2,6-dimethyloct-7-en-2-yl acetate

Endpoint summary

Description of key information

Dihydromyrcenyl acetate has a NOAEL >=868.7 mg/kg bw based on read across from Dihydromyrcenol, which was tested via feed in an OECD TG 422.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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:

The information is used for read across to Dihydromyrcenyl Acetate.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 March 1996

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies.
The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system 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

Route of administration:

oral: feed

Details on route of administration:

The oral route of administration was chosen to present conservative exposure in the test animal.

Details on oral 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.

Duration of treatment / exposure:

Females:
- Toxicity phase females: At least five weeks.
- Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
- Recovery groups: At least five weeks followed by a minimum 14 days recovery.
Males
- Toxicity phase males: Three weeks pre-pairing up to necropsy after minimum of five weeks.
- Recovery phase males: Three weeks pre-pairing up to necropsy after minimum of five weeks followed by a minimum 14 days recovery.

Frequency of treatment:

continuous dietary exposure

Dose / conc.:

15 000 ppm

Remarks:

Equivalent to an actual received dose of 868.7 mg/kg bw/day (males) or 892.7 mg/kg bw/day (females)

Dose / conc.:

5 000 ppm

Remarks:

Equivalent to an actual received dose of 291.7 mg/kg bw/day (males) or 325.2 mg/kg bw/day (females)

Dose / conc.:

1 500 ppm

Remarks:

Equivalent to an actual received dose of 89 mg/kg bw/day (males) or 97.2 mg/kg bw/day (females)

No. of animals per sex per dose:

Group 1: Control group and high-dose group
- Reproductive phase: 10 females
- Toxicity phase: 5 males, 5 females
- Recovery phase: 5 males, 5 females

Group 2: Low and mid-dose group
- Reproductive phase: 10 females
- Toxicity phase: 10 males, 5 females

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.)
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and week 6
- Dose groups that were examined: Toxicity, recovery and spare animals as well as all toxicity phase females and the first 5 toxicity phase males of groups 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 6, and week 2 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: Five lowest numbered surviving toxicity phase males and females in each group, all recovery phase animals
- Parameters:

Activated partial thromboplastin time (APTT)
Prothrombin time (PT)
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Reticulocyte % (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV) # females only
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt) # males only
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Reticulocyte % (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV) # females only
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt) # males only
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6, and week 2 of recovery
- Animals fasted: Yes
- How many animals: Five lowest numbered surviving toxicity phase males and females in each group, all recovery phase animals
- Parameters:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol) # females only
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6, and week 2 of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters using manual methods:
Clarity and Colour (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter # males only
- Parameters using Multistix reagent strips interpreted using the Clinitek®500 instrument:
Ketones (Keto)
Bile pigments (Bili)
Urobilinogen (Urob)
Blood pigments (UBld)
- Parameters using a Roche P Modular Analyser:
Protein (T-Prot)
Creatinine (T-Creat)
Glucose (T-Gluc)
Sodium (T-Na)
Potassium (T-K)
Chloride (T-Cl)
- Parameters using microscopic evaluation:
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during each week of treatment and recovery and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation
- Dose groups that were examined: all recovery animals in Group 1 and 4 and five lowest numbered surviving toxicity phase males and females in Group 2 and 3 during Week 6 of treatment.
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Oestrous cycles - reproductive phase females
Mating
Parturition observations and gestation length

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table below)
HISTOPATHOLOGY: Yes (see table below)

Tissue and regions examined:

