Registration Dossier

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:
2009-09-04 to 2010-06-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(1995)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyclohexane, 1,4-bis[(ethenyloxy)methyl]-(1,4-Cyclohexandimethanoldivinylether)
- Physical state: liquid, colorless, clear
- Analytical purity: 98.8%
- Lot/batch No.: 90916716K0
- Stability under storage conditions over the test period: guaranteed by the sponsor; the sponsor holds this responsibility.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 11 to 13 weeks
- Weight at study initiation:
-- mean group body weight for males 311 - 340 g
-- mean group body weight for females 186.5 - 220 g
- Fasting period before study: no, but withdrawal of food of about 16 to 20 hours at test ending, before necropsy
- Housing: individually in Makrolon cages, type M III, floor area of about 800 cm2
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in olive oil were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. The maximum period for which each preparation was used was less than 7 days.
For the preparation of the administration solutions the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with olive oil and subsequently thoroughly shaked until completely dissolved.
Details on mating procedure:
Males and females from the same dose group were mated 14 days after starting the treatment, overnight in a ratio of 1:1 for a maximum of 2 weeks.
In the high dose group, since one male died, another one was mated twice, with two different females of the same dose group.
The animals were paired by placing the female in the cage of the male mating partner. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the dosing preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in olive oil for a period of 7 days at room temperature were carried out prior to the start of the study. Given that the test substance is completely miscible with olive oil, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations. Of each sample, one additional reserve sample was retained.
The stability of test substance in olive oil was demonstrated for a period of 7 days at room temperature.
All measured values for the test substance were in the expected range of the target concentrations (90-110%), demonstrating the correctness of the diet preparations.
Duration of treatment / exposure:
The male animals were sacrificed 29 days after the beginning of the administration, and examined.
The female animals were sacrificed at the end of 4 days of lactation (PND 4).
Frequency of treatment:
Single treatment, daily, from test start until sacrifice
Details on study schedule:
The test substance was daily administered by gavage to the parental animals (F0 generation); the treatment lasted up to one day prior to sacrifice. Females being in labor received no treatment. The animals of the control group were treated in the same way as those of the test group,
with the vehicle only (olive oil). The daily volume administered was 4 mL/kg bw.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200 and 1000/600 mg/kg bw/day
Basis:
actual ingested
[as toxicity was observed in almost every high-dose animal, as well as reduction of food consumption (45% in males) and body weight loss (-13.2 g) right after testing start, the initial level of 1000 mg/kg bw/d was reduced to 600 from day 4 onwards.]
No. of animals per sex per dose:
Ten animals per sex and dose group were used.
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
MORTALITY AND CLINICAL SYMPTOMS, PARENTAL ANIMALS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Littering and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams; on week days (except public holidays) the littering behavior of the dams was further inspected in the afternoon.

BODY WEIGHT, PARENTAL ANIMALS
The body weight of the male and female parental animals was determined once a week; body weight change was calculated from these results.
The following exceptions for the females were mentioned:
- During the mating period the females were weighed on the gestation day 0, 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on day post parturition (PND 4).

FOOD CONSUMPTION, PARENTAL ANIMALS
Food consumption was determined once a week (over a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period.
- Food consumption of the F0 females with evidence of sperm was determined on gestation day 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and PND 4.

REPRODUCTION PARAMETERS, PARENTAL MALES
The pairing partners, the number of mating days until vaginal sperm was detected in female animals, and the gestational status of the females were recorded for the F0 breeding pairs.
For the males, the mating index (number of males with confirmed mating X 100 / number of males placed with females) and the fertility index (number of males proving their fertility X 100 / number of males placed with females) were calculated.
Referring to sperm parameters, sperm motility (microscopy), sperm morphology (microscopy after staining with eosin), sperm head count in the cauda epididymis and in the testis (microscopy with MAKLER chamber after homogenization), were evaluated. Sperm motility examinations and the preparation of the specimens for sperm morphology were carried out in a randomized sequence; sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and high-dose test group, only.

