6,10-dimethylundeca-3,5,9-trien-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997-01-22 to 1997-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, according to OECD guideline 407

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,10-dimethylundeca-3,5,9-trien-2-one

EC Number:

205-457-1

EC Name:

6,10-dimethylundeca-3,5,9-trien-2-one

Cas Number:

141-10-6

Molecular formula:

C13H20O

IUPAC Name:

6,10-dimethylundeca-3,5,9-trien-2-one

Details on test material:

- Name of test material (as cited in study report): Ro 02-2438/000
- Physical state: clear amber liquid
- Lot/batch No.: 14106
- Expiration date of the lot/batch: 1997-10-14
- Storage condition of test material: under nitrogen at room temperature, in the dark

Test animals

Species:

rat

Strain:

other: HsdBrl:WH (Wistar Hannover)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Shaw's Farm, Blackthorn, Bicester, OX6 0TP, UK
- Age at study initiation: 3 to 4 weeks old
- Weight at study initiation: 131-154 g (males), 98-122 g (females)
- Housing: in groups of 6, in grid-bottomed stainless steel cages
- Diet (e.g. ad libitum): a pelleted rodent died, SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diet Services, Witham, UK), ad libitum
- Water (e.g. ad libitum): mains tap water, ad libitum
- Acclimation period: 11 days prior to the starting of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 37-55
- Air changes (per hr): air conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

maize oil

Remarks:

BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily for each dose level
- Storage temperature of food: room temperature

VEHICLE
- Concentration in vehicle: 0, 10, 50, 200 mg/mL pseudoionone
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A standard analytical method by Gas Liquid Chromatography (GLC) was used to analyse the Pseudoionone in the Maize oil formulations. Except of 0.9 area-%, the Maize oil was not detected by the GLC; the detected Maize oil peaks were neglected. To determine the content of Pseudoionon in the Maize oil, Octadecane (Fluka 74705) was used as Internal Standard.

Duration of treatment / exposure:

28-days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 250, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

control: 12
50 mg/kg bw/d: 6
250 mg/kg bw/d: 6
1000 mg/kg bw/d: 12

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly thereafter up to and including the day of necropsy

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: Yes

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from the first 6 males and 6 females in each group during week 4 of treatment and from remaining animals towards the end of week 2 of the treatment-free period
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: first 6 males and 6 females in each group and then the remaining animals towards week 2 of the treatment-free period
- Following parameters were examined: cell morphology, erythrocyte count, haemoglobin concentration, leucocyte differential count, mean cell haemoglobing, mean cell haemoglobin concentration, mean cell volume, packed cell volume, platelet count, total leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from the first 6 males and 6 females in each group during week 4 of treatment and from remaining animals towards the end of week 2 of the treatment-free period
- Animals fasted: Yes
- How many animals: irst 6 males and 6 females in each group and then the remaining animals towards week 2 of the treatment-free period
- Following parameters were examined: A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, calcium, chloride, cholesterol, creatinine, gamma glutamyl transpeptidase, glucose, globulin, inorganic phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: from the first 6 males and 6 females in each group during week 4 of treatment and from remaining animals towards the end of week 2 of the treatment-free period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Following parameters were examined: bilirubin, blood pigments, glucose, ketones, leucocytes, microscopic examination of sediment deposit, nitrites, pH, protein, specific gravity, urobilinogen, volume