Abnormalities: Fix Histology Pathology
Adrenals: Weigh Fix Histology Pathology
Brain: Weigh Fix Histology Pathology
Caecum: Fix Histology Pathology
Colon: Fix Histology Pathology
Duodenum: Fix Histology Pathology
Epididymides: Weigh Fix Histology Pathology
Eyes: Fix Histology Pathology
Heart: Weigh Fix Histology Pathology
Ileum: Fix Histology Pathology
Jejunum: Fix Histology Pathology
Kidneys: Weigh Fix Histology Pathology
Liver: Weigh Fix Histology Pathology
Lungs: Fix Histology Pathology
Lymph nodes: Fix Histology Pathology
Ovaries: Weigh Fix Histology Pathology
Peyer’s Patch: Fix Histology Pathology
Prostate: Weigh Fix Histology Pathology
Sciatic nerve: Fix Histology Pathology
Seminal vesicles: Weigh Fix Histology Pathology
Skeletal muscle: Fix Histology Pathology
Skin with mammary glands: Fix Histology Pathology
Spinal cord: Fix Histology Pathology
Spleen: Weigh Fix Histology Pathology
Sternum (with marrow): Fix Histology Pathology
Stomach: Fix Histology Pathology
Testes: Weigh Fix Histology Pathology
Thymus Weigh Fix Histology Pathology
Thyroid (with parathyroid) Fix Histology Pathology
Trachea: Fix Histology Pathology
Urinary bladder: Fix Histology Pathology
Uterus: Weigh Fix Histology Pathology
Vagina: Fix Histology Pathology

Pathology procedures for remaining toxicity males and reproductive phase females
Abnormalities: Organs weighed, samples fixed or sections examined microscopically.
Epididymides: Organs weighed, samples fixed or sections examined microscopically.
Ovaries: Organs weighed, samples fixed or sections examined microscopically.
Prostate: Organs weighed, samples fixed or sections examined microscopically.
Seminal vesicles: Organs weighed, samples fixed or sections examined microscopically.
Testes: Organs weighed, samples fixed or sections examined microscopically.
Uterus: Organs weighed, samples fixed or sections examined microscopically.
Vagina: Organs weighed, samples fixed or sections examined microscopically.

Other examinations:

Offspring: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following sequence of statistical tests were used for grip strength, motor activity, body weight, food consumption, clinical pathology, litter size and survival indices, organ weight data:
- Bartlett's test for variance homogeneity (Bartlett 1937), followed by
Parametric analysis:
- Analysis of variance for any group differences
- Inter group comparisons using t-tests, error mean square from one-way analysis of variance
- F1 approximate test
- Williams' test for a monotonic trend
- Dunnett's test (Dunnett 1955, 1964) for non-monotonic trend
Non-parametric analysis
- Logarithmic and square-root transformations
- Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) for group differences
- Inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945)
- H1 approximate test for monotonicity of dose-response
- Shirley's test for a monotonic trend
- Steel's test (Steel 1959) for non-monotonic trend

For grip strength, motor activity, clinical pathology, litter size and survival indices:
- Fisher’s Exact tests (Fisher 1973)
- Pairwise comparisons for treatment groups against control group
- Generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991) for sex ratio
- Treatment group comparison to control using a Wald chi-square test
- Exact two-tailed Linear-by-linear test (Cytel 1995) for gestation length (asymptotic version used if needed)

For organ weight data:
- Analysis of covariance (Angervall and Carlstrom, 1963) for parametric methods

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs observed, throughout the study, that were considered to be attributable to treatment with 2,6-dimethyloct-7-en-2-ol.

Mortality:

no mortality observed

Description (incidence):

No mortalities occured related to treatment with 2,6-dimethyloct-7-en-2-ol.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
Unaffected by treatment

Females:
High dietary level group (15000ppm)
Week 1 treatment: 9/20 animals experienced weight loss
Week 2-5 treatment: weight gain restored to control level
Week 1 recovery: significant more weight gain when compared with controls
Week 2 recovery: weight gain restored to control level

Reproductive phase Females:
Day 13-20: 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.

Offspring:
Day 1-4: No effect
Day 4-7: reduced growth in male and female offspring (parents received 15000ppm)

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).

Overall mean achieved dosages for the period Week 1 to 6 of treatment (toxicity phase and recovery phase animals) were 89, 291.7 and 868.7 mg/kg/day for males receiving 1500,
5000 and 15000 ppm, respectively, and 97.2, 325.2 and 892.7 mg/kg/day for females receiving 1500, 5000 and 15000 ppm, respectively.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Observed by visual inspection
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, in Week 6 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):

Week 6, minor observations:

Males 15000 ppm:
- Significant increases in mean cell haemoglobin (p<0.05) and consequently mean cell haemoglobin concentration (p<0.01);
- Significant reduction in platelet counts (p<0.05)

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.

