(E)-3-methyl-5-cyclopentadecen-1-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1995-09-01 to 1995-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 407 and in compliance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: oral

Remarks:

other: rangefinder

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Preliminary study used as range-finder experiment for an OECD 407 test

Principles of method if other than guideline:

7 day oral (gavage) dose rangefinding study in the rat

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR (VAF plus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river (UK) Limited, Margate, England
- Age at study initiation: about 3-4 weeks old on arrival
- Weight at study initiation: males 144-159 g, females 114-134 g
- Fasting period before study: none
- Housing: in groups of 3, by sex, in grid-bottomed stainless steel cages, measuring 56x35x20 cm, suspended over cardboard-lined excreta trays.
- Diet (e.g. ad libitum): pelleted diet ad libitum (SQC Rat and Mouse Maintenance diet No. 1, Expanded, Special diets services, Witham, England).
- Water (e.g. ad libitum): main water ad libitum.
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22°C
- Humidity (%): 32-59%
- Air changes (per hr): air conditioned
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 1995-05-05

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % w/v

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was formulated for dosing as suspensions in the vehicle. Formulations were prepared freshly each day. A weighted quantity of test article was suspended in the appropriate weight of vehicle. Separate formulations were prepared for each dose level.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

A sample of each formulation prepared on day 1 (including the control formulation) was taken and stored frozen (<-18°C) pending possible future analysis.

Duration of treatment / exposure:

7 days

Frequency of treatment:

Once daily

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

None

Positive control:

None

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, on day 8 and at necropsy.

FOOD CONSUMPTION: due to a technical error foo was given to all animals ad libitum on one occasion during the treatment period and therefore no quantitative results were obtained.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, by observing the appearance of the tissues in situ from the cranial, thoracic and abdominal cavities.
ORGAN WEIGHT: for all animals: adrenal, brain, heart, kidneys, liver, ovaries, spleen, testes and thymus.
HISTOPATHOLOGY: no (tissues preserved but not processed further).

Other examinations:

None

Statistics:

None

Clinical signs:

no effects observed

Description (incidence and severity):

Minimal to moderate post dose salivation was noted on days 6 and 7 in males and females dosed at 750 and 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Unaffected by administration of the test article.

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 examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weights were higher in all treated males groups and females dosed at 750 and 1000 mg/kg bw/day (males, group 2 - 18%/23%; group 3 - 11%/14%, group 4 - 12%/17%; females, group 3 - 22%/21%, group 4 - 15%/14%, for absolute and relative, respectively).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic findings noted at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

other: Maximum Tolerated Dose

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

None

Conclusions:

Maximum tolerated dose = 1000 mg/kg bw/day

Executive summary:

Twenty-four rats of the Crl:CD(SD)BR strain were divided into 4 groups consisting of 3 males and 3 females. Three groups received ST 04 C 91, orally by gavage once daily, for 7 days, at dose levels of 500, 750 or 1000 mg/kg bw/day. The fourth group received the vehicle 0.5% (w/v) aqueous carboxymethylcellulose an acted as control.

Animals were observed daily, bodyweights were recorded on days 1 and 8. All animals were subjected to a detailed necropsy during which the weights of identified organs were recorded and a range of tissues was preserved but not processed further.

 

There were no deaths. Minimal to moderate post dose salivation was noted on days 6 and 7 in males and females dosed at 750 and 1000 mg/kg bw/day.

Bodyweights and bodyweight gains were those expected for animals of this age and strain.

Absolute and relative liver weights were higher in all treated males groups and females dosed at 750 and 1000 mg/kg bw/day. There were no treatment-related macroscopic findings noted at necropsy.

