-016, Clear colourless liquid, stored at room temperature under nitrogen in dark
- Substance Type: Solvent-extracted/hydrotreated paraffinic distillate - Other Lubricant Base Oils (Sufficiently Refined, IP 346 < 3%)

Test animals

Species:
mouse
Strain:
other:
Sex:
male
Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: 15 to 25 grams
- Fasting period before study: not reported
- Housing: individually housed (except for first week of acclimation) in suspended stainless steel cages
- Diet (e.g. ad libitum): Ad libitum; Purina Certified Rodent Pellets 5002
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: 1987-03-17 To: 1991-02-19

Administration / exposure

Type of coverage:
open
Vehicle:
other: Toluene
Details on exposure:
TEST SITE
- Area of exposure: interscapular area on back of each mouse
- % coverage: not reported
- Type of wrap if used: not reported
- Time intervals for shavings or clipplings: weekly


REMOVAL OF TEST SUBSTANCE
- Washing (if done): none
- Time after start of exposure: not reported


TEST MATERIAL
- Amount applied (volume or weight with unit): a single 37.5 microlitre dose
- Concentration (if solution): MRD-87-014 was prepared as a 30% (w/v) solution in Primol 185; MRD-87-012 was prepared as a 75% (w/w) solution in Primol 185; all other test substances were applied neat
- Constant volume or concentration used: no


USE OF RESTRAINERS FOR PREVENTING INGESTION: Not reported
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data reported.
Duration of treatment / exposure:
24 months or until observation of carcinoma at which time the animal was sacrificed
Frequency of treatment:
Twice per week
Doses / concentrations
Remarks:
Doses / Concentrations:
37.5 µL (150 mg/kg/day)/application
Basis:

No. of animals per sex per dose:
50 animals/dose
Control animals:
yes
Positive control:
The positive control used was benzo (a) pyrene and designated as MRD-86-951. Benzo (a) pyrene was prepared as a 0.05% (w/v) solution
in toluene at six month intervals. Another dose group was administered toluene.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined for viability twice daily on Monday through Friday, and at least once daily on Saturday, Sunday, and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for presence of dermal growths and irritation, as well as for clinical signs, were performed at initiation of dosing, weekly thereafter until study termination, and prior to sacrifice due to moribund condition.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Observations for presence of dermal growths and irritation, as well as for clinical signs, were performed at initiation of dosing, weekly thereafter until study termination, and prior to sacrifice due to moribund condition.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded the week prior to dosing (pre-dose), during the first week of the study, once every 4 weeks thereafter until study termination, and prior to sacrifice due to moribund condition.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; examinations of the external surface; all orifices; the thoracic, abdominal, and cranial cavities and their viscera; the cervical tissues and organs; and the carcass were conducted. (see table in results section)
HISTOPATHOLOGY: Yes; skin and grossly observable masses (see table in results section)

Animals were sacrificed when observed with a carcinoma, when in in the opinion of the Study Director, sacrifice of an animal was mandated by its moribund condition, or for human purposes. Animals were sacrificed by asphyxiation with carbon dioxide gas.
Other examinations:
No other examinations were performed.
Statistics:
The following analysis was performed at each time period using Group 721 (Primol 185-treated group) as the control: Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's test was performed to determine if the dose groups have equal variance. If the variances are equal the testing was done using parametric methods, otherwise nonparametric techniques were used. For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's test was used to determine which treatment groups differ significantly from control. For the nonparametric procedures, the test of equality of means was performed using the Kruskal-Wallis test. If significant differences among the means were indicated, Dunn's Summed Rank test was used to determine which treatment group differ significantly from control. The test for equal variance (Bartlett) was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance. Survivorship was compared across groups by use of the Kaplan-Meier life table technique as well as by the Kruskal-Wallis analysis and Cox's test. The time to tumour analysis was performed based on the Kaplan-Meier method, the Generalized Kruskal-Wallis and method, the Fisher Exact test, and the Weibull method.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
no effects observed
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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:
effects observed, treatment-related
Details on results:
The positive control, Group 951, had the lowest survivorship and greatest tumour incidence among the other groups. However, this reduced survivorship was directly related to the animals' termination following the first appearance of a carcinoma. The remaining 6 treated groups had equivalent survivorship and had no observed masses.

