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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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

1
Chemical structure
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
- Lot no.: RAW317-40
- Expiration data: June 2016
- Purity: 97.1%

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
- 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
Details on route of administration:
The food was provided to the rats as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice weekly with fresh portions from the freezer and topped up if needed.
Vehicle:
unchanged (no vehicle)
Details on oral 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
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 / concentrationsopen allclose all
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
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
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.


- Section schedule rationale (if not random):
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
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. Non-mated female 45 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

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
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".

Results and discussion

Results of examinations

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 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
Description (incidence and severity):
- Mean liver weight and kidney weight was increased in the males of the high dose group, reaching statistical significance for the relative organ weights.
- In females of the high dose group a trend towards increased mean liver weight and kidney weight did not reach statistical significance.
- There were no statistically significant weight changes in the other organs including the male reproductive organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The control group male that was found dead during the mating period did show enlarged and flabby kidneys. The stomach was empty of feed but contained some yellow fluid and the thymus was small, the prostate was red and the bladder contained hemorrhagic fluid.
As the male was not exposed to the test substance, these findings were considered incidental findings, not related to the treatment.
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.

Effect levels

Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical 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 thenominal. 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 rats, 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 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.

Haematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. No effects were observed on haematology 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, litter data or pup observations in any group.

-Based on the absence of adverse effects the NOAEL for parental effects was placed at the level of 15000 mg/kg diet (highest concentration tested; equivalent 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).