4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Endpoint summary

Description of key information

In conclusion, treatment with Kephalis by dietary inclusion in male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm revealed parental toxicity at 5000 and 15000 ppm. When corrected for mean test article intake, the parental NOAEL of 1500 ppm corresponds to 97-103 mg/kg for males and 127-175 mg/kg for females. Thus, the worse case scenario is for male animals with a NOAEL of 97 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:

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
3) OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
4) The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
5) Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the
European Union No. L142, May 2008.
6) OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity
Study in Rodents, October 2008.
7) The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day
oral toxicity study in rodents, July 2000.

List of protocol deviations

1. Deviations from the daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.

2. During the lactation period, no clinical observations were registered in the computer for the pups of litter 73 (Group 4) on Day 2.
Evaluation: Sufficient data were available for a thorough evaluation.

3. Inadvertently, the terminal body weight of animal no. 15 was not recorded, one eye of animal no. 4 and one mandibular lymph node of animal no. 42 were not available for histopathology.
Evaluation: Sufficient data were available for evaluation.

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Identification: Kephalis
Appearance: Pale yellow liquid
Batch: PE00097828
Purity/Composition: 85.2% (sum of two peaks)
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions: until 14 March 2015 (expiry date)
Purity/composition correction factor: No correction factor required
Test substance handling: Use amber glassware or wrap container in aluminium-foil
Chemical name (IUPAC), synonym or trade name: 4-(1-ethoxyvinyl)-3,3,5,5,-tetramethylcyclohexanone (main component)
CAS Number: 36306-87-3
EC Number: 252-961-2
Molecular formula: C14H24O2
Molecular weight: 224.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
Source: F0 Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11-12 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: Upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo.
Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 407 pups.
Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Animal Husbandry
Room number: A0.07 (A0.23 for motor activity measurements).
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hitemp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.

Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Diet Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Method: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. No correction was made for the purity or the specific gravity/density of the test substance.

Stability: Stability of diets for at least 8 days at room temperature in the concentration range of 1500-15000 ppm was confirmed in Project 505320.

Frequency: of preparation Diets were prepared once weekly.

Storage conditions: Diets were kept frozen at ≤ -15°C until needed, and at room temperature in the diet store room in the animal house thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (08 October 2014), according to a validated method (Project 505320). Samples of diets were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability of diet at room temperature over the concentration range used in this study was previously confirmed in Project 505320.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Random samples were taken from all groups and were stored at ≤-15°C for possible future analysis. Any remaining samples were discarded at finalization of the study report.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Oral, by inclusion in the diet - Ad libitum
The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test substance intake was estimated based on the body weight and food consumption values.

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study

Observations and examinations performed and frequency:

Parental animals

Mortality / Viability: At least twice daily.
Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations: The following tests were performed on the selected 5 animals/sex/group:
- hearing ability, pupillary reflex and static righting reflex (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum
and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Pups

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Sacrifice and pathology:

Pathomorphologic examination was performed on 80 Wistar (Han) rats (40 males, 40 females) which had been subjected to a combined 28 day oral (diet) toxicity study and reproduction/developmental toxicity screening test with the test item Kephalis.

The rats were assigned to four dose groups, each containing 10 males and 10 females. The test item was administered once daily by dietary inclusion at doses of 1500, 5000 and 15000 ppm (mg/kg diet) (dose Groups 2, 3 and 4 respectively). The rats of the control Group 1 received standard powder rodent diet without test item.

The males and females were exposed from 2 weeks prior to mating, during mating, and up to the day prior to necropsy for at least 28 days. All rats were necropsied. Histopathologic examination was performed on an extensive list of organs and tissues from 5 selected males and females of Group 1 and Group 4, thyroid glands, liver and spleen from selected Group 2 and 3 males and females, kidneys from selected Group 2 and 3 males. reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups as well as all organs with macroscopic findings from all rats. Additional slides of the testes were made of the 5 selected males of Groups 1 and 4 and all males suspected to be infertile or which died before mating and stained with
PAS/haematoxylin to assess spermatogenesis.

There were no test item related unscheduled deaths.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 6) was applied to compare the treated groups to the control group.
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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Parental animals
All females at 15000 ppm had pale feces during the second week of the treatment period. This was considered treatment related, but the effect was transient and in the absence of any overt signs of ill health was not considered adverse.

One control female had red coloration of the vagina over 2 days. Piloerection was noted for a single female for one day at 5000 ppm and for three females at 15000 ppm over 4-7 days. These signs were transient and occurred for only a few females and were therefore considered incidental in nature.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Parental animals
One male at 5000 ppm (no. 24) died while being anesthetized for blood sampling on the day of planned necrospy. This was not related to treatment with Kephalis.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals
Males at 15000 ppm had lower body weights and body weight gains beginning the second week of the premating period and persisting through Day 15 of the mating period. Females had lower body weights on Day 8 of the premating period and from Day 4 post coitum throughout the end of treatment (Day 4 of lactation). Body weight gains were also lower during this time, though the difference from controls were not always statistically significant.

Body weights and body weight gains were unaffected with treatment up to 5000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals
At 15000 ppm, absolute and relative food consumption were lower during the first week of treatment for animals of both sexes, but recovered thereafter for males. Relative food consumption was higher than controls for males during the mating period.
Absolute and relative food consumption remained lower for females throughout the post coitum and lactation periods (relative food consumption was not always significantly different from controls).

