Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Givaudan Corporation (Clifton, N J, USA).
- Purity: 98%

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CDR(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc., St Constant, Quebec, Canada)
- Age at study initiation: 41 - 44 days old
- Weight at study initiation: males 141-182 g and females 113-148 g
- Housing: Housed individually in stainless-steel cages
- Diet (e.g. ad libitum): Standard laboratory rodent chow (Certified Purina" Rodent Chow No. 5002)
- Water: Tap water ad libitum
- Acclimation period: At least 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature : 21 ± 3°C
- Humidity: 50± 20%
- Photoperiod : 12-hr light/dark cycle

Administration / exposure

Type of coverage:
open
Vehicle:
other: Phenylethyl alcohol
Details on exposure:
TEST SITE
- Area of exposure: Clipped unabraded surface of the backs of the rats daily
- % coverage: Approximately 25% of the body surface area

TEST MATERIAL
- Concentration (if solution): 3.75, 12.0, 37.5, 120.0 mg/mL for 7.5, 24, 75 or 240 mg/kg bw/day
- Constant volume or concentration used: Yes (Appropriate amounts of the test materials were dissolved in the vehicle to yield a constant dose volume of 2 ml/kg body weight across all groups). Dosing solutions were freshly made each week and adjusted each week to achieve the desired dose on a mg/kg bw/day basis.

VEHICLE
Phenylethyl alcohol (PEA), which was supplied by PPF International, Inc. (East Hanover, N J, USA).

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes (Elizabethan-type collars to prevent ingestion of the test substances)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose solutions were periodically checked for accuracy of formulation throughout the study but no specific data provided.
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
7.5 mg/kg bw/day (nominal)
Dose / conc.:
24 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Musk Ketone: 15 males and 15 females
PEA: 30 males and 30 females
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were observed twice a day for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were observed once a day for clinical signs. Once a week they were given a detailed physical examination.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The rats were observed once a day for dermal irritation.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed once a day.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
Blood samples were obtained from all rats that survived to the end of the study (except those selected for neuropathological evaluations) by venepuncture of the orbital sinus under light anaesthesia or from the abdominal aorta following an ip injection of sodium pentobarbital. Haematological were performed on blood from ten rats/sex in the vehicle control group, and in the groups given 24 or 240 mg musk ketone. The rats were starved overnight before blood sampling. The haematological parameters examined included haematocrit, haemoglobin, methaemoglobin, red blood cell (RBC) count, RBC morphology, platelet count, white blood cell count (total and differential) and Wintrobe's constants (calculated).

CLINICAL CHEMISTRY: Yes
Blood samples were obtained from all rats that survived to the end of the study (except those selected for neuropathological evaluations) by venepuncture of the orbital sinus under light anaesthesia or from the abdominal aorta following an ip injection of sodium pentobarbital. Clinical chemical evaluations were performed on blood from ten rats/sex in the vehicle control group, and in the groups given 24 or 240 mg musk ketone. The rats were starved overnight before blood sampling. The clinical chemical parameters examined were alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, calcium, chloride, cholesterol, creatinine, creatinine phosphokinase, glucose, y-glutamyl transferase, inorganic phosphate, lactic dehydrogenase, magnesium, total bilirubin, total protein, triglycerides and uric acid.

NEUROBEHAVIOURAL EXAMINATION: Yes
At the end of the treatment period, three rats/sex/dose (musk ketone) and seven rats/sex (control group) were randomly selected for neuropathological examination. Where possible, this selection was limited to those rats that showed clinical signs of neurotoxicity. Selected rats were deeply anaesthetized with sodium barbitone or pentobarbital and systemically perfused with fixatives according to standard procedures.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Except for those selected for neuropathological evaluation, all surviving rats were killed by an ip injection of sodium pentobarbital or by ether anaesthesia followed by exsanguination from the abdominal aorta. All of these rats, and any that died or were killed in extremis during the study, were examined at necropsy for gross pathological changes.

The following organs were weighed and retained at necropsy: adrenals, brain, epididymis (fixed in Zenker's fluid), heart, kidneys, liver, ovaries, pituitary, testes (fixed in Zenker's fluid), and thyroid and parathyroids. In addition, the
following tissues and organs were retained: abdominal aorta, bone marrow (femur), caecum, colon, duodenum, oesophagus, eyes, head, ileum, jejunum, lungs, mesenteric lymph nodes, mammary gland, pancreas, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, thymus, trachea, urinary bladder, uterus, vagina, all gross lesions and all tissue masses. All organs and tissues were fixed and preserved in neutral buffered 10% formalin, unless otherwise indicated.

HISTOPATHOLOGY: Yes
Histopathological examination was performed on the following selected organs and tissues from rats in the vehicle control groups and the groups given the 240 mg/kg bw/day musk ketone: heart, kidneys, liver, ovaries, skin, testes, thyroid and any tissues with gross abnormalities.
Statistics:
Differences in body weights, relative organ weights, and haematological and clinical chemical values were analysed by appropriate tests including chi-square test (with Yate's correction), Fisher's exact probability test, Bartlett's test ANOVA, and Kruskal-Wallis and Dunn's test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The signs and symptoms associated with the demise of the rats in all groups were generally non-specific. They included reduced muscle tone, abnormal respiration and, occasionally, ataxia.

Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from variable desquamation, and occasional atony of the skin, there were no significant dermatological changes at the treatment site that could be attributed to the test substances.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the vehicle control group, one male given 24 mg/kg bw/day musk ketone and one male given 240 mg/kg bw/day musk ketone died or were killed before the end of dosing (Table 2).

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Throughout the study, the body weights of males and females given the high dose of musk ketone and of the females given 75 mg/kg/day were significantly lower than those of the vehicle controls (Figs 2 and 3), whereas food consumption was similar to or greater than that of the controls. No effects of musk ketone on body weight or food consumption were observed at other doses.

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Throughout the study, the body weights of males and females given the high dose of musk ketone and of the females given 75 mg/kg/day were significantly lower than those of the vehicle controls (Figs 2 and 3), whereas food consumption was similar to or greater than that of the controls. No effects of musk ketone on body weight or food consumption were observed at other doses.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The males and females in the high-dose musk ketone groups showed variations in haematological parameters, notably decreases in haemoglobin, haematocrit, RBC counts and mean corpuscular volume (data not shown). However, all of the values fell within historical ranges for this strain of rat and were not considered to be of any biological significance. There was no correlation with the observed deaths.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no effects on clinical chemistry parameters (data not shown).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Neither during nor at the end of the study were specific treatment-related functional impairments observed that were attributable to musk ketone (data not shown).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The livers of females exposed to the high dose of musk ketone were increased in weight, compared with vehicle controls, but this was not associated with any pathological findings (Table 3).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross changes in any of the organs examined, including the testes, which could be attributed to treatment (data not shown)..
Neuropathological findings:
no effects observed
Description (incidence and severity):
There was no evidence of neurotoxicity in any of the rats exposed to musk ketone although detailed neuropathological examination of specially prepared sections of the central and peripheral nervous systems was carried out (data not shown).
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In musk ketone groups, microscopic lesions were variable and generally non-specific; there was no correlation with the observed deaths (data not shown)

Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
In a dermal repeated dose toxicity study in Sprague-Dawley Crl:CDR(SD) rat, the NOAEL for males and females was 75 mg/kg bw/day, based on the relative liver weight changes in females.
Executive summary:

In a repeat-dose dermal toxicity study (Ford and Api, 1990),  musk ketone (98%) was applied to the clipped unabraded skin of 15 Sprague-Dawley Crl:CDR(SD) rats/sex/dose at dose levels of 0, 24, 75, 240  mg/kg bw/day, 7 days/week during a 90-day period.

One female in the vehicle control group, one male given 24 mg/kg bw/day musk ketone and one male given 240 mg/kg bw/day musk ketone died or were killed before the end of dosing. Apart from variable desquamation, and occasional atony of the skin, there were no significant dermatological changes at the treatment site that could be attributed to the test substance. Throughout the study, the body weights of males and females given the high dose of musk ketone and of the females given 75 mg/kg/day were significantly lower than those of the vehicle controls, whereas food consumption was similar to or greater than that of the controls. No effects of musk ketone on body weight or food consumption were observed at other doses.

The males and females in the high-dose musk ketone groups showed variations in haematological parameters, notably decreases in haemoglobin, haematocrit, RBC counts and mean corpuscular volume. However, all of the values fell within historical ranges for this strain of rat and were not considered to be of any biological significance. There was no correlation with the observed deaths. There were no effects on clinical chemistry parameters.

The livers of females exposed to the high dose of musk ketone were increased in weight, compared with vehicle controls, but this was not associated with any pathological findings. There were no gross changes in any of the organs examined, including the testes, which could be attributed to treatment. In musk ketone groups, microscopic lesions were variable and generally non-specific; there was no correlation with the observed deaths.

Neither during nor at the end of the study were specific treatment-related functional impairments observed that were attributable to musk ketone. There was no evidence of neurotoxicity in any of the rats exposed to musk ketone although detailed neuropathological examination of specially prepared sections of the central and peripheral nervous systems was carried out.

The NOAEL for musk ketone in males and females was 75 mg/kg bw/day, based on the relative liver weight changes in females.

This dermal toxicity study in the rat is acceptable and satisfies the guideline requirement for a repeat-dose dermal toxicity study (OECD 411).