2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented experiment according to GLP and EC and OECD guidelines.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Wistar rat, outbred, SPF quality.
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 180-190g
- Fasting period before study: not reported
- Housing: Animals were housed 5 to a tage (same sex) in stainless steel suspended cages with wire mesh floors
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Kliba, Klingentalmuehle AG, 4303, Kaiseraugst, Switzerland). Each batch was analysed for contaminants and results were examined and archived.
- Water (e.g. ad libitum): Tap water, ad libitum. Results of chemical and contaminants analyses are examined and archived quarterly.
- Acclimation period: At least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method: The test substance was weighed into a glass flask an an analytical balance and the vehicle (w/w) added. Adjustment was made for specific gravity of
vehicle.
- Frequency of test substance formulation: Daily immediately prior to dosing.
- Homogeneity of test substance in vehicle: By the use of a magnetic stirrer. The test preparations formed solutions and were shaken prior to use.
- Storage instructions for test substance formulation: At ambient temperature.
- Analysis of formulations: Samples of formulations prepared during week 3 were analysed to check accuracy of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: depending on dose
- Amount of vehicle: 5 mL / kg bw
- Actual dose volume: Dose volumes were calculated weekly according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the accuracy of dose preparations revealed values within the range of +5% to +8% of nominal, which was considered acceptable for this
type of formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, approximately the same time each day, 7 days per week

Remarks:

Doses / Concentrations:
0 mg/kg bw/d
Basis:
actual ingested

Remarks:

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

Remarks:

Doses / Concentrations:
200 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
800 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5 males + 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 300, 600 and 1000 mg/kg/day) to provide a basis for selection of
dose levels for a study of langer duration.
All animals were noted with excessive salivation following dose administration. Males receiving 600 or 1000 mg/kg/day showed slight body weight gain. Body weight loss was observed in females receiving 1000 mg/kg/day. Food consumption was slightly decreased in animals receiving 1000 mg/kg/day. Liver weights were noted as slightly increased among animals receiving 1000 mg/kg/day.

Based an these observations, treatment levels for a study of 28 days duration were selected to be 0, 50, 200 and 800 mg/kg/day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily. Severity of observations were graded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly

WATER CONSUMPTION :
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes were examined following instillation of tropicamide solution (5 mg/ml) at week 4 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count, Red cell distribution width, Total leucocyte count, Differential leucocyte 1(rel) count/SEG (Neutrophils) E0 (Eosinophils), BASO (Basophils), LYMPH (Lymphocytes), MONO (Monocytes)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin (total), Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein (total), Protein (albumin)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ORGAN WEIGHTS:
The following organ weights (and terminal body weight) were recorded at termination: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Testes

GROSS PATHOLOGY:
All animals surviving to the end of the observation period (day 29) were deeply anaesthetised by ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, Aorta, Brain, Cecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Esophagus, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), Nasopharynx, Ovaries, Pancreas, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, Tongue, Trachea, Urinary bladder, Uterus, Vagina

HISTOPATHOLOGY:
Slides of adrenals, heart, kidneys, liver, spleen and stomach, collected at termination from all animals of the control and high dose group and all gross lesions of all animals were prepared and examined by a pathologist. Based an the treatment-related morphologic changes, livers were also examined from all rats of group 2 (50 mg/kg/day) and 3 (200 mg/kg/day). All abnormalities were described and included in the report.
All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. A selection of liver sections were additionally stained with Best carmine for glycogen.

Statistics:

The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based an a pooled variance estimate was
applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated an the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

red blood cell parameters

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

inorganic phosphate, glucose and total bilirubin

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver and kidney

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
During the 28 day observation period, there were no clinical observations noted that were considered to be indicative of toxicity. Excessive salivation was noted an a few occasions in control animals, during intermittent periods in animals receiving 50 mg/kg/day and over the 28 day observation period in animals receiving 200 or 800 mg/kg/day. Although an increased incidence and degree was noted at higher treatment levels, this finding was considered not to be a toxic effect of treatment, as excessive salivation is often noted following oral treatment by gavage and may be attributed to bad taste or irritant effect of the test
substance. Alopecia is a common finding in rats of this age and strain and was occasionally noted in control and treated rats. Therefore this finding was
considered not to represent a change of toxic significance. Based an their incidental nature, other changes in clinical appearance of treated rats were considered of na toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period.

FOOD CONSUMPTION
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopical changes noted at week 4 of treatment, that could be attributed to treatment with HR 91/917 247

HAEMATOLOGY
Total red blood cell numbers (RBC), haemoglobin (HB) values and haematocrit (HCT) values were noted to be borderline, but statistically significantly lower than controls in females receiving 800 mg/kg/day. No such changes were observed in females receiving 200 or 50 mg/kg/day or in treated males.