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The study was designed with 4 groups for each sex. Control and high dose groups comprised 12 males and 12 females. Low and intermediate dose groups comprised 6 males and 6 females. The observations analysed were bodyweights pre-dose (week 1), bodyweight gains over the interval weeks 1 to 5 and 5 to 7, food consumption over the interval weeks 1 to 4 and 5 to 6 and absolute and bodyweight related organ weights.
Sexes were analysed separately.
The data were subjected to an analysis of variance (ANOVA). The absolute residuals from this analysis were further subject to ANOVA to assess whether there were any between group differences in variances (Levene's test). If Levene's test indicated there was no evidence of inequality of variance (i.e. p>0.01) then pairwise tests of all treated groups versus control were performed using Williams' test. For the comparison of the high dose against the control, a two-sided test was performed. Statistical significance was declared at the two-sided 5 % level and also noted at the 1 % and 0.1 % levels. If the comparison of the high dose with the control was not significant then testing was stopped, otherwise the process continued (now using one-sided tests) with the low dose.
If Levene's test indicated that there was evidence of differences between group variances, then a non-parametric approach to the data analysis was used. This involved Shirley's non parametric equivalent of the Williams' test. The version of Shirley's test used incorporated the modification to the ranking procedure suggested by Williams (1986). The testing scheme with Shirley's test was the same as that described for Williams' test above, two-sided tests for the high dose, one-sided tests otherwise and terminating the testing process when a non-significant result was obtained. Note that no adjustment for unequal sample sizes was made as described in Williams (1972) since for all variables analysed the control group was of a similar or equal size to the treated group

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was recorded on a number of occasions pre- and post dosing. These findings were considered to be treatment related. There were no mortalities during this study.

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation was recorded on a number of occasions pre- and post dosing. These findings were considered to be treatment related. There were no mortalities during this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration of Pseudoionone to males at 1000 mg/kg bw/d was associated with a marked, statistically significant, reduction in bodyweight gain to the end of the treatment period.

Food efficiency:

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight increase in the group mean RBC and packed cell volume (PCV) value for females dosed at 1000 mg/kg bw/d. There was a small, statistically significant increase in activated partial thromboplastin (PTT) times in males dosed 250 and 1000 mg/kg bw/d.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Small increases in alanine aminotransferase (ALT): males at 250 and 1000 mg/kg bw/d.An increase in gamma glutamyl transpeptidase (GGT): both sexes at 1000 mg/kg bw/d. Slight increases in total protein, globulin and cholesterol: females at 1000 mg/kg bw/d.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and bodyweight related liver and kidney weights were increased for males and females given 1000 mg/kg bw/d at the end of the treatment period. Increased bodyweight related kidney weights were also observed in males given 250 mg/kg bw/d.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Salivation was recorded on a number of occasions pre- and post dosing. These findings were considered to be treatment related. There were no mortalities during this study.

BODY WEIGHT AND WEIGHT GAIN
Administration of Pseudoionone to males at 1000 mg/kg bw/d was associated with a marked, statistically significant, reduction in bodyweight gain to the end of the treatment period.

FOOD EFFICIENCY
There were no treatment-related effects on food consumption.

HAEMATOLOGY
A slight increase in the group mean red blood cell (RBC) and packed cell volume (PCV) value for females dosed at 1000 mg/kg bw/d. There was a small, statistically significant, increase in activated partial thromboplastin (PTT) times in males dosed 250 and 1000 mg/kg bw/d.

CLINICAL CHEMISTRY
Small, statistically significant increases in alanine aminotransferase (ALT) levels were observed in males dosed at 250 and 1000 mg/kg bw/d, and females dosed at 1000 mg/kg bw/day. An increase in gamma glutamyl transpeptidase (GGT) levels in both sexes was observed at 1000 mg/kg bw/d.
Slight increases in total protein (T. Prot), globulin (Glob) and cholesterol (Chol) levels were observed in females dosed at 1000 mg/kg bw/d. Triglyceride (Trigs) levels were reduced in males at this dose.

URINALYSIS
No effects related to the treatment

ORGAN WEIGHTS
Absolute and bodyweight related liver and kidney weights were increased for males and females given 1000 mg/kg bw/d at the end of the treatment period. Increased bodyweight related kidney weights were also observed in males given 250 mg/kg bw/d.