During Week 2 of recovery (examined only platelet counts and cell volume):
- Males 15000 ppm: evidence of recovery in platelet counts
- Females 15000 ppm: evidence of recovery in mean cell volume

None of these 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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Week 6 of treatment, minor observations:

Males 15000 ppm:
- glucose concentration significantly reduced (p<0.05),
- chloride concentration significantly reduced (p<0.05)
- albumin/globulin ratio significantly reduced (p<0.01).

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).

Bile acids were markedly reduced in males treated at 5000 or 15000 ppm and in females treated at 1500 or 5000 ppm however review of the individual data revealed that these data were variable across all study groups and consequently this was not considered to be a response of treatment with 2,6-dimethyloct-7-en-2-ol.

Recovery
Total protein, albumin concentration and albumin/globulin ratio was investigated in both males and females previously treated at 15000 ppm during Week 2 of recovery; in addition cholesterol was also investigated in females. All parameters showed evidence of recovery in both males and females previously treated at 15000 ppm, when compared with controls.

None of these 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.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Week 6, minor observations:
Males 15000ppm:
- low pH (not significant)
- significantly increased specific gravity (p<0.05)

Females 15000ppm
- increase in urinary volume (not significant)
- significant increase in total potassium (p<0.05)
- significant increase in total chloride levels (p<0.01)

Recovery week 2
- specific gravity levels were investigated (males only) demonstrating recovery in this parameter with values similar in males previously treated at 15000 ppm when compared with controls.

None of these 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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Motor activity assessments during Week 6 of treatment, did not reveal any response to treatment in animals receiving 2,6-dimethyloct-7-en-2-ol.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Week 6:
males (15000ppm)
- Liver: Absulute and reletive liver weight were increased
females (1500, 5000 and 15000ppm)
- 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 (15000ppm)

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examinations conducted at scheduled termination, following six weeks of treatment, two weeks of recovery or on Day 7 of lactation revealed no findings that could be attributed to treatment.

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 kidneys, liver and female reproductive tract.

Kidneys:
Treatment males:
- Hyaline droplets in the cortical tubular epithelium were evident in the kidneys from all males treated at 15000 ppm examined histopathologically and one male treated at 5000 ppm. In the most severely affected animal this change was accompanied by slight multifocal cortical tubular basophilia and minimal granular casts.
Recovery males:
- Following two weeks without treatment, hyaline droplets in the cortical tubular epithelium were no longer evident in the kidneys of males previously receiving 15000 ppm, indicating a degree of recovery from this change. However, granular casts were still evident in one male previously treated at15000 ppm and a higher incidence and severity of cortical tubular basophilia was seen in this group compared to Controls.

Note: The accumulation of hyaline droplets in the cortical tubular epithelium of the kidneys, associated with granular casts and tubular basophilia is consistent with the finding of alpha 2u-globulin nephropathy (Frazier et al., 2012). This is a male rat specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein. There was evidence of recovery from this change following 2 weeks respite from treatment.

Liver
Treatment males:
- Portal pigment was seen in the bile ducts of three males receiving 15000 ppm. These consisted of deposits of brown pigment, predominantly located in the lumen of smaller bile ductules. Application of special stains to additional sections of liver showed this pigment to be Schmorl’s, Perls’ and Fouchet’s negative.
Recovery males:
- Following two weeks without treatment, portal pigment was seen in the bile ducts of three males previously receiving 15000 ppm, and in one animal the severity was greater than seen at the end of the treatment period.

Note: In the absence of any effect on the survival or reproductive performance of the animals and, no indication of impaired liver function from the blood chemistry analyses, these findings were considered not to represent an adverse effect of treatment.

Female reproductive tract
Treatment females:
- 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.
Recovery females:
- Following a two week recovery period, the changes observed in the female reproductive tract at the end of the treatment period were no longer apparent.

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 oestrus cycles prior to pairing, mating performance and fertility of these females had been unaffected by treatment.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 868.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: NOAEL = highest dose tested

Remarks on result:

other: The alpha 2u-globulin nephropathy is a male rat specific finding associated and therfore considered not relevant for the NOAEL setting. The histopathologic findings for the liver are considered not to be an adverse effect of treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 892.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested

Key result

Critical effects observed:

no

Overall remarks to study effects observed:

- Minor treatment related effects were observed for body weight, histopathology, blood chemistry and urine analysis. However, as the animals showed to recover from these findings and neither the survival nor the clinical condition were affected by treatment, they are considered not to represent an adverse effect of the treatment.

