reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Endpoint summary

Description of key information

90 day Repeated dose toxicity, oral (OECD 408, GLP, encapsulated via diet)
- NOAEL male >= 296 mg/kg bw/day,

- NOAEL female >= 1136 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2019 - Feb. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 Jun 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under nitrogen
- Stability under test conditions: The stability of the test substance preparation (encapsulated material in the diet) over a period of 35 days was determined before the start of the administration period.

FORM AS APPLIED IN THE TEST (if different from that of starting material): encapsulated
For each concentration, the test substance and Placebo (encapsulated) was weighed out and mixed with a small amount of food. Thereby, the concentration of the vehicle (capsules) in all test groups and vehicle controls (placebo) of males or females were adjusted to be a comparable concentration as given in the high dose groups of males or females. In order to obtain the desired concentrations, these premixes were added to the corresponding amounts of food, depending on test group. The preparations were prepared in a frequency for which the stability in the carrier was demonstrated.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age when supplied: 34 ± 1 days
- Age at the start of administration period: 42 ± 1 days
- Weight at study initiation (range of means per group): male 164-166 g, female 123 - 126 g
- Fasting period before study: no
- Animal identification: Ear tattoo (animal number)
- Housing: 2 animals per cage, Polysulfonate (H-Temp) cages type 2000P (floor area about 2065 cm2) and polycarbonate cages type III (floor area about 800 cm2) for FOB and during measurement of motor activity. The cages and wire covers were supplied by TECNIPLAST, Hohenpeissenberg, Germany resp. by Ehret, Emmendingen, Germany.
- Enrichment: wooden gnawing blocks (Lignocel Block Large); J. Rettenmaier & Söhne GmbH Co KG, Rosenberg, Germany play tunnel large (Art. 14153); PLEXX B.V., Elst, Netherlands/dust-free wooden bedding
- Diet: Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water (from water bottles); ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
- Food analysis: The food used in the study was assayed for chemical and microbial contaminants. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p 27354 (EPA), serve as the guideline for maximum tolerable contaminants. The levels of phytoestrogens did not exceed 350 μg of genistein equivalents/g food, and the amounts of microorganisms did not exceed 1×105/g food.
- Drinking water analysis: The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of the testing facility as well as for bacteria by a contract laboratory. The Drinking Water Regulation serve as the guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h

Route of administration:

oral: feed

Details on route of administration:

Oral administration has been extensively used to establish the toxicological profile of the test substance and is suitable for comparison.

Vehicle:

other: microcapsules

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For each concentration, the test substance was weighted out and mixed with a small amount of food. In order to obtain the desired concentrations, these premixes were added to the corresponding amounts of food, depending on test group.


DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared at intervals based on the stability of encapulated test material in feed of 35 days
- Mixing appropriate amounts with (Type of food): Ground Kliba maintenance diet mouse/rat „GLP“ meal
- Storage temperature of food: room temperature; retain samples was stored at the Laboratory Mech. Toxicology (at –20°C).
- Homogeneity and concentration control analyses: At the beginning and towards the administration period, samples were taken for verification of homogeneity, i.e. 3 samples in the highest and lowest concentration (were used as a concentration control at the same time). Additionally, homogeneity was verified from all test groups (was used as a concentration control at the same time) on days 7, 14, 21, 42, 63.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The coefficients of variation in the homogeneity analysis were not within the specification of the test facility (RSD +/-5%) in more than half of the samples (70%). However, according to the general accepted specification for formulations with solid vehicles (RSD ≤15%) most of the samples (84%) can be considered as homogeneous. Based on the high number of samples analyzed it was assessed that the low number of samples (16%) having a high coefficient of variance (RSD ≥15%), outside of the general accepted criteria, did not affect the validity of the study.
The mean concentrations determined were not within the specification of the test facility (90-110%) in more than half of the samples (60%). However, according to the general accepted specification for formulations with solid vehicles (80-120%) most of the samples (92%) confirmed the aimed nominal concentrations of methylionone 70. Based on the high number of samples analyzed it was assessed that the low number of samples (8%) showing a higher or lower concentration outside of the general accepted criteria did not affect the validity of the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

