Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-08 till 2016-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March, 1996

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Limit test:

no

Test material

Test material information

1

Reference substance name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

EC Number:

944-405-9

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 13-0156
- Phystical state: clear, yellowish liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Specific details on test material used for the study:

- Substance name in the test report: FRET 13-0156
- Physical state: clear liquid
- Storage conditions: 15-25 °C, dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Outbred rats (RccHanTM:WIST)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Schulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: Males: 303.38 – 307.00 g; Females: 188.58 – 190.68 g
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of four per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating females found sperm-positive on the same date were considered a "lot") and by the animal number within each lot the mated females were housed in the order of animal number). After delivery, the cage containing the dam with litter was transferred to another cage rack, the location being determined by delivery date and animal number as described for mated females. On the day of FOB testing and motor activity assessment, animals were temporarily kept singly.
- Diet: ad libitum, from their arrival, the rats received a cereal-based (closed formula) powder rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England).
- Water: ad libitum
- Acclimation period: > 5 days

DETAILS OF FOOD AND WATER QUALITY: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: two times during the study (on 03 December 2015 and on 04 January 2016)
- Mixing appropriate amounts with: VRF1 (FG) diet
- Storage temperature of food: ≤ -18 °C

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 1 week had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From both batches of diets prepared in the study (03 December 2015 and 04 January 2016), samples were taken and stored in a freezer (≤ -18 °C). Analyses to determine the stability, homogeneity and content of the test substance in the test diets were conducted as indicated below.
- Content: the content of the test substance at each dietary level was determined in the batches prepared on 03 December 2015 and 04 January 2016.
- Homogeneity: the homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (03 December 2015), by analyzing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analyzed.
- Stability: to demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 03 December 2015 (low-dose diet, mid-dose diet and high dose diet) were analyzed at t=0 and reanalyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.

Duration of treatment / exposure:

Duration of study, following acclimatisation, is dependent on the female performance and is approximately 54 days, [at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 4 days lactation].

Frequency of treatment:

Continously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg diet

Remarks:

Equivalent to at least 84.56 and 104.34 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

5 000 mg/kg diet

Remarks:

Equivalent to at least 279.86 and 332.8 mg/kg bw/day for males and females, respecively

Doses / concentrations 3

Dose / conc.:

15 000 mg/kg diet

Remarks:

Equivalent to at least 870.22 and 794.48 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale: the doses are based on the results of a dose-range finding study.

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: in the morning hours and the afternoon. On Saturdays, Sundays and public holidays only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and then once weekly until sacrifice for males, or until delivery of the first lot for females.

BODY WEIGHT: Yes
- Time schedule for examinations: one day before the start of the treatment and at the start of the study (day 0). Males were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during gestation and on day 0 and 4 of lactation. One non-mated female was weighed once per week after the mating period. All animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the end of the premating period (day 13)
- Anaesthetic used for blood collection: Yes, CO2/O2
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to the end of the premating period (day 13)
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly prior to sacrifice
- Dose groups that were examined: 5 males/group and in 5 females/group with a litter
- Battery of functions tested: FOB and motor activity

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined on days 0 and 4 in [F1] offspring: total litter size, numbers of each sex, number of stillbirths, live-and dead pups and grossly malformed pups. Mean pup weight was calculated per sex and for both sexes combined.

GROSS EXAMINATION OF DEAD PUPS:
Yes, grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. At necropsy of the dams, at or shortly after day 4 of lactation, pups were examined externally for gross abnormalities and sacrificed by gradual CO2 anesthesia, followed by irreversible hypothermia.

Postmortem examinations (parental animals):

SACRIFICE
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia and then examined grossly for pathological changes.

GROSS NECROPSY
Gross necropsy consisted of ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), testes, epididymides, seminal vesicles (with coagulating glands), prostate, all gross lesions.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on or shortly after day 4 of lactation.
- Pups were examined for gross abnormalities

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 or p<0.01. Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods. Additional information is supplied in the "any other information on materials and methods".

Reproductive indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated/number of females placed with males) x 100
- male mating index = (number of males placed with females /number of females inseminated) x 100
- female fertility index = number of pregnant females*100/number of inseminated females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites) / number of corpora lutea] x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered.

Offspring viability indices:

- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100
- sex ratio day 0 = [(number of live male or female pups on day 0 / number of live pups on day 0] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male died during the mating period. The death of this rat was not treatment-related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food intake in the high-dose group females was statistically significantly lower in the first week of the premating period, but recovered thereafter. This was considered to be related to the palatability of the test substance.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Phospholipids were statistically significantly increased in both males and females of the highdose group and glucose plasma levels were statistically significantly decreased in females of the high dose group.
- In absence of a dose response relationship statistically significant differences in PO4 in males of the low-dose and mid-dose group were considered not related to treatment. ASAT and ALAT activity were statistically significantly decreased in males of the low-dose (ASAT) and high-dose group (ASAT and ALAT). No toxicological relevance was attached to these findings, because the results for ALAT activity were within the historical control range (see Table 7a) and for ASAT and ALAT activity an increase rather than a decrease in these parameters is considered to represent a toxic effect.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The control group male that was found dead during the mating period showed pelvic dilatation and tubular dilatation in the kidneys. In addition the kidneys were a bit autolytic. The stomach was moderately autolytic and the thymus was severely autolytic. The prostate showed mild mixed inflammation with hemorrhagic material in the lumina. The prostate was a bit autolytic.
As the male was not exposed to the test substance, these findings were considered incidental findings, not related to the treatment.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Details on results (P0)

Fertility was not affected by the treatment. In each group 12 females were placed with males for mating. The male and female mating index was 100 % in all groups. All females were mated except one female in the control group. There were no treatment-related differences in pre-coital time. Reproductive performance was not affected by the treatment. All mated females were pregnant and had live litters. In each group the duration of gestation was comparable and the gestation index was 100%. No differences were found on the mean number of corpora lutea and implantation sites between the groups and pre-implantation loss was not affected by the treatment. There were no treatment-related differences in prenatal or perinatal loss. There were no litters with stillborn pups. One dead pup was found in the control group. In the mid dose group 1 pup and in the high dose group 2 pups were missing (cannibalized).

Effect levels (P0)

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One pup in the control group and one pup in the high dose group showed a wound. Another pup in the control group showed cyanosis. There were no treatment-related signs in pups during the lactation period.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic examination could be performed on the pups that were missing during the lactation period. The one pup in the control group that was found dead was not macroscopically examined.

Effect levels (F1)

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical results - the analytical report concludes that:

- The test substance was homogeneously distributed in the diet at each dose level.

- The test substance was stable in the test diets for the low and high dose groups under the experimental conditions of 4-days storage in the animal room. In the mid dose test diets concentrations did not meet the acceptance criteria related to the actual measured t = 0 concentration. However, when related to the nominal concentration the difference is 3% and accepted.

- Upon storage in a closed container in a freezer for more than 5 weeks concentrations were 14-20% lower as compared to the actual measured concentrations and therefore FRET 13- 0156 was considered to be unstable upon storage in a freezer, resulting in a loss of maximal 20% as compared to the t=0 concentration.

- The concentration of the test substance was close to intended (90-110%) for all diets at all dose levels, except for the low dose diet (1500 mg/kg) prepared on 3 December that differed +14% from the nominal. This slight deviation from the acceptance criteria was considered acceptable.

Applicant's summary and conclusion

Conclusions:

Based on the absence of adverse effects on male and female fertility and reproductive performance, the NOAEL was determined to be ≥15000 mg/kg diet corresponding to an intake of at least 894 mg/kg body weight per day in females and at least 870 mg/kg body weight per day in males.

Executive summary:

In this GLP compliant study performed according to OECD guideline 422, the possible effects of the test substance on general toxicity and reproductive performance and development of pups were examined in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 1500, 5000 and 15000 mg/kg diet during a premating period of 2 weeks and during mating, gestation until day 4 of lactation. These dietary levels provided an overall mean intake of at least 104, 333 and 894 mg/kg body weight per day in females of the low-, mid- and high-dose group and at least 85, 280 and 870 mg/kg body weight per day in males of the low-, mid- and high-dose group.

 

The content and homogeneity of the test substance in the carrier were analysed and accepted. Stability testing showed a decrease in concentration after storage of the test substance in the carrier in the freezer for more than 5 weeks and slightly lower concentrations in the mid dose group after 4 days in the animal room. There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no relevant differences in body weights or food consumption during the premating period, during the post-mating period in males, or in dams during the gestation period and the lactation period.

 

Hematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. No effects were observed on hematology parameters. In males and females of the high-dose group phospholipids were increased compared to controls, glucose plasma levels were decreased in females of the high-dose group and liver weight was increased in males of the high-dose group. In absence of macroscopic and microscopic changes, these effects were considered treatment-related, but not adverse. Macroscopic/ Microscopic examination revealed no treatment-related effects. There were no effects of the test substance on fertility and reproductive performance.

 

Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOAEL for reproduction and developmental effects was placed at ≥ 15000 mg/kg diet (the highest concentration tested; equivalent to an overall intake of at least 894 mg/kg bw/d in females and at least 870 mg/kg bw/d in males).