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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 20, 2014 - December 15, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-ethoxy-2,3-difluoro-4-[(1s,4r)-4-propylcyclohexyl]benzene
EC Number:
605-718-3
Cas Number:
174350-05-1
Molecular formula:
C17H24F2O
IUPAC Name:
1-ethoxy-2,3-difluoro-4-[(1s,4r)-4-propylcyclohexyl]benzene
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia s.r.l, on May 9th, 2014
- Age at study initiation: 10 weeks
- Weight at study initiation: (P) Males:347.3-373.9 g; Females 250.0-272.9 g
- Fasting period before study: -
- Housing: single
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 20 air changes per hour filtered on HEPA
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methocel KM4 Premium 0.25 % in Milli-Q
Details on exposure:
VEHICLE
- Concentration in vehicle: 0.25 % in pure water (Milli-Q)
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): DT381554
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 7 evenings/week with a maximum of 14 evenings
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the formulated test item was checked twice during the dosing period (i.e., on formulations prepared on one day of the pre mating and post mating periods). The HPLC analytical method was validated. A stability and homogeneity study in 0.25 % Methocel® K4M Premium in water was performed.
Duration of treatment / exposure:
F0 Males were treated for 2 weeks before the start of the mating period and throughout the same. Males were further dosed after the mating period until the minimum total dosing period of 28 days was completed. F0 Females were treated for 2 weeks before the start of the mating period and throughout the same. Treatment continued throughout pregnancy and until Day 3 of lactation. A concurrent control group received 0.25 % Methocel® K4M Premium aqueous solution with the same dosing regimens. All groups consisted of 10 males and 10 females.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
80 (40m / 40f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28 day study with similar chemical
- Rationale for animal assignment (if not random): random

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Body Weight and Food and Water Consumption: Yes
- Time schedule for examinations: Males and females were weighed on the day before the start of dosing (Day -1 of the study), on the first day of dosing (Day 1 of the study) and then weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (Day 0 of lactation) and on Day 4 of lactation. Weights were reported individually for each adult animal. During the pre-mating period and pregnancy, food and water consumption were measured at least weekly and during lactation on Day 4.
Sperm parameters (parental animals):
A detailed qualitative examination of one testis and the equilateral epididymis was done in 10 male rats of the control (vehicle) group, as well as in 10 males of the high dose group, taking intoaccount the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and disorganization of the germinal epithelium.
Litter observations:
On Day 0 of lactation, the pups of each litter were identified with a mark made by appropriately coloring different sites of the body; this mark was renewed when necessary. Live pups were counted and sexed and litters weighed within 24 hours of parturition (Day 0 of lactation) and on Day 4 post partum. At birth and during the lactation period, all the young were individually observed for:
- Live and stillbirths;
- Mortality ensuing after live birth, ascertained daily. Whenever possible, any pup found dead was examined externally and internally in an attempt to determine the cause of death;
- Sex (on Day 0 of lactation by an external examination and measurement of the ano-genital distance and by internal examination, at sacrifice, on Day 4 of lactation);
- External abnormalities at birth;
- Pup weight (on Days 0 and 4 of lactation)
Females were killed on Day 4 of lactation together with their pups.
Postmortem examinations (parental animals):
Necropsy and gross pathological examination were performed on all rats.
Males and females were sacrificed by cervical dislocation after preliminary CO2 anesthesia.

