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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 2015 - 27 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for read across to Tetrahydromyrcenol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approx. 10 weeks
- Weight at study initiation: Males: 335-402 g / Females: 230-291 g
- Fasting period before study: Only fasted overnight before blood sampling for haematology and blood
chemistry investigations
- Housing: Polycarbonate body with a stainless steel mesh lid (changed at appropriate intervals) used
during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Polypropylene cages
used during pairing.
- Diet: Ad libitum (but removed overnight before blood sampling for haematology and blood chemistry
investigations)
- Water (e.g. ad libitum): Ad libitum (but removed overnight during urine collection)
- Acclimation period: Six days before start of treatment

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 Certified powdered diet; Potable water from
the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate
intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Animal arrival: 24 June 2015
F0 necropsy: Toxicity phase 13 August 2015, Recovery phase 28 August 2015, Reproductive phase
19 to 24 August 2015

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
used as stabiliser
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): SDS VRF1 certified powdered diet
- Storage temperature of food: Ambient temperature (nominally 21°C) for eight days. Deep-frozen (nominally -20°C) for 22 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The dietary of administration was chosen to simulate a condition of potential human exposure.
- Concentration in vehicle: test material to corn oil ratio 5:1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For Week 1 and Final Week of treatment, representative samples of test diet were taken and submitted for analysis using chromatography. Each diet sample was sub-sampled (10 g ± 0.5 g) in duplicate and analysed in accordance with the analytical procedure. The analysed concentrations of 2,6-dimethyloct-7-en-2-ol in the test formulations analysed during the study, associated procedural recovery data and the deviation of mean results from nominal values were between -6% and +3.3% which is within applied limits of +10%/-15%. This confirms the accuracy of formulation. Procedural recovery values remained within 99.2% and 100.7%, confirming the continued accuracy of the method. The difference of individual values from the mean was <5% confirming the precision of analysis.
Details on mating procedure:
- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray or sperm in the vaginal smear referred to as Day 0 of gestation
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Toxicity phase males: Three weeks pre-pairing up to necropsy after minimum of five weeks.
Frequency of treatment:
Continuous
Duration of test:
6 weeks (5 weeks pre-mating / mating / gestation and 1 week postnatal)
Doses / concentrationsopen allclose all
Dose / conc.:
15 000 ppm
Remarks:
Corresponding actual intake
Week 1 to 3 prior to pairing: 954.5 mg/kg bw/day
Days 0-19 of gestation: 975.8 mg/kg bw/day
Days 1-6 of lactation: 1449.4 mg/kg bw/day
Males: 868.7 mg/kg bw/day
Dose / conc.:
5 000 ppm
Remarks:
Corresponding actual intake
Week 1 to 3 prior to pairing: 349.8 mg/kg bw/day
Days 0-19 of gestation: 342.7 mg/kg bw/day
Days 1-6 of lactation: 599.2 mg/kg bw/day
Males: 291.7 mg/kg bw/day
Dose / conc.:
1 500 ppm
Remarks:
Corresponding actual intake
Week 1 to 3 prior to pairing: 108 mg/kg/day
Days 0-19 of gestation: 102.3 mg/kg/day
Days 1-6 of lactation: 202.4 mg/kg/day
Males: 89 mg/kg bw/day
No. of animals per sex per dose:
10 females per dose group
5 males per dose group
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: The dietary levels of 1500, 5000 and 15000 ppm were selected based on the results of a 21-day preliminary study (Envigo Study Number: CIZ0001). In that study, administration of 2,6-dimethyloct-7-en-2-ol to CD rats by dietary administration for 21 days at dietary levels of 1500, 7500 or 15000 ppm was well tolerated. No animals died and the clinical condition of the animals was satisfactory.
- Rationale for animal assignment: random (Note: On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. The groups were adjusted to reduce inter-/intra-group variation.)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
Reproductive phase females:
- Before feeding on the day that treatment commenced, (Week 0) and weekly before pairing, Days 0, 6, 13 and 20 after mating, Day 1, 4, and 7 of lactation, On the day of necropsy.

