6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Test Item 205601/B
Identification Methyl Pamplemousse
Appearance Colourless to pale yellow liquid
Batch PE00170602
Purity/Composition See Certificate of Analysis
Test item storage At room temperature protected from light
Stable under storage conditions until: 11 March 2017 (expiry date).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The accuracy of diet preparations is considered acceptable if the mean measured
concentrations are 80-120% of the target concentration. Homogeneity is demonstrated if the
coefficient of variation is ≤ 10%.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500
to 15000 ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for 1 day at
room temperature (Test Facility Study No.507072 (method development and validation
study)).
In addition, random back-up diet samples will be taken from diets used for analytical
sampling and stored at ≤-15ºC for possible future analysis. Any remaining samples at
finalization of the study report will be discarded.
If the analytical determinations are not performed on the day of preparation, the samples will
be stored at ≤-15ºC.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat: Crl:WI(Han) (outbred, SPF-Quality). Untreated, nulliparous, non-pregnant females and untreated
males will be used at the initiation of the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

This species and strain of rat has been recognized as appropriate for general and reproduction toxic ity studies. Charles River Den Bosch has general and reproduction/developmental historical data in
this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
48 females and 40 males.
Acclimatisation: At least 5 days prior to start of pretest (females) or treatment (males).
Environmental controls for the animal room are set to maintain 18 to 24°C, a relative humidity of 40
to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle may
be interrupted for study related activities. Any variations to these conditions will be evaluated and
maintained in the raw data.

Route of administration:

oral: feed

Details on route of administration:

Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The test item will be mixed without the use of a vehicle, directly with the required amount of powder feed. No correction will be made for the purity/composition of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses will be conducted on a single occasion during the treatment phase on samples as specified below, according to a validated method (Test Facility Study No. 507072):
The accuracy of diet preparations is considered acceptable if the mean measured concentrations are 80-120% of the target concentration. Homogeneity is demonstrated if the coefficient of variation is ≤10%.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15000ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for 1 day at room temperature (Test Facility Study No.507072 (method development and validation study)).
In addition, random back-up diet samples will be taken from diets used for analytical sampling and stored at ≤-15ºC for possible future analysis. Any remaining samples at finalization of the study report will be discarded.
If the analytical determinations are not performed on the day of preparation, the samples will be stored at ≤-15ºC.

Duration of treatment / exposure:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.

Frequency of treatment:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.

Dose / conc.:

1 500 ppm

Remarks:

1500 ppm in the diet corresponded to mean daily test item intake levels of 113 mg/kg bw/day in males, and 204 mg/kg bw/day in females.

Dose / conc.:

5 000 ppm

Remarks:

5000 ppm in the diet corresponded to mean daily test item intake levels of 386 mg/kg bw/day in males, and 615 mg/kg bw/day in females.

Dose / conc.:

15 000 ppm

Remarks:

15,000 ppm in the diet corresponded to mean daily test item intake levels of 1102 mg/kg bw/day in males, and 1458 mg/kg bw/day in females.

No. of animals per sex per dose:

48 females and 40 males.

Control animals:

yes, concurrent no treatment

Details on study design:

Method Oral, by inclusion in the diet.
Frequency Ad libitum.
Dietary Inclusion Levels The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.
Exposure period Males will receive test diet for a minimum of 28 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period.
Females will receive test diet for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least thirteen days after delivery, up to and including the day before scheduled necropsy.

Positive control:

NA

Observations and examinations performed and frequency:

All animals will be deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water will be available.
All animals surviving to the end of the observation period and all moribund animals will be deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded.
Necropsy will be conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which deliver PND 14-16.
Females which fail to deliver Post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss Within 24 hours of litter loss.
Spontaneous deaths4 As soon as possible after death and always within 24 hours.
Euthanized in extremis6 When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.
The numbers of former implantation sites will be recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
For females which fail to deliver a complete nest, uterine contents (i.e. any fetuses, placenta and implantation sites) will be fixed (if applicable), but will not be examined histopathologically in first instance.
Samples of the tissues and organs specified on the next page will be collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Sacrifice and pathology:

Pups, younger than 7 days will be euthanized by decapitation.
All remaining pups (PND 7-15) will be sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater,
The Netherlands) by intraperitoneal (ip) injection.
Pups found dead during the weekend will be fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) if not necropsied on the same day.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) will be collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter will be collected into one serum tube. For further details, see section 3.10: Blood sampling for thyroid hormone analysis.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples will be collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) will be drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood will be collected into serum tubes.