Clinical in-life observations revealed low incidences of many findings intermittently for all groups. These findings included but were not limited to alopecia, sores, scabs, swelling, emaciation and ocular abnormalities. Additionally, urine staining and protruding penis were noted at a high frequency in the majority of groups (excluding Group 951). All these findings were considered incidental and/or common in animals of this type and age and not treatment-related. However, a high frequency of dermal irritation including desquamation or exfoliation, pinpoint scabbing or eschar, edema, erythema and atonia was noted in Groups 951 and 722. These dermal responses were considered treatment-related. There were no statistically significant differences between the negative control (MRD-85-721) and any other treatment group mean body weight values at any of the weighing intervals. At postmortem gross examination, a high incidence of liver masses was noted in all groups with the exception of Group 951. Desquamation or exfoliation was frequently noted in groups 951 and 722, along with other dermal abnormalities noted at a lesser frequency. Abnormalities of the kidney, heart, lymph node, liver, lung, adrenal, spleen, urinary bladder, gastro-intestinal tract, and seminal vesicles were noted in the majority of groups. In addition, a high incidence of ano-genital staining and protruding penis was noted in all groups except Group 951. At microscopic evaluation, squamous cell neoplasms were present in only the treatment skin of 46 mice in Group 951 (positive control). Dermal irritation of variable degree characterized by acanthosis (focal to diffuse), and subepidermal inflammatory infiltrate was a common finding in the mice of Groups 951 and 722. Dermal irritation was slight in Group 722, and slight to moderate in Group 951. Dermal irritation was not present in mice of Groups 011, 012, 014, 016 and 721 (negative control). In other tissues, spontaneous disease lesions including neoplasms as well as inflammatory and degenerative changes, were observed and were of the usual type and incidence for animals of this strain and age, and were considered unrelated to treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Based on the lack of treatment-related histopathology or gross pathology changes observed in the study.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Macroscopic Observations
Incidence
Group Tumours Carcinomas Papillomas
MRD-85-721 (Primol 185, negative control) 0/50 0/50 0/50
MRD-86-951 (Benzo(a)pyrene, positive control) 46/50 45/50 1/50
MRD-87-722 (Toluene, vehicle) 0/50 0/50 0/50
MRD-87-016 0/50 0/50 0/50
 
Microscopic Observations
Incidence
Group Tumours Carcinomas Papillomas
MRD-85-721 (Primol 185, negative control) 0/50 0/50 0/50
MRD-86-951 (Benzo(a)pyrene, positive control) 45/50 44/50 1/50
MRD-87-722 (Toluene, vehicle) 0/50 0/50 0/50
MRD-87-016 0/50 0/50 0/50
Estimated Median Survivorship based on Weibull and Kaplan-Meier Method
Group Weibull Median Survivorship (days) K-M Median Survivorship (days) Number of deaths
MRD-85-721 (Primol 185, negative control) 549 523 48
MRD-86-951 (Benzo(a)pyrene, positive control) 335 324 50
MRD-87-722 (Toluene, vehicle) 563 546 48
MRD-87-016 535 553 50
Estimated Median Time to Observed Skin Mass based on Weibull and Kaplan-Meier Method
Group Weibull Median Time to Mass (days) K-M Median Time to Mass (days) Number of Masses
MRD-85-721 (Primol 185, negative control) 1158 - 0
MRD-86-951 (Benzo(a)pyrene, positive control) 322 291 45
MRD-87-722 (Toluene, vehicle) 1188 - 0
MRD-87-016 1134 - 0

Applicant's summary and conclusion

Conclusions:
The test material was dermally applied neat and did not cause local or systemic effects after a 24-month period.
Executive summary:

Justification for Read Across

This substance is similar to the feedstocks for most of current dewaxing operations that produce the finished paraffin and microcrystalline waxes. These lubricant base oil data may serve as the basis for a worst case assessment of the reproductive potential of paraffin and microcrystalline waxes and are summarised in this section.

In a dermal carcinogenicity study, male C3H/HeNCrlBR mice (50/group) were dermally exposed to a lubricant base oil (designated MRD-87-016) and the appropriate controls at a frequency of twice per week for 24 months or until observation of carcinoma at which time the animal was sacrificed. The dermal application of the test material produced considerable irritation in groups 951 (positive control) and 722 (vehicle control). This irritation was noted macroscopically as desquamation, exfoliation, atonia, eschar, erythema and/or edema. Microscopic examination revealed acanthosis and subepidermal inflammatory infiltrate. Treatment group 016 and the negative control group 721 were generally free of dermal irritation during most of the study and at histopathological examination. Forty-five of the fifty Group 951 animals (positive control) had confirmed squamous cell carcinomas at histopathology. All other groups were free of any skin neoplasms. With the exception of the positive control group, there were no statistical differences in time to tumour and tumour production between groups. Survivorship analysis indicated that the positive control displayed the lowest survivorship; however, this finding is related to the fact that animals were euthanized following the appearance of a carcinoma. Gross postmortem examination showed a high incidence of liver masses in all groups except 951. These liver masses as well as other lesions noted histopathologically were of the usual type and incidence in this strain and age and were considered to be unrelated to treatment. Therefore, the test material did not cause local or systemic effects when applied neat.

 

The NOAEL was determined to be 150 mg/kg/day, based on the lack of treatment-related histopathology or gross pathology changes observed in the study.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was carried out according to TG 453.