- The histological changes observed in the liver were not considered to be adverse, as no effect on the survival or reproductive performance of the animals was observed and no indication of impaired liver function could be seen from the blood chemistry analyses.

- The alpha 2u-globulin nephropathy observed in the kidneys of the male rat is a male-rat-specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein. Also the animals showed to recover from this effect.

Conclusions:

Under the conditions of this study, the NOAEL for systemic toxicity was determined to be >=15000 ppm, representing a mean achieved dose of 868.7 mg/kg/day for both males and females using the lower value of the two.

Executive summary:

The repeated dose toxicity of Dihydromyrcenol is assessed in an OECD TG 422 dietary toxicity study for at least five weeks. The fertility and developmental toxic effects are presented in the reproductive toxicity section. An additional recovery group was also included to assess for reversibility, persistence or delayed occurrence of systemic effects for at least 14 days post treatment. A similarly constituted control group was assigned to each phase, and received powdered SDS VRF1 Certified diet (with corn oil) throughout the same relative treatment period. The following parameters were studied: mortality, 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.

Results

Clinical signs: Dietary administration of 2,6-dimethyloct-7-en-2-ol at levels of 1500, 5000 and 15000 ppm was well tolerated with no mortalities related to treatment. Overall mean achieved dosages for the period Week 1 to 6 of treatment (toxicity phase and recovery phase animals) were 89, 291.7 and 868.7 mg/kg bw/day for males, and 97.2, 325.2 and 892.7 mg/kg bw/day for females, respectively. There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, ophthalmic examination and macroscopic pathology.

Other effects: Minor treatment related effects were observed in the dose groups for body weight, histopathology, blood chemistry and urine analysis. However, as these showed recovery and neither the survival nor the clinical condition were affected by treatment, they are considered not to represent an adverse effect of the treatment. The histological changes (portal pigment) observed in the liver of three male rats of the high dose group, were also not considered adverse, as no effect on the survival or reproductive performance of the animals was observed and no indication of impaired liver function could be seen from the blood chemistry analyses. Evidence of alpha 2u-globulin nephropathy was found in the kidneys of the male rat (5000 and 15000 ppm), this is a male-rat-specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein.

Under the conditions of this study, the NOAEL for systemic toxicity was determined to be >=15000 ppm, representing a mean achieved dose of 868.7 mg/kg/day for males and  females using the lowest dose of males for which human related effects were seen.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The information is based on read across from Dihydromyrcenol. The read across rationale is presented in the Repeated dose Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs observed, throughout the study, that were considered to be attributable to treatment with 2,6-dimethyloct-7-en-2-ol.

Mortality:

no mortality observed

Description (incidence):

No mortalities occured related to treatment with 2,6-dimethyloct-7-en-2-ol.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
Unaffected by treatment

Females:
High dietary level group (15000ppm)
Week 1 treatment: 9/20 animals experienced weight loss
Week 2-5 treatment: weight gain restored to control level
Week 1 recovery: significant more weight gain when compared with controls
Week 2 recovery: weight gain restored to control level

Reproductive phase Females:
Day 13-20: 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.

Offspring:
Day 1-4: No effect
Day 4-7: reduced growth in male and female offspring (parents received 15000ppm)

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).

Overall mean achieved dosages for the period Week 1 to 6 of treatment (toxicity phase and recovery phase animals) were 89, 291.7 and 868.7 mg/kg/day for males receiving 1500,
5000 and 15000 ppm, respectively, and 97.2, 325.2 and 892.7 mg/kg/day for females receiving 1500, 5000 and 15000 ppm, respectively.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Observed by visual inspection
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, in Week 6 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):

Week 6, minor observations:

Males 15000 ppm:
- Significant increases in mean cell haemoglobin (p<0.05) and consequently mean cell haemoglobin concentration (p<0.01);
- Significant reduction in platelet counts (p<0.05)

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.

During Week 2 of recovery (examined only platelet counts and cell volume):
- Males 15000 ppm: evidence of recovery in platelet counts
- Females 15000 ppm: evidence of recovery in mean cell volume

None of these 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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Week 6 of treatment, minor observations:

Males 15000 ppm:
- glucose concentration significantly reduced (p<0.05),
- chloride concentration significantly reduced (p<0.05)
- albumin/globulin ratio significantly reduced (p<0.01).