4 941 ppm

Remarks:

Males - Effective concentration of Methylionon 70 corresponds to 296 mg/kg bw/day

Dose / conc.:

1 281 ppm

Remarks:

Males - Effective concentration of Methylionon 70 corresponds to 86 mg/kg bw/day

Dose / conc.:

494 ppm

Remarks:

Males - Effective concentration of Methylionon 70 corresponds to 33 mg/kg bw/day

Dose / conc.:

14 640 ppm

Remarks:

Females - Effective concentration of Methylionon 70 corresponds to 1136 mg/kg bw/day

Dose / conc.:

4 392 ppm

Remarks:

Females - Effective concentration of Methylionon 70 corresponds to 341 mg/kg bw/day

Dose / conc.:

1 464 ppm

Remarks:

Females - Effective concentration of Methylionon 70 corresponds to 114 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes

yes, plain diet

Details on study design:

- Dose selection rationale: Based on the results of the range finding repeated-dose 28-day toxicity study in Wistar rats, administration via the diet, showing no effects in females up to the limit dose (120000 ppm, 1243 mg/kg bw/d) and decreased body weight (-12.4%) in males at 40000 ppm (406 mg/kg bw/d) test substance. Thereby, the difference to the control in body weight was first observed after 14 days of administration (-10.2%) and increased thereafter. The dose of 1218 mg/kg bw/d (120000 ppm) in males exceeded the maximum tolerated dose caused sever signs of systemic toxicity manifested in decreased body weight of -23.8% on study day 28. Based on the test substance-related adverse findings in the range finder study, the given concentrations in the diet were selected.

- Rationale for animal assignment (if not random): Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex.

- Fasting period before blood sampling for clinical biochemistry: at least 16 hours

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (Mortality, Moribundity, Abnormal clinically signs)
- Time schedule: twice daily on working days and once daily on Saturdays, Sundays and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the administration period (day 0) and subsequently once a week (in the morning)
- examined parameters: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals, on study day 0 (start of administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Food consumption was determined weekly over a period of 7 days from day 0-35 and from day 42 onwards over a period of 1 day and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the beginning of administration, the eyes of all animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent. At the end of the administration period the eyes of the animals of the control and highest dose group were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling by puncturing the retrobulbar venous plexus and examination was carried out in a randomized sequence at the end of administration period
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood sampling by puncturing the retrobulbar venous plexus and examination was carried out in a randomized sequence at the end of administration period
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined in a randomized sequence.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the administration period (start in the morning)
- Dose groups that were examined: all animals
- Battery of functions tested: home cage observation table [No.4] / open fields observation table [No.5] / Sensory motor tests/reflex tests table [No.6] / Measurement of motor activity

Sacrifice and pathology:

NECROPSY: The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes (Organ weights see table [No.7])

HISTOPATHOLOGY: Yes (see table [No.8])

Other examinations:

ESTROUS CYCLE DETERMINATION
Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.

Statistics:

Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- for body weight and body weight change: DUNNETT'S
- for rearing, grip strength of fore- and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters, weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS and WILCOXON

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed in any treated male or female dose group animals.

Control animals
- no difference between untreated control and vehicle controls were observed.
- 1 female (untreated control): injury in the neck region from study day 35 until day 47 and sparse fur at the flank both from day 84 until the scheduled sacrifice

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals
4941ppm
- Body weight: Significantly decrease on study day 28 (-6.0%) and day 84 (-7.0%). Non significant decrease of mean body weight on study day 91 (-6.9%).
- Body weight change: significantly lower on study day 7 (-11.4%) and day 28 (-12.2%). Non significant decrease of mean body weight change for d0-91 (-11.1%).
Effect observed is considered a treatment-related effect in comparison to control. The degree of body weight effect is close by (-6.9%, non-statistically significant) but still slightly below the common accepted level of adversity in body weight (-10%). The body weight change in males of this test group were -11.1% (not statistically significant). A body weight change of -10% is considered as maximal tolerated dose level in a 90d repeated dose toxicity studies.