Histopathology
Reproductive organs (ovary, epididymis, prostate, seminal vesicle, testis) and all tissues showing abnormality were fixed.
Detailed histological examination was performed on the ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the highest dose group and the control group.
Postmortem examinations (offspring):
Necropsy and gross pathological examination were performed on all rats.
Pups were sacrificed by a pain-free method (i.e.. overdose of sodium pentothal i.p.).
Dead pups and pups killed on Day 4 of lactation were examined externally for gross abnormalities.
Statistics:
Body weight, food and water consumption, mortality, clinical signs, dosing, reproduction data, and fetal weight data were recorded and stored in the PROVANTIS™ system.
Reproductive indices:
- Mating index: percent ratio between the animals with positive smear + females found to be pregnant but without positive smear and the animals mated.
- Fertility index: percent ratio of females having evident signs of pregnancy with respect to the females that had positive vaginal smear and females found to be pregnant but without positive smear.
- Pregnancy index: the percent ratio of females with live births with respect to the pregnant females.
- Pregnancy period: the duration of pregnancy was determined for all those dams that reached pregnancy term as being the time that elapsed between the positive vaginal smear and the start of parturition.
- Mean pre-coital interval: was calculated on the dams proved pregnant and was expressed for each group as the mean time lapse (in days) between the beginning of the mating period and the ascertainment that copulation occurred. The females found to be pregnant but without a positive smear were excluded from this calculation.
Offspring viability indices:
- The mean F1 body weight was obtained by averaging the mean weight of each litter at the various times.
- The F1 probability of survival was analyzed per group. Animals that died owing to accidental causes or were sacrificed at the end of lactation were statistically censored.
- The mean value per litter of live pups (number of males and females and total) was calculated at different times (Days 0, 4 of lactation).
- The following indices of each litter were calculated:
Birth index: (No. of live newborns at birth/No. of implantations) x 100
Viability index on Day 4: (No. live pups on Day 4 after birth/No. of live births) x 100
Group mean values were calculated from individual data in two ways:
Mean A: calculated on all the surviving females having evident signs of pregnancy, including those that presented 100 % post-implantation losses.
Mean B: calculated only on those females with viable fetuses at term.
The external, visceral and skeletal malformations and variations found were described for each litter.
All data were recorded and stored in PROVANTIS TM System.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No findings or behavioral changes were observed during the study in any experimental animal except for one male of the 75 mg/kg bw/day treated group that showed piloerection from Day 4 to Day 12 of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males treated orally every day for a period of 28 days at doses of up to 225 mg/kg bw/d did not show any compound-related effect on body weight at any time point (including pre-mating and up to the end of the treatment period). Variations were observed in the mean body weight gain of the 75 mg/kg bw/day males in comparison to the control males, indeed a lower body weight gain was seen in this group during the days 7-14 interval, followed by a statistically significantly higher mean value during the subsequent period (days 14-21 interval). Therefore these findings were considered not compound-related since opposite trends were observed at different time points.
During the pre-mating period, the mean body weight and the mean body weight gain of the treated females were comparable to those of the control females. During pregnancy, minimal decreases (about 5 %) were seen in the mean body weight of the 225 and 75 mg/kg bw/day females in comparison to that of the control females from Day 14 onwards; a statistically significantly lower value being reached at 225 mg/kg bw/d as mean body weight, on Day 14, and at 75 mg/kg bw/d, during the days 14-20 interval as mean body weight gain. During the 4-day lactation period, the same figures were observed, the minimal decreases (about 5 %) in the mean body weight of the 225 and 75 mg/kg bw/day females still being present on Day 4 of lactation. No effects were seen at 25 mg/kg bw/d.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Weekly measurements of food consumption during the pre-mating period did not show any differences between treated and control males.
During the 2-week pre-mating period no differences in food consumption were observed between treated and control females. The mean daily food consumption of 225 mg/kg bw/d and 75 mg/kg bw/d treated females during pregnancy was slightly lower than that of the control females, a statistically significant level being reached during the days 0-7 and 7-14 intervals at 225 mg/kg bw/d and during the days 7-14 interval at 75mg/kg bw/d. During the 4-day lactation period the mean food consumption of the 225 and 75 mg/kg bw/day females was still slightly lower than that of the control females No effects were seen at 25 mg/kg bw/d during pregnancy and lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Weekly measurements of water consumption during the pre-mating period did not show any differences between treated and control males.
During the 2-week pre-mating period no differences in water consumption were observed between treated and control females. During pregnancy the mean water consumption of the 225 mg/kg bw/day treated females was slightly higher than that of the control females during the days 14-20 interval, reaching a statistically significant level. No other differences in mean daily water consumption were seen between treated and control animals. During the 4-day lactation period the mean water consumption of treated females was similar to that of the control females.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination performed on the ovaries, testes and epididymes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the 225 mg/kg bw/d group and the control group did not evidence any toxicological pathomorphological lesions.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The reproductive performance was unaffected, in fact, no differences were seen in the mating and fertility indices (ratio between mated and paired animals and ratio between pregnant and mated animals, respectively) among the various experimental groups, no compound-related effects were seen in the mean pre-coital interval was similar in all groups. No differences were seen in the pregnancy length and in the pregnancy index (ratio between females with live births and pregnant females) between treated and control groups. No compound-related effects were seen on pre-implantation losses (namely corpora lutea minus implantations) and on post-implantation losses (namely implantations minus live birth) at any doses, and no compound-related differences were observed in the viability index calculated on Day 4. Changes seen at 75 mg/kg bw/d as regards pre and post natal losses were considered not compound related since not confirmed at the highest dose and since the values fell within the normal range of variability of the historical control data for the laboratory.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The ano-genital distance of pups (males and females) belonging to the treated group was similar to those of male and female control pups, respectively, either when recorded at birth or on Day 4 at necropsy.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The mean weight of male and female pups at birth and on Day 4 post partum did not show any statistically significant differences between the treated groups and the control group. The ano-genital distance of pups (males and females) belonging to the treated group was similar to those of male and female control pups respectively, either when recorded at birth or on Day 4 at necropsy and the external sex corresponded to the internal one, checked at sacrifice, in allpups. Necropsy of pups on Day 4 did not evidence any compound-related changes.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed at the NOEL.