GROSS PATHOLOGY
No treatment-related abnormalities were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related effects were observed

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral (gavage) administration of Pseudoionone to HsdBrl:WH (Wistar Hannover) rat for 28 days at a dose of 1000 mg/kg bw/d was associated with the following findings: pre- and post-dose salivation (intermittent), a reduction in bodyweight gain in males, an increase in liver and kidney weights and a few minor changes in haematology and blood chemistry parameters.
The majority of clinical chemistry findings, although statistically significantly different from the comparable control values, were within the ranges of historical background data quoted for control animals. In the absence of any corroborative histopathological changes these findings were considered not sufficient to be of toxicological significance.
Administration of 250 mg/kg bw/d was also associated with post-dose salivation on a number of occasions and slight increase in kidney weights of males.
There were no observations persisting until the end of, or occuring during, the treatment-free period.
In view of the in-life, bodyweight, haematology, biochemistry and organ weight findings in rats given Pseudoionone by oral gavage at doses of 250 or 1000 mg/kg bw/d, this study established the NOEL (no-observed-effect-level) to be 50 mg/kg bw/d.

Executive summary:

In a GLP and the OECD guideline 407 conform study, the purpose was to assess the toxicity of the test article, Pseudoionone, when administrated orally, by gavage, to the rat once daily for 28 days and to assess the persistence or delayed occurrence of any observed effects over a 14 day treatment-free period.

Seventy two rats of the HsdBrl:WH (Wistar Hannover) strain were divided into 4 groups. Three groups received the test article,

Pseudoionone by oral (gavage) administration, at dose levels of 50, 250 or 1000 mg/kg bw/d, daily for 28 days. A fourth group received the vehicle (maize oil) alone, and served as controls. The control and highest dose groups comprised of 12 males and 12 females. The low and mid dose groups comprised 6 males and 6 females. The 6 male and 6 female animals with the highest identification numbers in the control and high dose groups were maintained, undosed, for a treatment-free period of 14 days after the full dosing period. Animals were observed daily. Bodyweights were twice weekly and food consumption was recorded weekly throughout the treatment period. Blood and urine samples were obtained during week 4 of treatment and also towards the end of the treatment-free period.

At the end of the treatment and treatment-free periods designated animals were sacrificed by CO₂ asphyxiation and subjected to a full mecroscopic examination. Designated organs were weighed and selected tissues were examined microscopically.

There were no mortalities during the study.

Salivation was noted on a number of occasions pre- and post-dosing in males and females given 250 and 1000 mg/kg bw/d. This finding was considered to be related to treatment.

At the end of the treatment period the bodyweights and bodyweight gains of males treated with Pseudoionone, were markedly reduced at the top dose of 1000 mg/kg bw/d. Bodyweights of females were unaffected by treatment. The weight gained by males maintained for the treatment-free period was comparable between groups. There were no obvious treatment related effects on food consumption.

At the end of the treatment period, haematology investigations showed small increases in red blood cell count and packed cell volume in females dosed at 1000 mg/kg bw/d. A minor increase in packed cell volume was still evident after the treatment-free period but red blood cell counts were similar to controls. Activated partial thromboplastin time in males was increased at doses of 250 and 1000 mg/kg bw/d.

A number of enymatic changes were also observed during week 4 of treatment, which fell within the ranges of background variation seen in untreated animals of this age and strain.

Macroscopic examination at necropsy revealed no obvious treatment related abnormalities.

Absolute and bodyweight related liver and kidney weights were increased for males and females given 1000 mg/kg bw/d at the end of the treatment period. Increased bodyweight related kidney weights were also observed for males given 250 mg/kg bw/d. Liver and kidney weights of animals given 1000 mg/kg bw/d were similar to controls at the end of the treatment-free period.

Histopathological examination revealed no abnormalities which could be related to treatment.

In view of the in-life, bodyweight, haematology, biochemistry and organ weight findings in rats given Pseudoionone by oral gavage at doses of 250 or 1000 mg/kg bw/d, this study established the NOEL (no-observed-effect-level) to be 50 mg/kg bw/d.