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).

Bile acids were markedly reduced in males treated at 5000 or 15000 ppm and in females treated at 1500 or 5000 ppm however review of the individual data revealed that these data were variable across all study groups and consequently this was not considered to be a response of treatment with 2,6-dimethyloct-7-en-2-ol.

Recovery
Total protein, albumin concentration and albumin/globulin ratio was investigated in both males and females previously treated at 15000 ppm during Week 2 of recovery; in addition cholesterol was also investigated in females. All parameters showed evidence of recovery in both males and females previously treated at 15000 ppm, when compared with controls.

None of these 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.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Week 6, minor observations:
Males 15000ppm:
- low pH (not significant)
- significantly increased specific gravity (p<0.05)

Females 15000ppm
- increase in urinary volume (not significant)
- significant increase in total potassium (p<0.05)
- significant increase in total chloride levels (p<0.01)

Recovery week 2
- specific gravity levels were investigated (males only) demonstrating recovery in this parameter with values similar in males previously treated at 15000 ppm when compared with controls.

None of these 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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Motor activity assessments during Week 6 of treatment, did not reveal any response to treatment in animals receiving 2,6-dimethyloct-7-en-2-ol.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Week 6:
males (15000ppm)
- Liver: Absulute and reletive liver weight were increased
females (1500, 5000 and 15000ppm)
- 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 (15000ppm)

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examinations conducted at scheduled termination, following six weeks of treatment, two weeks of recovery or on Day 7 of lactation revealed no findings that could be attributed to treatment.

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 kidneys, liver and female reproductive tract.

Kidneys:
Treatment males:
- Hyaline droplets in the cortical tubular epithelium were evident in the kidneys from all males treated at 15000 ppm examined histopathologically and one male treated at 5000 ppm. In the most severely affected animal this change was accompanied by slight multifocal cortical tubular basophilia and minimal granular casts.
Recovery males:
- Following two weeks without treatment, hyaline droplets in the cortical tubular epithelium were no longer evident in the kidneys of males previously receiving 15000 ppm, indicating a degree of recovery from this change. However, granular casts were still evident in one male previously treated at15000 ppm and a higher incidence and severity of cortical tubular basophilia was seen in this group compared to Controls.

Note: The accumulation of hyaline droplets in the cortical tubular epithelium of the kidneys, associated with granular casts and tubular basophilia is consistent with the finding of alpha 2u-globulin nephropathy (Frazier et al., 2012). This is a male rat specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein. There was evidence of recovery from this change following 2 weeks respite from treatment.

Liver
Treatment males:
- Portal pigment was seen in the bile ducts of three males receiving 15000 ppm. These consisted of deposits of brown pigment, predominantly located in the lumen of smaller bile ductules. Application of special stains to additional sections of liver showed this pigment to be Schmorl’s, Perls’ and Fouchet’s negative.
Recovery males:
- Following two weeks without treatment, portal pigment was seen in the bile ducts of three males previously receiving 15000 ppm, and in one animal the severity was greater than seen at the end of the treatment period.

Note: In the absence of any effect on the survival or reproductive performance of the animals and, no indication of impaired liver function from the blood chemistry analyses, these findings were considered not to represent an adverse effect of treatment.

Female reproductive tract
Treatment females:
- 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.
Recovery females:
- Following a two week recovery period, the changes observed in the female reproductive tract at the end of the treatment period were no longer apparent.

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 oestrus cycles prior to pairing, mating performance and fertility of these females had been unaffected by treatment.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 868.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: NOAEL = highest dose tested

Remarks on result:

other: The alpha 2u-globulin nephropathy is a male rat specific finding associated and therfore considered not relevant for the NOAEL setting. The histopathologic findings for the liver are considered not to be an adverse effect of treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 892.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested

Key result

Critical effects observed:

no

Overall remarks to study effects observed:

- Minor treatment related effects were observed for body weight, histopathology, blood chemistry and urine analysis. However, as the animals showed to recover from these findings and neither the survival nor the clinical condition were affected by treatment, they are considered not to represent an adverse effect of the treatment.

- The histological changes observed in the liver were not considered to be adverse, as no effect on the survival or reproductive performance of the animals was observed and no indication of impaired liver function could be seen from the blood chemistry analyses.

- The alpha 2u-globulin nephropathy observed in the kidneys of the male rat is a male-rat-specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein. Also the animals showed to recover from this effect.

Conclusions:

Under the conditions of this study, the NOAEL for systemic toxicity was determined to be >=15000 ppm, representing a mean achieved dose of 868.7 mg/kg/day for both males and females using the lower value of the two. This result is read across to dihydromyrcenyl acetate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

Information is based on read across from Dihydromyrcenol, which results in a Rel. 2

Additional information

No repeated dose toxicity data are available for Dihydromyrcenyl acetate. The results of a repeated dose study toxicity study with the analogue Dihydromyrcenol is presented below followed by the read-across rationale.

Dihydromyrcenol Repeated dose toxicity

The repeated dose toxicity of Dihydromyrcenol is assessed in an OECD TG 422 dietary toxicity studyfor at least five weeks. The fertility and developmental toxic effects are presented in the reproductive toxicity section.An additional recovery group was also included to assess for reversibility, persistence or delayed occurrence of systemic effects for at least 14 days post treatment. A similarly constituted control group was assigned to each phase, and received powdered SDS VRF1 Certified diet (with corn oil) throughout the same relative treatment period. The following parameters were studied: mortality, 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.

Results

Clinical signs: Dietary administration of 2,6-dimethyloct-7-en-2-ol at levels of 1500, 5000 and 15000 ppm was well tolerated with no mortalities related to treatment. Overall mean achieved dosages for the period Week 1 to 6 of treatment (toxicity phase and recovery phase animals) were 89, 291.7 and 868.7 mg/kg bw/day for males, and 97.2, 325.2 and 892.7 mg/kg bw/day for females, respectively. There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, ophthalmic examination and macroscopic pathology.

Other effects: Minortreatment related effects were observed in the dose groups for body weight, histopathology, blood chemistry and urine analysis. However, as these showed recovery and neither the survival nor the clinical condition were affected by treatment, they are considered not to represent an adverse effect of the treatment.The histological changes (portal pigment) observed in the liver of three male rats of the high dose group, were also not considered adverse, as no effect on the survival or reproductive performance of the animals was observed and no indication of impaired liver function could be seen from the blood chemistry analyses.Evidence ofalpha 2u-globulin nephropathy was found in the kidneys of the male rat (5000 and 15000 ppm), this is a male-rat-specific finding associated with the binding of xenobiotics to this protein and interference with the normal lysosomal uptake and degradation of the protein.

Under the conditions of this study, the NOAEL for systemic toxicity was determined to be >=15000 ppm, representing a mean achieved dose of 868.7 mg/kg/day for males and  females using the lowest dose of males for which human related effects were seen.

The repeated dose toxicity of Dihydromyrcenyl acetate (CAS 53767-93-4) using read across from Dihydromyrcenol (CAS 18479-58-8)

 

Introduction and hypothesis for the analogue approach

Dihydromyrcenyl acetate comprises a C8 alk-1-ene chain with at C2 an acetate ester group and two methyl groups at C3 and C7. For this substance no data on repeated dose toxicity are available. In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the repeated dose toxicity of Dihydromyrcenyl acetate, the analogue approach is selected because for the key metabolite of Dihydromyrcenyl acetate that is Dihydromyrcenol information on repeated dose toxicity is available which can be used for read across.

Hypothesis: Dihydromyrcenyl acetate has the same repeated dose toxicity compared to Dihydromyrcenol

Available information: For Dihydromyrcenol a repeated dose study was conducted according to OECD guideline 422, in which animals were exposed via the diet to 1500, 5000 and 15000 ppm. The NOAEL for systemic toxicity is 15000 ppm (lowest mean achieved doses is 868.7 mg/kg/day for males).

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for repeated dose oral toxicity.

Purity / Impurities

The purity and impurities of the target chemical do not indicate repeated dose oral toxicity potential other than indicated by the parent substance. The impurities are all below < 10%.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection: Dihydromyrcenyl acetate will metabolise when exposed via all routes to Dihydromyrcenol, its key metabolite, besides acetic acid. The systemic exposure will therefore be to Dihydromyrcenol and therefore this latter substance is used for Dihydromyrcenyl acetate.