Absolute and relative food consumption was also lower for females at 5000 ppm during the first week premating and Days 0-11 post coitum, though differences from controls were not always statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Parental animals
Several changes in haematology parameters distinguished animals treated at 15000 ppm from controls. Males had relevant statistically significant increases in reticulocyte counts; their red blood cell distribution width (RDW) values were also correspondingly higher. As this was related to changes in spleen weight and microscopic findings, it was considered to be toxicologically relevant. High dose males also had significantly lower haemoglobin, haematocrit and mean corpuscular
haemoglobin concentration (MCHC) counts. Females at this dose level had significantly increased prothrombin time (PT) and activated partial thromboplastin time (APTT, not statistically significant) and lower monocytes, eosinophils, haemoglobin and haematocrit counts. The red blood cell distribution width was also lower than controls (not statistically significant). While these data remained within the range of available historical control data or were attributable to a high control value (haematocrit for
males and females), taken together with relevant changes in organ weights and microscopic findings, they were considered to be toxicologically relevant.

The statistically significant decrease in MCHC seen for males at 1500 ppm occurred in the absence of a dose response and was not considered toxicologically relevant at this level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals
At 15000 ppm, males had significantly higher cholesterol and creatinine (creatinine was also increased for males at 1500 and 5000 ppm). Females at this dose level had significantly higher potassium levels and lower levels of inorganic phosphate and aspartate aminotransferase (ASAT; also lower for females at 5000 ppm).
The remaining statistically significant differences seen for males at 15000 ppm were not considered toxicologically relevant as they remained within the range of available historical control data and/or were secondary to slightly low values obtained for controls (including total protein, albumin and calcium, see APPENDIX 5 for historical control data). Similarly, the statistically significant increase in urea seen for males at 5000 ppm occurred in the absence of a dose-dependent distribution and was not considered to be toxicologically relevant.

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals
Liver weights (absolute and relative) were significantly higher for animals of both sexes at 15000 ppm and for males at 5000 ppm. Relative liver weights were approximately 60% and 37% higher than controls for high dose males and females, respectively, and were 31% higher for males at 5000 ppm. Both sexes at 15000 ppm had significantly increased relative kidney relative weights compared to controls. Weights were 39% higher for males and 14% higher for females. Absolute kidney weights were also higher for males, though the difference from controls was not statistically significant was mainly attributable to the high value for male no. 32. High dose males had additional statistically significant differences in organ weights including lower terminal body weights, increased spleen (absolute and relative; 31% increase in relative weights), thyroid weights (relative only, 50% higher than controls), testes and epididymides (both relative only) weights.

The changes in testes and epididymides weights were not considered to be toxicologically relevant as they were due to the lower body weights, there were no effects noted during the microscopic evaluation and there was no treatment related effect on reproductive performance. The statistically significant increase in absolute and relative seminal vesicle weights noted for males at 1500 ppm were not considered to be toxicologically relevant as no dose dependent effects were seen and there were no treatment related microscopic findings noted in the reproductive organs.

All other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 15000 ppm two macroscopic findings noted correlated with relevant microscopic findings and included pale discoloration and enlargement of the kidney seen for a single male and accentuated lobular pattern of the liver seen for a single female. These correlated with a combination of hyaline droplet accumulation and granular casts in the kidney of the male and hepatocellular hypertrophy in the liver of the female and were thus considered toxicologically relevant.

All other macroscopic findings noted for control and/or treated animals were incidental in nature and included diaphragmatic hernia of the liver, yellowish, soft nodule on the epididymis, pelvic dilation of the kidneys, watery-clear cyst on the ovary and accessory liver (left lateral lobe). The findings remained within the range considered normal for rats of this age and strain, did not show a doserelated incidence trend and were therefore not considered to be toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

97 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: parental No Observed Adverse Effect Level (NOAEL)

Critical effects observed:

no

Accuracy and homogeneity of diet preparations were demonstrated by analysis. Parental results: At 5000 and 15000 ppm, toxicity was primarily characterized by adverse effects in the liver and kidneys (both sexes) and in the spleen (males). Relevant microscopic findings included hepatocellular hypertrophy in the liver. In the spleen, extramedullary hematopoiesis was dose dependently increased for males and decreased for females. In the kidneys, males had increased hyaline droplet accumulation and increased tubular basophilia (15000 ppm only) and granular casts were present. For animals at 15000 ppm, these findings were accompanied by relevant changes in the liver (also for males at 5000 ppm), kidney and spleen weights, changes in haematology and clinical biochemistry parameters indicative of altered function of these organ systems and lower body weights and food consumption. Relevant macroscopic findings were noted for only two animals, including discoloration and enlargement of the kidney (one male) and accentuated lobular pattern of the liver (one female). These corresponded to relevant histological findings (hyaline droplet accumulation and granular casts in the kidney and hepatocellular hypertrophy) and were thus considered toxicologically relevant. At 1500 ppm, similar microscopic findings were noted, but at a much lesser incidence and severity and were not considered adverse.

No treatment related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance and functional observations).

Conclusions:

In conclusion, treatment with Kephalis by dietary inclusion in male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm revealed parental toxicity at 5000 and 15000 ppm. When corrected for mean test article intake, the parental NOAEL of 1500 ppm corresponds to 97-103 mg/kg for males and 127-175 mg/kg for females. Thus, the worse case scenario is for male animals with a NOAEL of 97 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

97 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1 study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available