Female animals
14640ppm
- Body weight change value was significantly lower only on day 7 (-29.4%).
- No test substance-related findings were observed on body weights
The high dose in females is the limit dose and did not show a treatment-related effect on the body weight.

No test substance related, adverse effects were observed in other dose group male/female animals.

Vehicle control
- no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male animals
4941ppm
- Intense food spilling, so that the food consumption value could not be determined properly
- Food consumption was significantly increased on study day 7 (+27.2%); considered as treatment-related but not adverse.

No test substance related, adverse effects were observed in other dose group male animals.

Female animals
14640ppm
- Intense food spilling, so that the food consumption value could not be determined properly
- Food consumption was significantly increased on study day 7 (+35.5%); considered as treatment-related but not adverse.

4392ppm
- Intense food spilling, so that the food consumption value could not be determined properly
- Food consumption was significantly increased on study day 7, 14 and 21 ranging from +40.0% to +56.9%; considered as treatment-related but not adverse.

1464ppm
- Food consumption was significantly increased on study day 21, 28 and 35 ranging from +17.6% to +33.2%; considered as treatment-related but not adverse.

Vehicle control (males)
- Food consumption was significantly decreased on study day 77 (-7.9%).
Vehicle control (females)
- Food consumption was significantly decreased on study day 35 (-6.2%).

Several values were excluded since the determined food consumption were unrealistic high and above the threshold indicating spilling according the standard operating procedure of the test facility. The remaining values below that threshold are partially still higher than normally expected for the rat strain in the age used in this study, which might also be caused by spilling. The encapsulation of the test substance was necessary since the palatability of direct formulation of the test item into the diet was not given. The encapsulation can reduce the odor of a test item but cannot seal it completely. Therefore, the rats still search in the food racks for diet with lower odor and spill parts of the diet out of the food containers. Therefore, the increase food consumptions were considered as treatment-related but not adverse.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes with regard to water consumption were observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals
4941ppm
- relative, large unstained cell (LUC) counts were significantly increased (0.4%), but the values were within the historical control range (males, LUC 0.1-0.6 %).

Female animals
14640ppm
- red blood cell (RBC) counts were significantly decreased (-2.93% vs. ctrl), but the alteration was not dose-dependent.

1464ppm
- red blood cell (RBC) counts were significantly decreased (-5.46% vs ctrl.), but the alteration was not dose-dependent.

All mentioned changes were regarded as incidental and not treatment-related.
No test substance related, adverse effects were observed in other dose group male/female animals.

vehicle control (males)
- significantly higher red blood cell (RBC) counts compared to the food control group, but the RBC values in the placebo group were also within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
4941ppm
- higher triglyceride values than in the vehicle group (+45.76% vs ctrl), not statistically significant
1281ppm
- higher triglyceride values than in the vehicle group (+32.16% vs ctrl), not statistically significant
494ppm
- higher triglyceride values than in the vehicle group (+36.96% vs ctrl), not statistically significant

Females
14640ppm
- statistically significant increase in triglyceride values (+48.18% vs ctrl; 0.61 mmol/L), but within the historical control range, control triglyceride mean was below this range (females, triglycerides 0.44-0.82 mmol/L)

No test substance related, adverse effects were observed in other parameters and other dose groups animals.

vehicle control (males)
- significantly lower triglyceride values compared to the food control group (-42.92%) (below the historical control range)
vehicle control (females)
- significantly lower urea (-17.24%) and creatinine values (-13.8%) compared to the food control group, but within historical control ranges.