CLINICAL CHEMISTRY
Inorganic phosphate values in males receiving 800 mg/kg/day were noted to be slightly, but statistically significantly increased when compared to control males. Inorganic phosphate values among females receiving 800 mg/kg/day or animals receiving 200 or 50 mg/kg/day remained similar to control values.
Glucose levels were noted as slightly, but statistically significantly decreased in males receiving 800 or 200 mg/kg/day. No differences from control levels were apparent in glucose levels of males receiving 50 mg/kg/day and treated females.
A slight, but statistically significant increase of total bilirubin was noted in the serum of females receiving 800 mg/kg/day, when compared to control females. Total bilirubin values of males receiving 800 mg/kg/day or animals receiving 200 or 50 mg/kg/day remained in the same range as control animals.
Other biochemical parameters that were recorded as statistically significantly different from control animals, remained within the range normally seen for rats of this age and strain and were considered to have occurred fortuitously.

ORGAN WEIGHTS
Liver weights in males and females receiving 800 mg/kg/day were statistically significantly increased in comparison with controls (males: 38.7%; females 17.0%). Among animals receiving 200 or 50 mg/kg/day no differences from liver weights of control animals were noted.
Relative kidney weights in males receiving 800 or 200 mg/kg/day were slightly, but statistically significantly higher than those of control males (200 mg dose group: 11.7%, 800 mg dose group: 13.2%). Relative kidney weights of males receiving 50 mg/kg/day or treated females remained comparable to control weights.
Compared to controls, statistically significantly increased adrenal weights were noted in females receiving 800 or 50 mg/kg/day before and after correction for body weight (50 mg dose group: 18.2% and 12.5% increase for absolute and relative adrenal weight, respectively; 800 mg dose group: 16.7% and 20.0% increase for absolute and relative adrenal weight, respectively) . However, as there was no clear treatment-related distribution and the control value was at the lower range of that normally seen in rats of this age and strain, these differences were considered not to reflect a toxicologically relevant change.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. All macroscopically observed changes, including renal pelvis dilatation, watery cyst in the left ovary, cyst in the left uterus harn and hernia diaphragmatica of the left median liver lobe, occurred incidentally in control or treated rats and considered not to exceed normal background variation for rats of this age and strain. These changes were therefore considered not to be of toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related changes were noted in the liver. Periportal hepatocellular hypertrophy, accompanied by fine hepatocellular vacuolation, was noted in 4 out of 5 males receiving 200 mg/kg/day (3 animals were scored "1 - minimal", 1 animal was scored "2 - slight") and all males receiving 800 mg/kg/day (2 animals scored "1 - minimal", 3 animals scored "2 - slight"). The vacuolated hepatocytes were negative for glycogen upon Best carmine staining. Among 3 out of 5 females receiving 800 mg/kg/day, liver changes were limited to slight (2 animals) or minimal (1 animal) hepatocellular hypertrophy. There were no other microscopic findings noted that were considered to be treatment related. The small number of changes recorded in treated animals were within the range commonly seen for rats of this age and strain.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed upon microscopic examination of the liver at 200 mg/kg bw.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Findings in males at this dose level are not considered adverse.

Critical effects observed:

not specified

Discussion

Analysis of concentrations of test substance formulations were within the range of +5% to +8% of nominal, which was considered to be acceptable for this type of formulation.

Polyethylene glycol (PEG) was used as the vehicle, and was dosed at 5 mL/kg bw. This corresponds to 4440 mg/kg bw. In a study by Hermansky (Hermansky et. al., 1995, Food & Chemical Toxicology, 33(2); p. 139 -149), referenced in the registration dossier of PEG, the following observations are made:

"Small increase in absolute and/or relative kidney weights observed. Although no microscopic changes were observed in the kidneys or urinary bladder, a slight, reversible renal toxicity may have resulted in male rats treated by gavage with 2800 mg/kg bw and rats of both sexes treated by gavage with 5600 mg/kg bw PEG 400/d)."

As a consequence, as the vehicle PEG is dosed at amounts exceeding its NOAEL, some of the effects observed in the current 28d study can be attributable to the vehicle, rather than to the tested substance.

The principal organ affected in this study was the liver. An increased liver weight was observed in males and females receiving 800 mg/kg/day. Nevertheless, only in the male animals the increase exceeded 20%. Although no macroscopically observed changes were apparent, microscopic examination revealed periportal hepatocellular hypertrophy, which was accompanied by fine hepatocellular vacuolation in males.

The findings at 200 mg/kg bw were seen in male rats only and are not of toxicological relevance when factors as dose relationship, historical controls and literature on histopathological findings were taken into account. Therefore, the NOAEL as determined from this study is 200 mg/kg bw/d.

Conclusions:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 200 mg/kg/day was established.

Executive summary:

In this subacute 28 -day toxicity study, the test substance was administered daily by gavage to SPF-bred Wistar rats. The study comprised of four groups. All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and an the day before necropsy. During week 4 of treatment, both eyes of all animals were examined. On the day of termination blood was collected from each animal for clinical laboratory investigations. Subsequently, macroscopic observations and organ weights were recorded. A histopathalogical examination was performed an adrenals, heart, kidneys, liver, spleen and stomach.