REPRODUCTION PARAMETERS, PARENTAL FEMALES
The pairing partners, the number of mating days until vaginal sperm could be detected, and gestational status were recorded.
For the females, the mating index (number of females mated x 100 / number of females placed with males), the fertility index (number of females pregnant X 100 / number of females mated), and the gestation index (number of females with live pups on the day of birth X 100 / number of females pregnant) were calculated.
Referring to delivery data, the total number of pups delivered and the number of liveborn and stillborn pups were noted and the live birth index
(number of liveborn pups at birth X 100 / total number of pups born) was calculated.
Moreover, after sacrifice of the female animals, the implantation sites were counted and the post implantation loss (number of implantations - number of pups delivered X 100 / number of implantations) was calculated for each individual pregnant animal.

NECROPSY, PARENTAL ANIMALS
All F0 parental animals were sacrificed (decapitation under isoflurane anesthesia) for the purpose of necropsy and were subjected to gross pathology; particular attention was given to the reproductive organs. One male of the high dose group was found dead on day 7 and one female had to be sacrificed in extremis on day 30.
The weight of the anesthetized animals prior to sacrifice was determined, and following organs were weighed: testes, epididymides and ovaries.
Immediately after organ weighing, the right testis and cauda epididymides were taken from the F0 males of all test groups for evaluation of the sperm parameters (see above).
The following organs/tissues of the parental animals were fixed (4% neutral buffered formaldehyde solution or modified Davidson's solution): all gross lesions, adrenal glands, testes (modified Davidson's solution), epididymides (modified Davidson's solution), pituitary gland, prostate gland, seminal vesicles with coagulation glands, ovaries, uterus, oviducts and vagina.
After fixation, the samples were processed, sectioned and hematoxylin-eosin stained on slides for histopathological assessment by light microscopy. Gross lesions were correlated with histopathological findings. All gross lesions were examined. Examination of the testes, epididymides and ovaries was done for all males and females of the control and high dose groups.

PUPS AND LITTER PARAMETERS
All pups delivered from the F0 dams were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups. Check for dead or moribund pups was made twice daily on week days and once on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and pups dying during the lactation period (PND 0 to PND 4) were determined. The number of live pups/litter was calculated on the day of birth and on PND 4. Furthermore, the viability index (number of live pups on day 4 after birth X 100 / number of liveborn pups on the day of birth) was calculated.
The live pups were examined daily for clinical symptoms including gross morphological findings.
The sex of the pups was determined on PND 0 (anogenital distance) and was confirmed at necropsy of the pups (PND 4); The sex ratio was calculated (number of live male or female pups on PND 0/4 X 100 / number of live male and female pups on PND 0/4).
The pups were weighed one day after birth (PND 1) and on PND 4; body weight change was calculated from these results.
Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed less than 25% of the mean weight of the respective control pups).

NECROPSY OF THE PUPS
All surviving pups (after sacrifice on PND 4), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
Food consumption, body weight and body weight change (parental animals and pups), number of mating days, duration of gestation, number of pups delivered/litter, implantation sites, and post implantation loss were statistically assessed by simultaneous comparison of all dose groups with the control using the DUNNETT-test (two-sided) for the hypothesis of equal means.

Mating index fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, and number of litters with affected pups at necropsy were assessed by pairwise comparison of each dose group with the control using FISHER'S EXACT test for the hypothesis of equal proportions.

The proportions of affected pups per litter with necropsy observations were assessed by pairwise comparison of the dose group with the control using the WILCOXON-test (one-sided) for the hypothesis of equal medians.