Statistics:

The following statistical methods will be used to analyse the data:
• If the variables can be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one
t-test) based on a pooled variance estimate will be applied for the
comparison of the treated groups and the control groups for each sex.
• The Steel-test (Ref. 3; many-to-one rank test) will be applied if the data cannot be assumed to follow
a normal distribution.
• The Fisher Exact-test (Ref. 4) will be applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) will be applied to motor activity data to
determine intergroup differences. In case intergroup differences are seen, the Wilcoxon test (Ref. 6)
will be applied to compare the treated groups to the control group.
All tests will be two-sided and in all cases p < 0.05 will be accepted as the lowest level of significance.
Group means will be calculated for continuous data and medians will be calculated for discrete data
(scores) in the summary tables. Test statistics will be calculated on the basis of exact values for
means and pooled variances. Individual values, means and standard deviations may be rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter,
yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, body weight gain and body weights of males were statistically significantly lower from Day 1 of the mating period. Mean body weights were 8% lower compared to
controls at the end of treatment. Females at 15,000 ppm also showed slightly lower body weight gain being statistically significantly at Days 7-14 of the post-coitum period.
Thereafter, their weight gain tended to return to control values. Mean body weights were less than 5% lower compared to controls.
It should be remarked that the control group means at post-coitum Days 17 and 20 were somewhat depressed by the lower weight gain of two females (nos. 44 and 47) which had only one implantation site and delivered no offspring. Thus, the difference in weight gain between 15,000 ppm females delivering offspring and controls was somewhat larger than indicated by the group means in the summary table.
Body weight and body weight gain of 15,000 ppm females during the pre-mating and lactation periods were considered not to be affected by treatment.
Other statistically significant variations in body weight gain at 1500 and 5000 ppm occurred in the absence of a dose-related response, and were therefore considered to be unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 5000 and 15,000 ppm, food intake of females was lower during the post-coitum and lactation period, up to 20 or 40% lower than controls at 5000 and 15,000 ppm respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Based on palatability issues of the test substance.

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, statistically significantly higher platelet counts and delayed prothrombin time (PT) were recorded for females. Means remained within the range considered normal for rats of this age and strain7
.
The statistically significantly reduced prothrombin time in males at 5000 and 15,000 ppm was not considered to be related to treatment due to the lack of a dose-related response.

Description (incidence and severity):

At 15,000 ppm, lower glucose and higher chloride were recorded for females. Means remained within the range considered normal for rats of this age and strain. For glucose, the control mean was relatively high
At 1500 and 5000 ppm, clinical biochemistry parameters were similar to control means.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were not considered to be affected by treatment.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, a statistically significantly lower hind limb grip strength was noted in males (relative difference from controls: aproximately 40%). Mean hind limb grip strength was below the normal range for rats of this strain and age.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were noted in the weights (absolute and relative to body weight) of the liver of males at 15,000 ppm and in the kidneys of males at 5000 and 15,000 ppm.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the spleen (females), a minor increase in incidence and severity (up to slight degree) of hemosiderin pigmentation was recorded at 5000 and 15,000 ppm. The minimal degree of
pigmentation recorded in some females of the control and 1500 ppm groups was within background pathology for female rats of this age and strain.
In the liver (both sexes), hepatocellular hypertrophy of the centrilobular area was recorded at a minimal degree in two males and two females at 15,000 ppm.
In the kidneys (males), a combination of increased incidence and severity (up to marked degree) of hyaline droplet accumulation, tubular basophilia and granular casts was recorded
at 5000 and 15,000 ppm. At 1500 ppm, treatment-related renal findings were limited to an increased incidence of hyaline droplet accumulation (up to slight degree).
The remainder of the recorded microscopic findings were within the range of background
pathology encountered in rats of this age and strain. There was no test item-related alteration
in the prevalence, severity, or histologic character of those incidental tissue alterations

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Parental NOAEL: 1500 ppm1, based on renal changes in males at 5000 ppm (combination of hyaline droplet accumulation, tubular basophilia and granular casts, and associated higher renal weight). Given that the renal changes are considered to represent a male rat specific response, a NOAEL of 5000 ppm3 may be considered for risk assessment purposes based on the significantly lower food intake of females at 15,000 ppm.

Executive summary:

Parental NOAEL: 5000 ppm (386 mg/kg bw/day).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available