OTHER EXAMINATIONS: During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumpt ion, ophthalmic examination, haematology (peripheral blood), blood chemistry, urinalysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.
Ovaries and uterine content:
Not studied
Fetal examinations:
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Statistics:
Not relevant
Indices:
- Post-implantation survival index (%) = Total number of offspring born / Total number of uterine implantation sites x 100
- Live birth index (%) = Number of live offspring on Day 1 after littering / Total number of offspring born x 100
- Viability index (%) = Number of live offspring on Day 7 / Number of liver offspring on Day 1 after littering x 100
Historical control data:
Not reported for offspring

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reproductive phase Females: body weight gain was significantly reduced from Day 13-20 of gestation in females treated at 5000 or 15000 ppm.
Lactation phase Females: Day 1-7: body weight gain was significantly reduced in females treated at 15000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During gestation, food intake in females treated at 15000 ppm food intake remained slightly low when compared with controls, from Day 0 to Day 3; thereafter food intake improved and was similar to that of controls. During lactation, food intake was slightly low in females treated at 5000 ppm with a markedly low food intake observed in females treated at 15000 ppm. This effect persisted throughout lactation (until Day 7).
Food efficiency:
not specified
Description (incidence and severity):
On occasions throughout the treatment period, animals treated at 15000 ppm were observed to consume more water than the animals treated at 0, 1500 or 5000 ppm. This effect is considered to be due to the palatability of the test material and not an adverse response to treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination revealed no findings that could be attributed to treatment with 2,6- dimethyloct-7-en-2-ol.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
None of the findings were conclusively linked to any of the treatment related microscopic pathology changes, and since all of the affected parameters evaluated during Week 2 of recovery showed recovery and, the survival, clinical condition and reproductive performance of the animals was unaffected by treatment, they are considered not to represent an adverse effect of treatment.

Females 5000 or 15000 ppm:
- Mean cell haemoglobin concentration was significantly increased (p<0.05 in both groups)
- Mean cell volume was significantly reduced in females treated at 15000 ppm
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
None of the findings were conclusively linked to any of the treatment related microscopic pathology changes, and since all of the affected parameters evaluated during Week 2 of recovery showed recovery and, the survival, clinical condition and reproductive performance of the animals was unaffected by treatment, they are considered not to represent an adverse effect of treatment.

Females 1500, 5000 or 15000 ppm:
- glucose concentration significantly reduced (p<0.05, p<0.01 and p<0.01 respectively)
- potassium concentration significantly reduced (5000 or 15000 ppm) (p<0.01 both)
- albumin/globulin ratio reduced (15000 ppm)
- cholesterol increased (15000 ppm)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
None of the findings were conclusively linked to any of the treatment related microscopic pathology changes, and since all of the affected parameters evaluated during Week 2 of recovery showed recovery and, the survival, clinical condition and reproductive performance of the animals was unaffected by treatment, they are considered not to represent an adverse effect of treatment.