No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in high dose females triglyceride values were significantly increased, but the values were within the historical control range, whereas the control triglyceride mean was below this range. Further, urea and creatinine values in females of the placebo group were significantly lower compared to the food control group but within historical control ranges. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
In males of the placebo group, triglyceride values were significantly lower compared to the food control group and also below the
historical control range. However, the triglyceride values in all male treated groups were higher than in the placebo group and therefore more like those of the food control group, and they were not significantly changed. Therefore, the significant lower triglyceride values in males of the placebo group compared to the food controls was considered to be not relevant.

In all dosed males and females as well as in the placebo control group compared to the food control group, no treatment-related alterations of T3, T4 and TSH levels were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related adverse effects were observed in home cage / open field observations, sensorimotor tests/ reflexes and quantitative parameters assessed. Most findings were equally distributed between test-substance treated and the control groups, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Motor activity
Male animals
4941ppm
- significantly higher values at interval 10 compared to the vehicle control
- significantly higher values at interval 11 compared to the vehicle control
1281ppm
- significantly higher values at interval 11 compared to the vehicle control
494ppm
- significantly higher values at interval 11 compared to the vehicle control

Female animals
14640ppm
- significantly higher values at interval 5 compared to the vehicle control
1464ppm
- significantly higher values at interval 5 compared to the vehicle control

No test substance related, adverse effects were observed in other dose group female animals.

Vehicle vontrol (males)
- significantly higher values at interval 11 compared to the untreated control

Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for treated animals. The changes observed were regarded to be incidental and not related to treatment. The overall motor activity was not affected in all test groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals
4941ppm
- statistically significant changes in absolute organ weights of heart (-8% vs ctrl), prostate (-17% vs ctrl) and seminal vesicle (-14% vs ctrl)
- statistically significant changes in relative organ weights of liver (+10% vs ctrl.)

Female animals
14640ppm
- statistically significant change in absolute organ weights of liver (+11% vs ctrl.)
- statistically significant changes in relative organ weights of liver (+15% vs ctrl.)

4392ppm
- statistically significant changes in relative organ weights of liver (+10% vs ctrl.)

1464ppm
- statistically significant change in absolute organ weights of liver (+14% vs ctrl)
- statistically significant changes in relative organ weights of liver (+13% vs ctrl.)

No test substance related, adverse effects were observed in other dose group animals or other organs assessed.

Vehicle control (females)
- statistically significant changes in relative organ weights of adrenal glands (-13% vs ctrl.) and heart (-8% vs ctrl.)

All absolute significantly changed organ weights were within historical control values. The relative liver weight in high dose males was slightly above historical control values, but as the absolute liver weight was not significantly changed and there were no histopathologic findings observed, it was regarded to be unrelated to treatment, but most likely due to the slight and not significant decrease in terminal body weight in this group.
The relative liver weight in low and high dose females were slightly above historical control data, whereas the relative liver weight in mid dose females were within historical control values. Due to no findings in histopathology and no dose response relationship it was not regarded to be treatment related.
The decreased relative weights in adrenal glands and heart of females of the Placebo group were within historical control values and regarded to be an incidental finding.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: calculated from food consumption and analytically confirmed dose in food

Sex:

male

Basis for effect level:

other: No adverse test substance related effects observed at the highest dose tested

Dose descriptor:

NOAEL

Effect level:

1 136 mg/kg bw/day (actual dose received)

Based on:

other: calculated from food consumption and analytically confirmed dose in food

Sex:

female

Basis for effect level:

other: No adverse test substance related effects observed at the highest dose tested

Critical effects observed:

no

Result tables are provided in the attached background material.

The mean daily test substance intake over the entire study period was calculated to be 32, 91, 181 mg/kg bw/d in low, mid, high dose males and 129, 458, 1612 mg/kg bw/d in low, mid, high dose females, respectively, when considering a content of about 18.3% Methylionone 70 in the encapsulated material.

Because of intense food spilling by mid and high dose male and female animals, food consumption values could not be determined properly for these test groups. Thereby, the calculated substance intake considering the food consumption values is likely to be biased. The exclusion of several food consumptions values resulted in a limited numbers of food consumption values below the exclusion criteria used for the calculations of substance intake. Therefore, a more likely food consumption was estimated based on historical control data of food consumption for this rat strain and age leading to a lower test substance intake in the range of the aimed exposure levels in this study.

The estimated food consumptions in the test groups were based on following considerations:

Using the mean of mean food consumption per body weight and day within a 91-day exposure period from 9 OECD 408 studies performed in the respective test facility, the substance intake was calculated. This approach is more likely to represent the food consumption in the test groups and the test substance intake in this study by excluding the bias of spilling in a different way. Comparing the results of calculated substance intake, the higher differences are observed in the higher dose levels where the spilling was more pronounced.

This correction led to a mean daily test substance intake of 33, 86, 296 mg/kg bw/d in low, mid, high dose males and 114, 341, 1136 mg/kg bw/d in low, mid, high dose females, respectively.

Conclusions:

The administration of Methylionon (encapsulated) via the diet to male and female Wistar rats for 3 months did not cause any test substance-related, adverse signs of toxicity in male and
female animals up to a dose of 296 mg/kg bw/d and 1136 mg/kg bw/d, respectively.
Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 296 mg/kg bw/d for male and 1136 mg/kg bw/d for female rats.

Executive summary:

In a repeated dose 90-day oral toxicity study, the encapsulated test substance was administered over a period of 3 months via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 2700, 7000 and 27000 ppm capsules (low, mid and high dose males, respectively), as well as 8000, 24000 and 80000 ppm (low, mid and high dose females, respectively). The doses correspond to 494 ppm (33 mg/kg bw/d), 1281 ppm (86 mg/kg bw/d) and 4941 ppm (296 mg/kg bw/d) Methylionone 70 in males, respectively, as well as to 1464 ppm (114 mg/kg bw/d), 4392 ppm (341 mg/kg bw/d) and 14640 ppm (1136 mg/kg bw/d) Methylionone 70 in females, respectively.

With regard to clinical examinations, signs of general systemic toxicity in Wistar rats of both sexes were not observed in males up to 296 mg/kg bw/d, the highest concentration tested, as well as in females up to the limit dose (1136 mg/kg bw/d). The highest tested concentration of the test substance in the diet for males were chosen because 120000 ppm capsules (1243 mg/kg bw/d Methylionone 70) in male animals of the range-finding repeated dose 28d study exceeded the maximum tolerated dose and 40000 ppm capsules (406 mg/kg bw/d Methylionone 70) caused an increasing effect on body weight (from -10.2% on study day 14 up to -12.4% on study day 28). In this study, 296 mg/kg bw/d Methylionone 70 caused a decrease in the mean male body weight values on study days 28 (-6.0%) and 84 (-7.0%). At the end of the administration period the degree of effect was close by (-6.9%, non-statistically significant) but still slightly below the common accepted level of adversity in body weight (-10%). However, the body weight change in males of this test group were -11.1% (not statistically significant). A body weight change of -10% is considered as maximal tolerated dose level in a repeated dose 90d toxicity studies. Therefore, it was considered that the highest dose tested in males was so close to a maximum tolerated dose, that further testing was not indicated, especially regarding animal welfare.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound in males of 296 mg/kg bw/d and in females of 1136 mg/kg bw/d.

Regarding pathology, there was an increase in relative liver weight in low (114 mg/kg bw/d) and high dose (1136 mg/kg bw/d) females ppm which were slightly above historical control data. Due to the fact, that the absolute values were not above historical control data, the relative liver weight of mid dose animals was within historical control values, no dose-response relationship was present and no histopathologic findings were observed, this weight increase was regarded to be not adverse, if treatment-related at all. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

In conclusion, the administration of Methylionon (encapsulated) via the diet to male and female Wistar rats for 3 months did not cause any test substance-related, adverse signs of toxicity in male and female animals up to a dose of 296 mg/kg bw/d and 1136 mg/kg bw/d, respectively. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 296 mg/kg bw/d for male and 1136 mg/kg bw/d for female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