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:
For the test item 225 mg/kg bw/d was considered the NOAEL for reproduction and developmental toxicity.
Executive summary:

In this study the assessment and evaluation of the toxic characteristics of a chemical test item was performed in Crl:CD (SD) rats according to OECD 421. The test material was administered to the animals daily by oral route at 25, 75 and 225 mg/kg bw/day as follows:
F0 males were treated for 2 weeks before the start of the mating period and throughout the same. Males were further dosed after the mating period until the minimum total dosing period of 28 days was completed.
F0 females were treated for 2 weeks before the start of the mating period and throughout the same. Treatment continued throughout pregnancy and until Day 3 of lactation.
A concurrent control group received 0.25 % Methocel® K4M Premium aqueous solution with the same dosing regimens.
All groups consisted of 10 males and 10 females. All animals were observed for survival and clinical signs; in addition to the observations on parents, any abnormal behavior of the offspring was recorded. Body weight and food and water consumption were recorded at scheduled times during the premating, pregnancy and lactation periods.
On Day 0 of lactation pups were counted, sexed (by an external examination and measurement of the ano-genital distance, confirmed later by an internal examination at sacrifice) and weighed. All pups were individually observed for live and stillbirths; mortality ensuing after live birth was ascertained daily. Any pup found dead was examined externally and internally in an attempt to determine the cause of death.
Males were sacrificed at the end of the treatment period (Day 29 of the study), females were sacrificed on Day 4 of lactation with their litters. Females showing no evidence of copulation were killed 24-26 days after the last day of the mating period.
The uteri of apparently non-pregnant females were stained using the Salewski method and examined for the presence of implantation sites.
At the time of sacrifice all animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system of adult animals. The number of implantation sites and corpora lutea was recorded in females. The testes and epididymides of all male adult animals were weighed. Detailed histological examination was performed on the ovaries, testes and epididymes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the highest dose group and the control group.


All animals survived the treatment period. No clinical signs were observed in males and females at any time point except in one male of the 75 mg/kg bw/day group that showed piloerection for a few days during the treatment period.
No compound-related changes were seen in body weight, or food and water consumption of male animals during the 28-day treatment period.
At 75 and 225 mg/kg bw/d a slightly lower mean daily food consumption was observed in female animals during the first weeks of gestation that resulted in minimal changes (about 5 % less than controls) in mean body weight of these two groups at the end of gestation and up to end of the study (Day 4 of lactation). No changes were seen in the mean daily water consumption of treated females except for a slightly higher consumption at 225 mg/kg bw/d at the end of gestation.
No changes were seen in body weight, or food and water consumption of female animals at 25 mg/kg bw/d.
At necropsy of the adult animals no macroscopic changes were seen and no differences were noted in the mean weight of testes and epididymides of treated males in comparison with the controls.
Histological examination performed on the ovaries, testes and epididymes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the 225 mg/kg bw/d group and the control group did not evidence any toxicological pathomorphological lesions.
The reproductive performance was unaffected, in fact, no differences were seen in the mating and fertility indices (ratio between mated and paired animals and ratio between pregnant and mated animals, respectively) among the various experimental groups, no compound-related effects were seen in the mean pre-coital interval was similar in all groups.
No differences were seen in the pregnancy length and in the pregnancy index (ratio between females with live births and pregnant females) between treated and control groups.
No compound-related effects were seen on pre-implantation losses (namely corpora lutea minus implantations) and on post-implantation losses (namely implantations minus live birth) at any doses, and no compound-related differences were observed in the viability index calculated on Day 4.
Changes seen at 75 mg/kg bw/d as regards pre and post natal losses were considered not compound related since not confirmed at the highest dose and since the values fell within the normal range of variability of the historical control data for the laboratory.
The mean weight of male and female pups at birth and on Day 4 post partum did not show any statistically significant differences between the treated groups and the control group. The ano-genital distance of pups (males and females) belonging to the treated group was similar to those of male and female control pups respectively, either when recorded at birth or on Day 4 at necropsy and the external sex corresponded to the internal one, checked at sacrifice, in all pups.
Necropsy of pups on Day 4 did not evidence any compound-related changes.


In conclusion, the test material given orally to male rats for 28 consecutive days and to females during the pre-mating, mating and gestation periods and up to day 3 of lactation at the doses of 0, 25, 75 and 225 mg/kg bw/day only induced minimal changes, not dose-related, in food consumption and body weight of the 75 and 225 mg/kg bw/day females.
Reproductive performance was unaffected and no changes were evidenced in the reproductive organs at any dose. Offspring development and survival were considered not to be affected by the test-item, since changes seen at 75 mg/kg bw/d in the pre and post natal losses and in the postnatal survival were not confirmed at the highest dose and since the values fell within the normal range of variability of the historical control data for the laboratory.
On the basis of the above findings, 225 mg/kg bw/d was considered the NOAEL for reproduction and developmental toxicity.