2-ethoxy-1,3-dimethylcyclohexane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

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)

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:

Identification IFF 05-0293
Appearance Clear liquid
Batch RAW317-37
Purity/Composition 97.7%
Test substance storage At room temperature protected from light
Stable under storage conditions until 31 March 2016 (expiry date)

Test animals

Species:

rat

Details on species / strain selection:

Rat: Crl:WI(Han) (outbred, SPF-Quality).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant
females and untreated animals were used at initiation of the study.
Source F0 Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment Approximately 10-12 weeks.
Number of F0-animals 40 females and 40 males.
Acclimatization F0 At least 5 days prior to start of treatment

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Method Oral gavage, using a plastic feeding tube. Formulations were placed on a
magnetic stirrer during dosing.
Dose volume 5 mL/kg body weight. Actual dose volumes were calculated according to
the latest body weight.
Frequency Once daily for 7 days per week, approximately the same time each day
with a maximum of 6 hours difference between the earliest and latest
dose.
Exposure period 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 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). Female nos. 56 and 64 (Group 2
and 3, respectively) were not dosed on Day 1 of lactation as these
females were littering at the time of dosing. The omission of one day of
dosing over a period of several weeks was not considered to affect the
toxicological evaluation

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 was confirmed, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who
had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Remarks:

oral gavage

Details on analytical verification of doses or concentrations:

according to a validated method (Project 510061). 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%.
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 (Project 507920).

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 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). Female nos. 56 and 64 (Group 2
and 3, respectively) were not dosed on Day 1 of lactation as these
females were littering at the time of dosing. The omission of one day of
dosing over a period of several weeks was not considered to affect the
toxicological evaluation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day
with a maximum of 6 hours difference between the earliest and latest
dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

Mortality / Viability At least twice daily.
Clinical signs At least once daily from treatment onwards up to the day prior to
necropsy, detailed clinical observations were made in all animals after
dosing. 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, recorded as the mean of three
measurements per animal (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 are
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 (all before blood sampling). These tests were performed after
observation for clinical signs (incl. arena observation, if applicable) at no
specific time point, but within a similar time period after dosing for the
respective animals.
Body weights Males and females were weighed on the first day of exposure (prior to
first 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 for
evidence of premature delivery. 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.

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. Animals showing pain, distress or discomfort, which was
considered not transient in nature or was likely to become more severe,
were sacrificed for humane reasons based on OECD guidance document
on humane endpoints (ENV/JM/MONO/ 2000/7).
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):

Clinical Laboratory Investigations: Haematology and Clinical Biochemistry
Pathology: Necropsy, organ weight and histopathology

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded1. The stomach of
pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.
If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse 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. 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.

Reproductive indices:

Mating index (%) = Number of females mated/Number of females paired x 100
Fertility index (%)= Number of pregnant females / number of females paired x 100
Conception index (%) = Number of pregnant females / number of females mated x 100
Gestation index (%) = Number of females bearing live pups /number of pregnant females x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Percentage live males at First Litter Check =Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check = Number of live female pups at First Litter Check /Number of live pups at First Litter Check x 100
Percentage of postnatal loss = Number of dead pups before planned necropsy / number of live pups at first litter check x 100

Offspring viability indices:

Viability index = Number of live pups before planned necropsy / number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were noted that were considered toxicologically relevant.
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 (no. 79) 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 (Ref. 5) and this single incidence is regarded
unrelated to the treatment with IFF 05-0293.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

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.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.
− Liver: Hepatocellular hypertrophy was recorded up to slight degree in males and females
starting at 150 mg/kg.
− Kidneys (males): An increased incidence and severity of hyaline droplet accumulation was
recorded in males starting at 50 mg/kg. This was accompanied by a slightly increased severity
of tubular basophilia at 500 mg/kg and with granular casts in one male at 50 mg/kg and two
males at 500 mg/kg.
− 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.
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.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Details on results (P0)

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation
sites were unaffected by treatment.
For female nos. 59 and 70 the number of pups was slightly higher than the number of implantations.
This was considered caused by normal resorption of these areas as these enumerations were
performed on Day 6 or 7 of lactation.
All females (except for one control couple (male no. 10/female no. 50) showed evidence of mating. All
females were pregnant and delivered offspring, except for two control couples (male nos. 6 and 9 /
female nos. 46 and 49). For these control couples that did not have offspring, no abnormalities were
seen in the reproductive organs, which could account for their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be
attributed to the test item and spermatogenic staging profiles were normal for all males examined.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

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):

Clinical signs
Incidental clinical symptoms of surviving pups consisted of scabs, wounds, and blue spots on the
snout or abdomen. The control pup sacrificed in extremis showed a wound on the chest and right front
leg. The nature and incidence of these clinical signs remained within the range considered normal for
pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Mortality
One pup of the control group was sacrificed in extremis, and one pup each at 50 and 500 mg/kg were
found dead or missing during lactation. The missing pup was most likely cannibalised. No toxicological
relevance was attributed to these dead/missing/sacrificed 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:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The pup found dead at 500 mg/kg had no milk in the stomach. The nature and incidence of this finding
remained within the range considered normal for pups of this age, and was therefore considered to be
of no toxicological relevance.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

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 500 mg/kg in an combined 28 day repeated dose toxicity study with reproductive/developmental screening assay according to OECD TG 422.

Executive summary:

IFF 05-0293 was administered by daily oral gavage to male and female Wistar Han rats at dose levels

of 50, 150 and 500 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to

termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating,

during post-coitum, and at least 4 days of lactation (for 40-53 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this

study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and

implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

Three control females did not deliver offspring; two were not pregnant and one control female showed

no evidence of mating, No abnormalities were seen in the reproductive organs, which could account

for their lack of offspring. It was considered that sufficient control data were available to allow for a

valid interpretation of the study data, also considering the absence of reproductive and developmental

toxicity up to the highest dose tested in this study.

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg).

No treatment-related changes were noted in any of the developmental parameters investigated in this

study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup

development consisting of mortality, clinical signs, body weight and macroscopy).

In conclusion, treatment with IFF 05-0293 by oral gavage in male and female Wistar Han rats at dose

levels of 50, 150 and 500 mg/kg revealed adverse parental effects in males only, consisting of

degenerative lesions in the kidneys at 50 mg/kg and higher. No reproduction and developmental

toxicity was observed for treatment up to 500 mg/kg.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL (males): - below 50 mg/kg (based on degenerative kidney lesions (i.e. granular

casts and tubular basophilia, related to hyaline droplet accumulation)

- At least 500 mg/kg, when male rat specific kidney findings are

disregarded.

Parental NOAEL (females): at least 500 mg/kg

Reproduction NOAEL: at least 500 mg/kg

Developmental NOAEL: at least 500 mg/kg