296 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

In the chosen key study for repeated dose toxicity, i.e. a repeated dose 90-day oral toxicity study according to OECD TG 408 and GLP (BASF 2020; 50C0609/17C165), Methylionone 70 (dissolved in sunflower oil and encapsulated in Glycerin/Alginate capsules) was administered over a period of 3 months via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 2700, 7000 and 27000 ppm capsules (low, mid and high dose males, respectively), as well as 8000, 24000 and 80000 ppm capsules (low, mid and high dose females, respectively). The doses of capsules administered correspond to 494 ppm (33 mg/kg bw/d), 1281 ppm (86 mg/kg bw/d) and 4941 ppm (296 mg/kg bw/d) Methylionone 70 in males, respectively, as well as to 1464 ppm (114 mg/kg bw/d), 4392 ppm (341 mg/kg bw/d) and 14640 ppm (1136 mg/kg bw/d) Methylionone 70 in females, respectively.

With regard to clinical examinations, signs of general systemic toxicity in Wistar rats of both sexes were not observed in males and females up to the highest dose tested (296 mg/kg bw/d males; 1136 mg/kg bw/d females). The highest tested concentration of Methylionone 70 for males in the diet was chosen based on the results of a range-finding repeated dose 28 day study, showing that 120000 ppm capsules (1243 mg/kg bw/d Methylionone 70) exceeded the maximum tolerated dose and 40000 ppm capsules (406 mg/kg bw/d Methylionone 70) caused an increasing effect on body weights in males (from -10.2% on study day 14 up to -12.4% on study day 28). In the main 90 day study, the adverse findings of decreased body weights of -12% observed in males at 40000 ppm capsules (406 mg/kg bw/d) of the corresponding range finding study were not observed. The high dose (296 mg/kg bw/d) tested in the main 90 day study caused a decrease in the mean male body weight values on study days 28 (-6.0%) and 84 (-7.0%). At the end of the administration period the degree of the effect was close by (-6.9%, non-statistically significant) but still slightly below the common accepted level of adversity in body weight (-10%). However, the mean body weight change in males of this dose group was -11.1% (not statistically significant). A body weight change of -10% is considered a maximal tolerated dose level in repeated dose 90 day toxicity studies. Therefore it is considered, that the highest dose tested in males was close to a maximum tolerated dose, and further testing is not indicated especially in the context of animal welfare.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound in males of 296 mg/kg bw/d and in females of 1136 mg/kg bw/d.

Regarding pathology, there was an increase in relative liver weight in low (114 mg/kg bw/d) and high dose (1136 mg/kg bw/d) females, which were slightly above historical control data. Due to the fact, that the absolute values were not above historical control data, the relative liver weight of mid dose animals was within historical control values, no dose-response relationship was present and no histopathologic findings were observed, this weight increase was regarded to be not adverse, if treatment-related at all. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

In conclusion, the administration of Methylionon (encapsulated) via the diet to male and female Wistar rats for 3 months did not cause any test substance-related, adverse signs of toxicity in male and female animals up to a dose of 296 mg/kg bw/d and 1136 mg/kg bw/d, respectively. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 296 mg/kg bw/d for male and 1136 mg/kg bw/d for female rats.

 

Further repeated dose toxicity data are available for the analogue substance alpha-iso-methylionone (CAS No.127 -51 -5). Two 90-day oral subchronic studies under GLP were performed according to OECD Guideline 408 (Dunster, 2006 and Politano 2012). Ten male and ten female Sprague-Dawley rats (Crl:CD(SD)IGS BR) were daily administered by gavage with 5, 30, 500 mg/kg bw/d at a volume of 4 ml/kg bw in olive oil. While no toxic effects were noted at 5 mg/kg bw/d, a greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of in nine of ten males at 30 mg/kg bw/day. Administration of 500 mg/kg bw/d led to centrilobular or generalised hepatocyte enlargement in four out of ten males and nine out of ten females. Also, greater incidences of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium in nine out of ten males. Two male rats treated at this dose level exhibited associated higher grades of tubular basophilia, whereas a higher incidence of follicular cell hypertrophy in the thyroid was seen in relation to treatment for seven out of ten males. In addition, a higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for all males treated at this dose level.

