Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

alpha-iso-methylionone

IUPAC Name:

alpha-iso-methylionone

Test material form:

liquid

Details on test material:

Alpha-Iso-Methylionone
CAS: 127-51-5
Molecular weight: 206.32 g/mol
Molecular formula: C14H22O
Canonical SMILES: CC1=CCCC(C1C=C(C)C(=O)C)(C)C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD IGS BR strain

Details on species / strain selection:

Male and female Sprague-Dawley Crl:CD IGS BR strain rats were obtained from Charles River (UK) Ltd. (Margate, Kent)

Sex:

male/female

Details on test animals or test system and environmental conditions:

After 6 days of acclimatization, the animals were randomly allocated to treatment groups using a total randomization method and the group mean bodyweights were thereafter determined to ensure similarity between the test groups. Animals were housed according to sex in two groups of three and one group of four. At the initiation of treatment males weighed 131 to 172 g, females weighed 122 to 155 g, and were 6 to 8 weeks old. The study room was supplied with at least 15 air changes per hour. Temperature ranged from 19 to 23C and humidity from 40 to 70%. A 12:12-hour light-dark cycle period was used. Food was available ad lib, as was water. Wooden chew blocks and cardboard fun tunnels were provided for environmental enrichment.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The dose volume was 4 mL/kg/d.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations were stored in darkness at 4C and were tested for stability, homogeneity, and concentration. The formulations were observed to be stable for at least two weeks and to be within acceptable concentration limits (mean nominal concentrations of +/- 3%).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0 (vehicle), 5, 30 and 500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of a 14-day dose range finding (DRF) study. In the DRF, 3 rats per sex per dose were treated at 0 and 1000 mg/kg bw/day. No deaths were observed, however increased salivation, some slightly reduced body weights, and some pale kidneys were observed at 1000 mg/kg. Therefore, 500 mg/kg bw/day was selected as top dose in the definitive study.

Positive control:

Not included.

Examinations

Observations and examinations performed and frequency:

The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters included hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration), total leukocyte count, differential leukocyte count, platelet count, reticulocyte count, prothrombin time, and activated partial thromboplastin time. Blood chemistry parameters included urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorous, aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, and total bilirubin.

Sacrifice and pathology:

All rats were sacrificed on day 91. The following organs were weighed at all dose levels: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg: adrenals, thoracic aorta, bone and bone marrow (femur including stifle joint and sternum), brain (cerebrum, cerebellum, and pons), cecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum (including Peyer patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), esophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, and uterus.

Statistics:

Homogeneity of means: ANOVA or ANCOVA and Bartlett test.
Transformed data: Williams test (parametric) or the Shirley test (nonparametric).
Data showing no dose-dependency, but homogeneity of the mean: Dunnett (parametric) or Steel (non-parametric).
Pairwise tests (student t test parametric or Mann-Whitney U test nonparametric) were used on a case-by-case basis.
Histopathology data: Chi-square analysis and Kruskal-Wallis one-way nonparametric analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity.One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical.

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological effects were observed at any dose level.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

2

Dose descriptor:

LOAEL

Effect level:

<= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

3

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Applicant's summary and conclusion

Conclusions:

Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.

Executive summary:

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 5, 30 and 500 mg/kg/day for a total of 90 days. Dose levels were selected based on the results of a dose range-finding (DRF) study. In the DRF, three rats per sex per dose level were treated at 0 and 1000 mg/kg/day for 14 days. No unscheduled deaths were observed in the DRF, and clinical signs were limited to increased salivation at 1000 mg/kg. Male rats treated at 1000 mg/kg weighed slightly less than the controls on the 4th day of study. At necropsy, 2 males and 1 female treated at 1000 mg/kg showed pale kidneys. Based on these data, doses of 0, 5, 30 and 500 mg/kg/day were selected in the definitive 90-day study. The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters includedhemoglobin, erythrocytecount, hematocrit, erythrocyte indices (meancorpuscular hemoglobin, mean corpuscular volume, and meancorpuscular hemoglobinconcentration), total leukocyte count,differential leukocyte count, platelet count, reticulocytecount, prothrombin time, and activated partial thromboplastintime. Blood chemistry parameters included urea,glucose, total protein, albumin, albumin/globulin ratio, sodium,potassium, chloride, calcium, inorganic phosphorous, aspartateaminotransferase (AST), alanine aminotransferase, alkalinephosphatase, creatinine, total cholesterol, and total bilirubin.All rats were sacrificed on day 91.Thefollowing organs were weighedat all dose levels:adrenals, brain, epididymides, heart, kidneys, liver, ovaries,spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg:adrenals, thoracic aorta, bone and bonemarrow (femur including stifle joint and sternum), brain (cerebrum,cerebellum, and pons), cecum, colon, duodenum,epididymides, eyes, gross lesions, heart, ileum (including Peyerpatches), jejunum, kidneys, liver, lungs (with bronchi), lymphnodes (cervical and mesenteric), mammary glands, muscle (skeletal),esophagus, ovaries, pancreas, pituitary, prostate, rectum,salivary glands (submaxillary), sciatic nerve, seminal vesicles,skin (hind limb), spinal cord (cervical, midthoracic, and lumbar),spleen, stomach, testes, thymus, thyroid/parathyroid, tongue,trachea, urinary bladder, and uterus.All animals survived to planned death. Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity. One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical. No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed. Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed. No adverse effects on food intake, food efficiency, or water intake were observed during the study period. The ophthalmoscopic examination revealed no treatment-related effects at 500 mg/kg. No treatment-related effects on haematology were observed. Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study. No gross pathological effects were observed at any dose level. Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders). The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group. Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.