 

Administration of ST 04 C 91 orally by gavage was associated with increased absolute and relative liver weights in all treated males and females dosed at 750 and 1000 mg/kg bw/day. In the absence of any findings at necropsy, it was concluded that 1000 mg/kg bw/day would be a suitable high dose level for a longer term study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(5E)-3-Methyl-5-cyclopentadecen-1-one

Cas Number:

101151-17-1

Molecular formula:

C16 H28 O

IUPAC Name:

(5E)-3-Methyl-5-cyclopentadecen-1-one

Constituent 2

Reference substance name:

(5Z)-3-Methyl-5-cyclopentadecen-1-one

Cas Number:

21944-94-5

Molecular formula:

C16 H28 O

IUPAC Name:

(5Z)-3-Methyl-5-cyclopentadecen-1-one

Constituent 3

Reference substance name:

(4E)-3-Methyl-4-cyclopentadecen-1-one

Cas Number:

62221-84-5

Molecular formula:

C16 H28 O

IUPAC Name:

(4E)-3-Methyl-4-cyclopentadecen-1-one

Test material form:

liquid

Details on test material:

- Description: Clear, colourless liquid
- Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR (VAF plus)

Details on species / strain selection:

Standard strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river (UK) Limited, Margate, England
- Age at study initiation: about 28 days old on arrival
- Weight at study initiation: males 150-186 g, females 132-158 g
- Fasting period before study: none
- Housing: in groups of 6, by sex, in grid-bottomed stainless steel cages
- Diet (e.g. ad libitum): pelleted diet ad libitum, unless period of deprivation associated with laboratory investigations
- Water (e.g. ad libitum): ad libitum, unless period of deprivation associated with laboratory investigations
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 41-61%
- Air changes (per hr): air conditioned
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 1995-09-01 To: 1995-10-26

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was formulated daily for dosing as suspensions in the vehicle (0.5 % w/v CMC). Separate formulations were prepared for each dose level. the weighted quantity of test article was suspended in the appropriate volume of vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0.5 % w/v
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation were assessed on one day in weeks 1 and 4 for test article.
A gas chromatographic method of analysis (Quintiles, FCH003) was developed. The detector response to ST 04 C 91 was found to be linear over the injection concentration range of 25.36 µg/mL to 177.52 µg/mL. The concentrations of ST 04 C 91 in study formulations in weeks 1, 3 and 4 were within 10% of their nominal concentration, except for week 3, group 3 and week 4, group 4 formulations which were 81% and 87% of nominal, respectively. The test article formulations generated were therefore considered to be suitable for the purpose of the study. Due to an administration error the data produced for this study has been misplaced. During retrial 1 the formulations indicated that the recoveries were lower than the original analysis. The sub-samples for analysis were, however, stored prior to analysis and it was apparent that these low volume samples did not appear to re-suspend using a magnetic stirrer. These samples were of insufficient volume to permit re-suspension using a Silverson mixer. It was considered that the physical instability of low volume samples may, at least in part, have accounted for the lower recoveries recorded in retrial 1. This problem was discussed with Dr. John Davies of HSE on 1996-11-01 who was satisfied with the outcome.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 animals/sex/dose +6 satellite animals/sex in control and high dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in light of the study "7-d oral (gavage) dose rangefinding study in the rat". 24 rats of the Crl:CD(SD)BR strain were divided into 4 groups consisting of 3 males and 3 females. Three groups received the test material orally by gavage once daily, for 7 days at dose levels of 500, 750 or 1000 mg/kg bw/day. The fourth group received the vehicle 0.5% (w/v) aqueous CMC and acted as control. Administration of ST 04 C 91 orally by gavage was associated with increased absolute and bodyweight related liver weights in all treated males and females at 750 and 1000 mg/kg bw/day. In the absence of any findings at necropsy, it was concluded that 1000 mg/kg bw/day would be a suitable high dose level for a longer term study. Due to slightly elevated liver weights in male animals at the lower dose levels, the low dose level for the subsequent study should be less than 500 mg/kg bw/day.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: The 6 male and 6 female with the highest identification numbers in groups 1 and 4
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then weekly thereafter.

