2-ethoxy-1,3-dimethylcyclohexane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

with reproduction/developmental toxicity sceening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2015 - December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

endpoints 7.8.1 and 7.8.2

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)

Version / remarks:

March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test.

Version / remarks:

July 1995

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.

Version / remarks:

July 2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-ethoxy-1,3-dimethylcyclohexane

EC Number:

944-610-3

Cas Number:

286472-48-8

Molecular formula:

C10H20O

IUPAC Name:

2-ethoxy-1,3-dimethylcyclohexane

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 05-0293
- Phystical state: clear, colorless liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: RAW317-37
- Expiration date of the batch: 31 March 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

- Purity: 97.7%
- Specific gravity/density: 0.85 g/mL
- Stability in vehicle (corn oil): Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 10-12 weeks
- Weight at study initiation: males 278-327 grams, females 184-221 grams
- Fasting period before study: no
- Housing: during premating in groups of 5 animals/sex/cage; during mating females were caged together with males on a one-to-one-basis; during post-mating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed; during lactation pups were kept with the dam until termination; all in Macrolon plastic cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY:
No data of weghalen?

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 October 2015 To: 14 December 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (0.92) and density of the test item
(0.85 g/mL). No correction was made for the purity/composition of the test item

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg bw

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 (23 October 2015), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-53 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:

once daily for 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Dose levels were based on results of a 10-day dose range finding study in 3 females:
Clinical appearance included hunched posture and piloerection and/or salivation, uncoordinated movements, abnormal gait, noted intermittently during the observation period at 500 and 1000 mg/kg bw/d. Body weight was normal at 500 mg/kg bw/d and max 3% lower compared to day 1 level at 1000 mg/kg bw/d. Food consumption was slightly reduced at 1000 mg/kg bw/d. No abnormalities were noted at macroscopy. Liver weight was slightly increased at 1000 mg/kg bw/d and kidney weight was normal.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling)
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: General reproduction data, litter and pup examination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations:

the following organ weights from 5 animals/group were recorded:
Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles including coagulating glands, thyroid including parathyroid

from all remaining males: epididymides and testes

from all remaining females: ovaries and uterus (including cervix)

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 (many-to-one 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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals at 50 mg/kg and higher was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Swelling of the chest was noted for one female at 500 mg/kg from Week 5 of treatment onwards. This was considered to be related to the presence several black-brown, hard nodules in the subcutis of the axillary region at necropsy. At microscopic examination this was diagnosed as an adenocarcinoma of the mammary gland. Adenocarcinomas of the mammary gland can be seen as a spontaneous tumor in young female Wistar (Han) rats and this single incidence is regarded unrelated to the treatment with IFF 05-0293.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The higher body weight gain of females at 50 mg/kg over Days 1-8 of the premating period and of females at 50 mg/kg and higher over Days 1-4 of the lactation period were considered not to represent an effect of treatment. No dose-related trend was apparent, and control weight gain of females during lactation was considered to be slightly low compared to values expected for rats of this age and strain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The higher absolute and relative food intake of females at 50 mg/kg over Days 1-4 of the lactation period occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as they occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
− Higher total protein in males at 50, 150 and 500 mg/kg (no dose-related trend).
− Higher creatinine in males at 500 mg/kg.
− Higher calcium in males at 500 mg/kg.
− Higher chloride in females at 500 mg/kg.
Any other statistically significant changes in clinical biochemistry parameters were considered
unrelated to treatment as they occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant higher liver weights were noted in males at 150 and 500 mg/kg bw/d (absolute and relative to body weight) and in females at 500 mg/kg bw/d (relative to body weight). Mean relative liver weights were approximately 32% and 15% higher than the control mean at 500 mg/kg bw/d for males and females, respectively. At 150 mg/kg bw/d, mean relative liver weight of males was approximately 17% higher than the control mean.
Statistically significant higher adrenal gland weights were present in males (absolute and relative to body weight) at 150 and 500 mg/kg bw/d. Mean relative adrenal gland weight was approximately 29% higher than the control mean at both dose levels.
All other organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The recorded macroscopic findings were within the range of background findings encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were present in the thyroid gland (males), liver (both sexes), kidneys (males) and adrenal glands (females):
− Thyroid gland: Hypertrophy of follicular cells was recorded at a slightly increased severity in males at 150 and 500 mg/kg bw/d.
− Liver: Hepatocellular hypertrophy was recorded up to a slight degree in males and females starting at 150 mg/kg bw/d.
− Kidneys (males): An increased incidence and severity of hyaline droplet accumulation was recorded in males starting at 50 mg/kg bw/d. This was accompanied by a slightly increased severity of tubular basophilia at 500 mg/kg bw/d and with granular casts in one male at 50 mg/kg bw/d and two males at 500 mg/kg bw/d.
− Adrenal glands (females): An increased incidence and severity (up to slight degree) of vacuolation of the zona glomerulosa was recorded in females at 500 mg/kg bw/d.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed up to and including the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: kidney effects (tubular basophilia, granular casts, hyaline droplet)

