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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

252-961-2

EC Name:

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

Cas Number:

36306-87-3

Molecular formula:

C14H24O2

IUPAC Name:

4-(1-ethoxyethenyl)-3,3,5,5-tetramethylcyclohexan-1-one

impurity 1

Reference substance name:

(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

Molecular formula:

C14H24O2

IUPAC Name:

(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

impurity 2

Reference substance name:

(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one

IUPAC Name:

(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one

impurity 3

Reference substance name:

(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

IUPAC Name:

(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

impurity 4

Reference substance name:

1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene

IUPAC Name:

1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene

impurity 5

Reference substance name:

3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene

IUPAC Name:

3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene

Test material form:

liquid

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.

Test animals

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.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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.

Details on mating procedure:

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.

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).
Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Oral, by inclusion in the diet - Ad libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Doses / concentrations 3

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Examinations

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.

Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for 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. Palpation was used to aid in confirmation of pregnancy. 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.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

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. If possible, defects or cause of death were evaluated.
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.

Postmortem examinations (parental animals):

All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food and thus were exposed to the test diet until the day of necropsy.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Necropsy was conducted on the following days:
Condition Day of necropsy
Females which delivered Lactation Days 5-7.
Females which failed to Post-coitum Days 26
deliver1 (no. 46)
Males Following completion of the mating period (a minimum of 28 days of dose administration)

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (from Klinipath, Duiven, The Netherlands).
All pups were sexed and descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated.

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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):

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):

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):

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):

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
One control female was not pregnant.
For female nos. 49 (control), 60 (1500 ppm), 70 (5000 ppm) and 74 (15000 ppm) the number of pups were slightly higher than the number of implantations and/or corpora lutea. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Days 5-7 of lactation.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The only clinical sign noted was scabs on the snout, seen for a single control pup (litter no. 49). This was incidental.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were three, one and three pups in the 1500, 5000 and 15000 ppm groups, respectively, that died or went missing during the first days of lactation. Missing pups were most likely cannibalised. No pups died or went missing in the control group. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups at 15000 ppm were statistically significantly lower than for pups in the control groups. The slightly lower pup body weights were considered secondary to lower maternal body weights at this level. As the differences from controls were slight and the weights remained within the available historical control data range (see APPENDIX 5), they were not considered to be adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Cannibalism was an incidental macroscopic finding seen for one pup found dead at 1500 ppm (litter no. 57). There were no other macroscopic findings noted for any pup.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The mean number of living pups/litter at the first litter check were 10.2, 10.9, 10.7 and 9.5 pups in the control, 1500, 5000 and 15000 ppm groups, respectively.

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

other: Macroscopy

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

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. No reproductive or developmental toxicity was noted up to 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. The reproductive and developmental NOAELs of at least 15000 ppm corresponds to 925-1051 mg/kg for males and 1095-1518 mg/kg for females.