Females 15000 ppm:
- increase in urinary volume (not significant)
- significant increase in total potassium (p<0.05)
- significant increase in total chloride levels (p<0.01)
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Females (1500, 5000 and 15000 ppm):
- Uterus, cervix, oviducts: absolute and adjusted combined weight showed a dose-proportional (but not statistically significant) reduction in weight (95%, 81% and 66% of control, respectively)
- Kidney: marginally low absolute and adjusted kidney weight (15000 ppm)
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with 2,6-dimethyloct-7-en-2-ol were seen in the female reproductive tract. All females receiving 15000 ppm were in diestrus, whereas Control females and females receiving 1500 or 5000 ppm were more often in proestrus or metoestrus. Uterine luminal dilatation was not evident in any of the females receiving 15000 ppm. The apparent difference in stage of oestrus is of uncertain significance. Note: This finding may be related to the test article related bodyweight effects seen in females of this dose group during treatment, but was considered to be of uncertain relationship to treatment since the efect was reversible and the oestrus cycles prior to pairing, mating performance and fertility of these females had been unaffected by treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
gestation
Effect level:
>= 975.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL = highest dose tested
Dose descriptor:
NOAEL
Remarks:
lactation
Effect level:
>= 1 449.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL = highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no difference in offspring body weight gain from Day 1-4 of age when compared with the control group. Growth thereafter (Day 4-7) was lower in male and female offspring from parents receiving 15000 ppm. This low growth of the offspring was considered not to represent an adverse effect of treatment since the survival, clinical condition and macropathology findings for the offspring were not affected by treatment. There was no effect of treatment in offspring from parents receiving 5000 or 1500 ppm.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Mortality was observed in litters of all test groups (including control), but this is not considered treatment related.
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
gestation
Effect level:
>= 975.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
received via dams
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested
Dose descriptor:
NOAEL
Remarks:
lactation
Effect level:
>= 1 449.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
received via dams
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Dihydromyrcenol did not show adverse developmental effects in an OECD TG 422 up to the maximum dose of 15000 ppm (equivalent to 975.8 mg/kg bw/day).
Executive summary:

In this endpoint the findings of the reproductive/developmental toxicity screening are reported as part of an OECD TG 422 oral toxicity study in rats following dietary administration of Dihydromyrcenol. A similarly constituted control group was assigned, and received the vehicle, powdered SDS VRF1 certified diet with corn oil, throughout the same relative treatment period. Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 7 of lactation. The F1 generation were killed on Day 7 of age, but received no direct administration of the substance; any exposure was in utero or via the milk. Males were treated daily for three weeks before pairing. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, ophthalmic examination, haematology (peripheral blood), blood chemistry, urinalysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio,bodyweightand macropathology for all offspring were also assessed.

Results

There were no adverse maternal effects observed attributed to treatment on: clinical condition, sensory reaction, grip strength, motor activity, ophthalmic examination and macroscopic pathology.  In addition oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival, sex ratio or external development were unaffected. In reproductive phase females, food intake was low in females treated at 15000 ppm throughout pre-pairing until Day 3 of gestation; thereafter food intake improved throughout the gestation phase. However, body weight gain was significantly reduced from Day 13-20 of gestation in females treated at 5000 or 15000 ppm. For females treated at 15000 ppm, this effect persisted throughout lactation (Day 1-7) when food consumption was low, which was likely responsible for the low growth of the offspring from Day 4 to Day 7 of age. For females treated at 5000 ppm, body weight gain during lactation was similar to the control group despite food intake being slightly low.

Other effects: Several minor differences in haematology, blood chemistry and urinalysis parameters were recorded; none were conclusively linked to any of the treatment related microscopic pathology and since the survival, clinical condition and reproductive performance of the animals was unaffected by treatment, they are considered not to represent an adverse effect of treatment.

In the female reproductive tract, all females receiving 15000 ppm were observed to be in diestrus. This was associated with a reduced incidence of luminal dilatation of the uterus, correlating with the stage of oestrous reported in the vagina.  This finding may be related to the test article related bodyweight effects seen in females of this dose group during treatment, but was considered to be of uncertain relationship to treatment since the oestrus cycles prior to pairing, mating performance and fertility of these females had been unaffected by treatment.

Developmental toxicity: Survival, litter size and sex ratio were not adversely affected by parental exposure. There was no difference in offspring body weight gain from Day 1-4 of age when compared with Controls; growth thereafter (Day 4-7) was lower in male and female offspring from parents receiving 15000 ppm. This low growth of the offspring was considered not to represent an adverse effect of treatment since the survival, clinical condition and macropathology findings for the offspring were not affected by reatment. There were no changes detected at examination of the offspring dying before or at scheduled termination which indication any effect of the test material.

Conclusion: the study indicates that there were no developmental adverse effects attributed to treatment with the substance by dietary administration. The NOAEL for developmental toxicity was therefore determined to be the highest dose tested of 15000 ppm, representing a mean achieved dose of 975.8 mg/kg bw/day via dams during gestation.