Structural similarities and differences:BothDihydromyrcenyl acetate and Dihydromyrcenol have the same backbone: i.e. an alkene chain with one methyl groupon C2 and on C6. Dihydromyrcenol has acetate as a functional group and Dihydromyrcenol an alcohol.

Toxico-kinetic, Absorption: Dihydromyrcenol acetate and Dihydromyrcenol will have similar absorption characteristics. The acetate will be somewhat (s)lower because it has a slightly higher molecular weight (198 and 156) and log Kow (4.9 and 3.25), respectively.

Metabolism: Dihydromyrcenyl acetate will metabolise into its alcohol via all routes of exposure because of the availability of carboxyl esterases in all tissues (e.g. Toxicological handbooks,see also Belsito et al., 2008, Wu et al., 2010). This metabolism was experimentally in vitro shown for a similaracetate ester, Geranyl acetate extra (trans-3,7-dimethyl-2,6-octadiene-1-yl-acetate). In plasma, liver S9 fraction of rats, gastric juice simulant and intestinal-fluid simulant including pancreas lipase. Geranyl acetate extra was hydrolysed within 0.5 hour almost completely in rat plasma, liver S9 fraction of rats and intestinal fluid simulant as demonstrated by the decrease of Geranyl acetate extra and formation of the hydrolysis product Geraniol extra. In gastric fluid simulant the degradation was about 50% after 2 hours.The ester bond present in the acetate ester of Dihydromyrcenyl acetate is expected tohydrolyse at a comparable rate into the respective alcohol and acetic and therefore the systemic exposure of Dihydromyrcenyl acetate will be to Dihydromyrcenol.

Toxico-dynamics:There will be no differences in reactivity because the systemic exposure will be Dihydromyrcenol and acetic acid. Acetic acid is a normal constituent of the body and not affecting the reactivity of the substance.

Conversion of the NOAEL: A conversion based on molecular weight to Dihydromyrcenyl acetate from Dihydromyrcenol is not considered necessary because the Dihydromyrcenol presents a conservative value having the lower molecular weight.

Uncertainty of the prediction: There are no remaining uncertainties other than those addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.

Conclusions for repeated dose toxicity

For Dihydromyrcenyl acetate no repeated dose toxicity information is available but for Dihydromyrcenol such information is present, which can be used for read across.When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. This documentation is present in the current document. For Dihydromyrcenol a repeated dose toxicity study was performed according to OECD guideline 422 in which no adverse effects were found resulting in a NOAEL >=868.7 mg/kg bw. This information can directly be used for read across to Dihydromyrcenyl acetate.

Final conclusion: Dihydromyrcenyl acetate has a NOAEL of >=868.7 mg/kg bw

Data matrix supporting the read across to Dihydromyrcenyl acetate from Dihydromyrcenol

NAME	Dihydromyrcenyl acetate	Dihydromyrcenol
	Target	Source
Chemical structure
Chemical formula	C12H22O2	C10H20O
CAS	53767-93-4	18479-58-8
EINECS	258-751-7	242-362-4
REACH registration	2018	Registered
Molecular weight	198.0	156.3
Physico-chemical data
Physical state
Melting point,^oC	<-20°C (IFF, 2017)	<-20°C (measured)
Boiling point,^oC	221.2°C (IFF, 2016)	193°C (measured)
Vapour pressure, Pa	14.4 Pa at 24°C (IFF, 1999)	20 Pa at 25°C (measured)
Water solubility, mg/l	6.1 mg/L at 24°C (IFF, 1999)	939 mg/L at 20°C (measured)
Log Kow	Log Pow: 4.9 (IFF, 2017)	Log Pow: 3.25 (measured)
Human health
Acute oral toxicity	LD50: >5000 mg/kg bw (OECD TG 401)	LD50: 3600 mg/kg bw ) (OECD TG 401)
Repeated dose toxicity	>=868.7 mg/kg bw (Read across)	>=868.7 mg/kg bw (using the test substance intake of the males) (OECD TG 422)

 

Justification for classification or non-classification

Based on the current data-set, the substance does not need to be classified for effects after repeated exposure according to EU CLP (EC No. 1272/2008 and its amendments).