The authors stated that the kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect may not be indicative of a hazard to human health and for the purposes of hazard evaluation the no observed adverse effect level for males should be regarded as 30 mg/kg/day. Besides this, hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature. Therefore, the no effect level (NOEL) was estimated as 5 and 30 mg/kg bw/d for males and females, respectively. NOAELs were observed to be 30 mg/kg bw/d for males and females. Due to the structural similarities, the same result could also be expected for methylionone, however, these findings were not confirmed in the present key subchronic repeated dose toxicity study with Methylionone 70.

 

Dermal

No data on repeated dose toxicity data of Methylionone (EC 215-635-0) after dermal application are available. The analogous substance alpha-iso-methylionone (CAS No.127 -51 -5) has been subjected to a 90-day subchronic dermal toxicity (2 rat studies).

In the first study (Becci 1980), Sprague–Dawley rats (15/sex/dose) were administered 50, 170, 580, or 2000 mg/kg body weight/day of neat alpha-iso-methyl-ionone (no dosing vehicle) via clipped skin for a period of 90 days. Clinical, laboratory and gross and histopathological evaluations were conducted.

On the skin at the application site, there was a dose-dependent increase in the severity of erythema, and eschar formation. Since erythema and eschar formation occurred in all treatment groups, a NOAEL for this effect could not be established.

All further findings were overlaid with effects resulting from severe skin damages, inflammation and infection at the site of application, making the interpretation of the data complicated. It remained unclear, to what extend the findings were endogenous or reflect the response to stress caused by tissue damage/necrosis and inflammation/infection.

In a subsequent 90-day study (Moreno 1981), alpha-isomethyl ionone was applied dermally daily to groups of 5 male and 5 female Sprague–Dawley rats at a daily dose of 10 mg/kg as a 1% solution in phenethyl alcohol (PEA). No dermal reaction to treatment was noted at any time during the study. The hematology, clinical chemistry, and urinalysis parameters evaluated were comparable to the controls. A slight, but significant increase in serum alkaline phosphatase activity was reported in males. The relationship of this finding to treatment was considered questionable. There was no evidence of a treatment-related effect on body weight gain, necropsy observations, organ weights, or on the results of the microscopic examination. As a result, the NOAEL for the skin appeared to be 10 mg/kg, the only dose tested, but the inclusion of only 5 animals per sex and of only one dose precludes statistical analyses of the data. The lack of dermal reactions in this study contrasts to the severe skin effects in the earlier study as expected with the application of a far lower and less concentrated solution (1% in PEA).

It is concluded that there are currently no entirely adequate studies available to assess the subchronic dermal toxicity of methyl-ionone (compare Belsito 2007).

Since the total interpretation of the results is difficult and the predominant effect is massive skin damage, these dermal studies were not used for the derivation of the systemic DNELs after long term exposure.

 

Inhalation

In an inhalative repeated-dose toxicity study, eight complex fragrance mixtures consisting of approximately 200 ingredients (close to one-half of the ingredients present at a level of 1% or more) were tested (Fukayama, 1999). Methyl ionone was present in seven out of the eight mixtures and was tested in syrian hamster and CD and Sprague-Dawley rats. They were whole-body exposed to the mixtures at 5, 9 or 50 mg/m³ for 4 h/day, 5 days/week for 6 or 13 weeks. The concentrations of fragrance in the chamber used were monitored and the found particle size ranged from 0.5 to 7.5 µm. In two of the 7 fragrances the highest determined exposure levels were 12.8 and 219.5 µg/m³. No gross pathological or histopathological findings related to test material exposures were observed, so that the value of 50 mg/m³ could be determined as the NOEL. However, because different mixtures of substances were used in this study, the estimated values could not be taken into account for assessment.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at relevant doses upon subchronic oral exposure in rats. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.