Remarks on result:

other:

Remarks:

NOAEL for males can be considered as 500 mg/kg if kidney effects disregarded since that is not relevent for human

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Relevant for humans:

no

Any other information on results incl. tables

Accuracy of preparation: The concentrations analysed in the formulations at 50, 150 and 500 mg/kg bw/d were in agreement with target concentrations, with mean accuracies of 93, 98 and 96%, respectively. No test item was detected in the control group.

Homogeneity: The formulations at 50 and 500 mg/kg bw/d were homogeneous with coefficient of variation of 1.3 and 7.7%, respectively.

Applicant's summary and conclusion

Conclusions:

For IFF 05-0293 a parental NOAEL for systemic effects of 500 mg/kg bw/d was derived, based on the absence of test substance related effects up to and including the highest dose tested.

Executive summary:

IFF 05-0293 was administered via oral gavage to male and female Wistar Han rats at doses of 50, 150 and 500 mg/kg bw/d in corn oil (10 rats/sex/dose level) according to OECD 422 Guideline. Concurrent controls (10 rats/sex) received the vehicle. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-53 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No mortality or toxicologically related clinical signs were noted. No treatment-related effects on body weight, food consumption, functional behaviour, haematology, and macroscopy were observed.

An adverse histopathological lesion was recorded in the kidneys of males in all treatment groups. In these males, an increased incidence and severity (up to slight) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to likely represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. In this study the increased hyaline droplet accumulation in males was accompanied by indicators of renal tubular damage in the form of granular casts at minimal to slight degree (50 and 500 mg/kg bw/d). This combination of findings was considered to be non-adverse.

Histopathology showed a minimal or slight hepatocellular hypertrophy of the centrilobular areas of the liver for both sexes at 150 and 500 mg/kg bw/d. This histopathological observation was accompanied by higher liver weights in males at 150 mg/kg bw/d (relative weight approximately 17% higher) and in males and females at 500 mg/kg bw/d(relative weight approximately 32% and 15% higher, respectively). These findings occurred without any degenerative histopathological changes or corroborative changes in clinical biochemistry parameters and were therefore considered to be an adaptive, non-adverse finding. Other histopathological changes that were considered to be related to treatment but were considered not adverse in nature were recorded in the adrenal and thyroid glands. Follicular cell hypertrophy as recorded in the thyroid gland of males at 150 and 500 mg/kg bw/d with a minor increase in severity (slight degree) was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded. An increased incidence and severity of vacuolation of the zona glomerulosa of the adrenal glands was seen in females at 500 mg/kg bw/d. Given the slight nature of vacuolation of the zona glomerulosa of the adrenal glands, and absence of any degenerative findings, this was considered to be non-adverse. Additionally, higher adrenal gland weights were present in the opposite sex at 150 and 500 mg/kg bw/d, and were approximately 29% higher at both dose levels. In absence of any morphological support, the higher adrenal gland weight was considered not adverse in nature. Statistically significant changes in clinical biochemistry parameters consisted of higher total protein in males at 50 mg/kg bw/d and higher, higher creatinine and calcium in males at 500 mg/kg bw/d and higher chloride in females at 500 mg/kg bw/d. These changes were considered not toxicologically relevant since no (clear) dose-related trend was noted, changes were generally minor in nature (within the range considered normal for rats of this age and strain), and no apparent relation to morphological lesions described above was seen.

Based on these results a parental NOAEL for systemic effects of 500 mg/kg bw/d was derived.