FOOD CONSUMPTION:
The food consumed by each cage of animals was recorded weekly throughout the treatment and treatment-free periods and group mean weekly intakes were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes - Both eyes of all animals were examined before treatment started.
- Time schedule for examinations: in week 4
- Dose groups that were examined: the eyes of all control and high dose animals were examined (Group 1 and 4).
- Examinations: using an indirect ophtalmoscope and then, if necessary, a direct ophtalmoscope after previous instillation of a mydriatic agne (Minims - 2% w/v Homatropine hydrobromide, Smith and Nephew Pharmaceuticals Limited, Romfort, England).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once in week 4
- Anaesthetic used for blood collection: No data, as the blood samples were taken by tail venepuncture.
- Animals fasted: Yes, overnight
- How many animals: all 72 rats
- Parameters checked in Table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4 for clinical chemistry and in week 5 for coagulation; and at the end of recovery period for groups 1 and 4
- Animals fasted: Yes, overnight
- How many animals: all 72 rats
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: end of week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)

Other examinations:

None

Statistics:

ANOVA was performed on all parameters, using treatment group as the factor in the analysis. Residuals from this preliminary analysis were examined for heterogeneity of variance using Levenes tests. If Levenes test was significant at the 1% level then the particular variable concerned was subjected to a non-parametric analysis. Otherwise, Williams test was performed to compare the high dose with control at the two-sided 5% level. If this test was statistically significant then comparisons of the subsequent doses against control were performed at the one-sided 5% level until a non-significant difference is found, at which point the testing was stopped. The highest dose at which the difference from control was non-significant was deemed to be the no effect level.
If Levenes test indicated that there were differences in the treatment group variances, then a Kruskal-Wallis ANOVA was performed to assess overall differences amongst the treatment groups, followed by Shirley’s non-parametric version of William’s test, which was based on mean ranks rather than the arithmetic means. Note that the Shirley’s test was the modified version described in Williams which should enhance the power of the tests of the lower doses against control, and was performed in the same hierarchical manner as Williams test above.
Variables that were measured pre-study (for example body weight prior to the first administration of treatment) were analysed by first performing ANOVA, and then assessing the heterogeneity of the treatment group variances using Levenes test and performing a non-parametric ANOVA analysis, if appropriate. This provided an overall test for the comparability of the treatment groups, and was followed by all possible pairwise tests using the protected Student t-test (ot its non-parametric equivalent).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs noted; hairloss in one female at 250 mg/kg bw/day, 2 males and 3 females dosed at 1000 mg/kg bw/day. A female (N° 70) dosed at 1000 mg/kg bw/day) had a swollen foot. This was considered coincidental and unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No deaths during the treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Both were unaffected by administration of test material. After the 2 weeks treatment free period bodyweight gains were slightly reduced in the high dose group females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Not affected by administrations of test material.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Did not reveal any treatment-related findings or abnormalities.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Activated partial prothrombin time (PTT) levels were increased outside the normal ranges found in this laboratory for males dosed at 500 and 1000 mg/kg bw/day (21% and 34% respectively). Fibrinogen levels were also increased (15%) in males dosed at 1000 mg/kg bw/day. Following a 2 week treatment free period PTT levels were within the normal ranges and fibrinogen levels were reduced, showing that recovery had occurred.
Females dosed at 1000 mg/kg bw/d had reduced prothrombin time during week 5, which were not observed at the end of the treatment free period.
The remaining statistically significant changes in neutrophils, lymphocytes, monocytes, basophils and prothrombin time were considered to be unrelated to treatment as the changes were minor and all group mean values were within the normal ranges found in this laboratory.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During week 4, non dose related increases in cholesterol were noted in all treated females (250 mg/kg bw/day - 15%, 500 mg/kg bw/day - 10% and 1000 mg/kg bw/day - 25%) and males dosed at 1000 mg/kg bw/day (28%). At 1000 mg/kg bw/day, two female animals had cholesterol levels above the normal range but at lower levels other animals only showed values towards the upper limit of the normal range. After a 2 week treatment free period group 4 animals showed cholesterol levels were within 10% of the control values.
All remaining statistically significant changes noted were within the normal ranges found in this laboratory and therefore were considered to be unrelated to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects on urine composition or cellularity.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and bodyweight related liver weights were statistically significantly higher in all treated males and female groups. This was however considered to be due to the control values being lower than expected for animals of this age and strain. After a 2 week treatment free period group 4 values for absolute and bodyweight related liver weights were within 10% of the controls.
A statistically significant increase was apparent in absolute ovary weights for females dosed at 1000 mg/kg bw/day. This was considered to be unrelated to treatment as the group mean value was within the normal ranges found in this laboratory.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings.