At 50 mg/kg/day: no treatment-related changes were observed.

At 200 mg/kg/day, the following effects were observed in males only: decreased serum glucose, increased kidney weights and microscopically observed periportal hepatocellular hypertrophy, accompanied by fine hepatocellular vacuolation. These findings are not of toxicological relevance when factors as dose relationship, historical controls and literature on histopathological findings are taken into account.

At 800 mg/kg/day a slight decrease in red blood cell numbers, haemoglobin values and haematocrit values was noted in females only. Also, increased clinical biochemistry values were found, including inorganic phosphate in males and total bilirubin values in females. Decreased clinical biochemistry parameters included glucose in males. An increased liver weight was noted in males and females and increased kidney weight was noted in males only. Microscopically observed periportal hepatocellular hypertrophy was noted in males and females. In males this was accompanied by fine hepatocellular vacuolation.

In conclusion, a NOAEL of 200 mg/kg bw is determined based on the outcome of the study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

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

Only one study is available assessing the effects of menthon glycerin-1,2-ketal upon repeated exposure. In this subacute 28-day toxicity study, 50, 200 or 800 mg/kg bw/d of the test substance was administered daily by gavage to SPF-bred Wistar rats. Body weight and food consumption were measured weekly and the day before necropsy. On the day of termination, blood was collected from each animal, and macroscopic and histopathological observations and organ weights were recorded.

At 50 mg/kg/day: no treatment-related changes were observed.

At 200 mg/kg/day, the following effects were observed in males only: decreased serum glucose, increased kidney weights and microscopically observed periportal hepatocellular hypertrophy, accompanied by fine hepatocellular vacuolation. These findings are not of toxicological relevance when factors as dose relationship, historical controls and literature on histopathological findings are taken into account. Moreover, some of the observed effects were likely to be related to the high dose of vehicle (PEG), which was administered at doses above its NOAEL.

At 800 mg/kg/day a slight decrease in red blood cell numbers, haemoglobin values and haematocrit values was noted in females only. Also, increased clinical biochemistry values were found, including inorganic phosphate in males and total bilirubin values in females. Decreased clinical biochemistry parameters included glucose in males. An increased liver weight was noted in males (+38%) and females (+17%) and increased kidney weight was noted in males only. Minimal to slight microscopically observed periportal hepatocellular hypertrophy was noted in males and females. In males this was accompanied by fine hepatocellular vacuolation.

In conclusion: a NOAEL of 200 mg/kg bw/d is determined based on the outcome of the study.

A 2 year repeated dose study with DL-menthol (Campbell, 1979) is added to this dossier for the purpose of the read-across justification. In this study, groups of 50 (male and female) Fischer 344 rats received DL-menthol via the diet, 7 days per week for 103 weeks. The nominal concentrations in the diet were 0.375% and 0.75%. Mean body weights of the dosed rats were slightly lower than those of corresponding controls. No other clinical signs related to administration of DL-menthol were noted in any dosed groups of rats. Survival at the end of the bioassay was at least 62% in all dosed and control groups of animals, and sufficient numbers of animals were at risk for the development of late-appearing tumours.
In male rats, no tumours occurred at incidences which were considered to be associated with the administration of DL-menthol.
In female rats, no tumours occurred at higher incidences in dosed groups than in control groups. Fibro adenomas of the mammary gland occurred at lower incidences in the low-dose (10/49) and high-dose (7/49) groups than in the control group (20/50), and alveolar/bronchiolar adenomas or carcinomas of the lung occurred only in the controls (3/50). It is concluded that under the conditions of this bioassay, DL-menthol was neither toxic nor carcinogenic for Fischer 344 rats. As no effects are observed the NOAEL in this study is > 375 mg/kg bw/d for DL-menthol. Extrapolation of this dose descriptor to the target chemical results in a NOAEL > 548 mg/kg bw/d for menthone 1,2-glycerolketal.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well documented study according to GLP and internationally accepted guidelines.

Justification for classification or non-classification

As described in section 3.9.2.9 of Annex I to EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP), the guidance values for classification for specific target organ toxicity following repeated exposure (STOT-RE) Category 2, based on a 28d study are: 30 -300 mg/kg bw/d.

However, the available study does not have a dose group tested at 300 mg/kg bw/d. Therefore, both the observations from the 200 and 800 mg/kg bw/d dose groups are compared to the criteria. The effects observed in the 200 mg/kg bw/d test group are not considered adverse. The effects observed at the 800 mg/kg bw/d dose group, though adverse, do not indicate functional organ impairment, nor are they comparable to any of the indicators provided in section 3.9.2.7.3 of Annex I to CLP. As a consequence, it can be concluded that classification of the substance for specific organ toxicity following repeated exposure (STOT-RE) is not required.

Likewise, the observed effects do not require classification as an R48 substance for danger of serious damage to health by prolonged exposure according to the criteria described in section 3.2.3 of Annex VI to Directive 67/548/EEC (Dangerous Substances Directive).