The weight parameters at necropsy were assessed by means of the non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the p-value was =< 0.05, a pairwise comparison of each dose group with the control group was done using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

MORTALITY AND CLINICAL SYMPTOMS
One male animal of the high dose group 3 (1000/600 mg/kg bw/d) was found dead on day 7 of the study after showing unsteady gait after treatment, hypothermia, blood in bedding, salivation after treatment and piloerection. Gross pathology examinations revealed erosion/ulceration in the glandular stomach and discoloration of contents in the urinary bladder. Histopathology revealed a severe tubular nephrosis which was likely to be causative for a renal failure and the death of this male. One female animal of the same group was sacrificed because it was unable to deliver on GD 23. Gross pathology revealed “erosion/ulceration” in the glandular stomach. No association of the delivery problem to the treatment was assumed.
MALES:
In the high-dose group unsteady gait after treatment (weeks 0 - 2), chromodacryorrhea and piloerection (week 0) were observed. All of these findings were considered to be test substance-related signs of systemic toxicity.
In all three groups, nearly all males showed salivation after treatment. This transient salivation for a few minutes immediately after each treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
FEMALES:
High-dose females showed piloerection (week 0); in one case, unsteady gait after treatment (weeks 0 - 1) and chromodacryorrhea (week 0) was further reported. All of these findings were considered to be test substance-related signs of systemic toxicity.
Nearly all treated females showed salivation after treatment. This transient salivation for a few minutes immediately after each treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. During gestation and lactation, salivation still was observed after treatment.
One high-dose dam lost all pups. Two high-dose dams did not properly nurse their pups (pups had no or less milk in the stomach).

BODY WEIGHT, PARENTAL ANIMALS
The body weights of the high-dose males (1000/600 mg/kg bw/d) were statistically significantly lower between weeks 1 - 3 (up to 10%) and the body weight change was statistically significantly decreased between weeks 0 - 3 (about 68% below control). The high-dose males actually lost weight (-13.2 g) during the first treatment week. A body weight loss was also noted for the high-dose F0 females (-0.3 g) during the first treatment week; body weights and body weight gain were comparable to the concurrent controls during the remaining study.
Mean body weights and body weight changes of the parental males and females in the low- and mid-dose groups (40 and 200 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study.

FOOD CONSUMPTION, PARENTAL ANIMALS
Food consumption of the males and females of the high dose group (1000/600 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (about 45% for the males, and about 21% for the females). It became comparable to the concurrent controls during the remaining treatment period including gestation and lactation.
Food consumption of the mid-dose males (200 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (about 13%) whereas the low-dose males (40 mg/kg bw/d) did not show any test substance-related changes of food consumption during the whole treatment period. The mid and low-dose females did not show any test substance-related changes of food consumption during the whole treatment period.

REPRODUCTION PARAMETERS, MALES
For all males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the males within the scheduled mating interval. Two low-dose males (40 mg/ kg bw/d) did not generate F1 pups; since no histomorphological correlate was found to explain these apparent infertilities and since there was no dose-relationship, the finding was considered to be incidental.
Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

REPRODUCTION PARAMETERS, FEMALES
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.5 and 3.7 days without any relation to dosing.
All sperm positive rats delivered pups or had implants in utero except for two low-dose females that did not become pregnant; no histomorphological correlate was found to explain these apparent infertilities.
The fertility index varied between 80% in the 40 mg/kg bw/d group and 100% (control, 200 and 1000/600 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.1 days); the gestation index was 90% in the high-dose group and 100% in the control, low- and mid-dose group.
Implantation, prenatal development and delivery were not affected by the treatment since neither the mean number of implantation sites nor the post-implantation loss or the average litter size showed any statistically significant differences between the groups.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% (test group 3) and 100% (control and test groups 1 and 2). Moreover, the number of stillborn pups was comparable between the groups. Thus, the test substance did not adversely affect reproduction and delivery of the parental females.

GROSS PATHOLOGY, PARENTAL ANIMALS
In the high dose group (1000/600 mg/kg bw/d), enlarged liver was seen in 9/10 males, enlarged kidneys and discoloured kidneys were respectively found in 2 males.
Necropsy of the decedent male revealed erosion/ulceration in the glandular stomach and discoloured contents in the urinary bladder. The moribund sacrificed female was found to have erosion/ulceration in the glandular stomach.
Further findings were incidental and without treatment-relationship.