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

No other information

Applicant's summary and conclusion

Conclusions:

The NOAEL for males and females was determined as 1000 mg/kg bw/day, based on a marginal increase in female cholesterol levels which was considered unlikely to be of any toxicological significance. All effects were minor, reversible and not supported by corroborative changes.

Executive summary:

In a subacute oral toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, the test material diluted in carboxymethylcellulose was administered to 6 Crl: CD (SD) BR (VAF plus) rats/sex/dose by gavage at dose levels of 0, 250, 500 and 1000 mg/kg bw/day. Two groups of 6 animals/sex received vehicle or the highest dose concomitantly to the other groups (same treatment duration) and were retained for a 2-week post-treatment observation period (recovery). Control rats were given the vehicle alone.

A seven-day preliminary study was performed at the dose of 0, 500, 750 and 1000 mg/kg bw/d. Increased absolute and bodyweight related liver weights were observed in all treated males and females at 750 and 1000 mg/kg bw/day. In the absence of any findings at necropsy, it was concluded that 1000 mg/kg bw/day would be a suitable high dose level for a longer term study. Due to slightly elevated liver weights in male animals at the lower dose levels, the low dose level for the subsequent study should be less than 500 mg/kg bw/day.

 

Clinical signs were observed daily whereas body-weight and food consumption was measured weekly. Ophtalmoscopy was performed on both eyes before the start of treatment and on control and high dose groups during week 4. Urine samples were collected from all animals during week 4. At the end of the treatment period (for all groups) or after the recovery period (for the control and high-dose group), the animals were sacrificed and further observed for haematology, blood chemistry, gross macroscopy, organ weight and histopathology.

  

No mortality was observed in any group. No signs of ill health, behavioural change or reaction to treatment were noted in all groups (treated and control). At necropsy, all organs and tissues appeared normal during the macroscopic observations. There were no treatment-related macroscopic, microscopic and ocular findings.

Activated partial prothrombin time (PTT) levels were increased outside the normal ranges found in this laboratory for males dosed at 500 and 1000 mg/kg bw/day (21% and 34% respectively). Fibrinogen levels were also increased (15%) in males dosed at 1000 mg/kg bw/day. Following a 2 week treatment free period PTT levels were within the normal ranges and fibrinogen levels were reduced, showing that recovery had occurred.

During week 4, non dose related increases in cholesterol were apparent in all treated females and in males dosed at 1000 mg/kg bw/day; however only two females dosed at 1000 mg/kg bw/d were outside references ranges. After a 2 week treatment free period group 4 animals showed cholesterol levels were within 10% of the control values.

Absolute and bodyweight related liver weights were statistically significantly higher in all treated males and female groups. This was however considered to be due to the control values being lower than expected for animals of this age and strain. After a 2 week treatment free period group 4 values for absolute and bodyweight related liver weights were within 10% of the controls. A statistically significant increase was apparent in absolute ovary weights for females dosed at 1000 mg/kg bw/day. This was considered to be unrelated to treatment as the group mean value was within the normal ranges found in this laboratory.

The NOAEL for males and females was determined as 1000 mg/kg bw/day, based on a marginal increase in female cholesterol levels which was considered unlikely to be of any toxicological significance. All effects were minor, reversible and not supported by corroborative changes.

Based on the results of this study, the test material is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.