ORGAN WEIGHTS, PARENTAL ANIMALS
Compared to controls (= 100%) the terminal body weight of the males of the 200 and 1000/600 mg/kg bw/d group was statistically significantly decreased (respectively: 94%; p≤0.05 and 87%; p≤0.01)
Compared to controls (= 100%) only the relative testes weight of the males of the high dose group was statistically significantly increased (112%; p≤0.01). Since no histomorphological correlate was observed in the testes, the increase was regarded to be due to the decreased terminal body weight and not to the test substance.

HISTOPATHOLOGY, PARENTAL ANIMALS
No treatment-related findings were observed in the genital organs of males (testes and epididymides) and females (ovaries).
In males of high dose group (1000/600 mg/kg bw/d), treatment-related findings were reported for the kidneys (3/10) and liver (9/10). In fact, the kidneys showed a severe tubular nephrosis with presence of intratubular crystals (minimal), multifocal inflammation (polymorphonuclear granulocytes), severe tubular regeneration and slight to moderate hyperplasia of transitional cells in the papilla and pelvis. In the one animal that died, the kidneys showed a severe tubular nephrosis associated with a high amount of crystal deposits in cortex, medulla and pelvis, as well as inflammation and tubular regeneration. In the liver, minimal to slight centrilobular hypertrophy of hepatocytes was noted.
The severe tubular nephrosis may have been causative for a renal failure and the death of the decedent male. In the moribund sacrificed female only erosions and ulcers were found in the glandular stomach.
All other findings noted were considered to be incidental and/or spontaneous in origin.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(Reproductive Performance and Fertility)
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
(Systemic Toxicity)
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects indicative of systemic toxicity were seen in the males of the low dose group.
Dose descriptor:
NOAEL
Remarks:
(Systemic Toxicity)
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects indicative of systemic toxicity were seen in the females of the low and mid dose groups.
Dose descriptor:
LOAEL
Remarks:
(Systemic Toxicity)
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Remarks:
(Systemic Toxicity)
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: At 1000/600 mg/kg bw/d: Unsteady gait after treatment during treatment weeks 0-2; Chromodacryorrhea, piloerection during treatment week 0; Statistically significantly decreased food consumption (-21%) during treatment weeks 0 – 1.

Results: F1 generation

Details on results (F1)

PUPS AND LITTER PARAMETERS
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index indicating pup mortality during lactation (PND 0 - 4) was distinctly reduced (59%; p≤0.01) in the high-dose group, resulting from significantly higher numbers of died (15 versus 1 in control; p≤0.01) and cannibalized pups (23 versus 0 in control; p≤0.01). In the remaining groups the viability index varied between 98% (test group 2), 99% (control) and 100% (test group 1) without showing any association to the treatment.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mean body weights of the high-dose pups (1000/600 mg/kg bw/d) were statistically significantly below control on PND 1 and PND 4. The average difference to the control was 25% on PND 1 and increased to 32% on PND 4. The high-dose pups gained about 44% less weight than the controls.
The mid-dose pups (200 mg/kg bw/d) weighed still 15% less than the control on PND 4; however, the difference was only statistically significant in the females. The same is true for the body weight gain, which was about 22% below control, but being statistically significant only in the females.
No statistically significant changes on F1 pup body weights and body weight gain were observed in the low-dose group (40 mg/kg bw/d).
Furthermore, the number of "runts" was higher in the high dose group.

NECROPSY OF THE PUPS
Necropsy of the pups revealed empty stomach in 17% of the high-dose pups, which was secondary to disturbed maternal care and therefore due to treatment.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(Developmental Toxicity)
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects indicative of developmental toxicity were seen in the pups of the low dose group.
Dose descriptor:
LOAEL
Remarks:
(Developmental Toxicity)
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion