Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
From 24 May 2005 to 28 June 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines (observed deviation was not considered to have compromised the validity of the study and results), adequate coherence between data, comments and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
food consumption of all male groups was not recorded after mating until sacrifice in error
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3500 (Reproduction/Developmental Toxicity Screening test)
Deviations:
yes
Remarks:
as above
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: (P) x 11 wks
- Weight at study initiation (mean): (P) Males: 414 g; Females: 250 g; (F1) Males: 23 g; Females: 9 g
- Fasting period before study: no
- Housing: suspended wire-mesh cages with a metal tray containing autoclaved sawdust (females were transferred to polycarbonate cages containing autoclaved wood shavings as nesting material on approximately day 14 post-coitum)
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: for a period of 16 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50% +/- 20%
- Air changes (per hr): 12 cylces/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2 June 2005 To: 19 July 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% tween 80, prepared using purified water and tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 2, 10 and 50 mg/mL, according to the specific study procedure. The test item was administered as a suspension in the vehicle. The test item dosage formulations were prepared for use up to nine days and stored at +4°C, protected from light, with Teflon gaskets inside the bottle caps.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not indicated
- Concentration in vehicle: 2, 10 and 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurs or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- Mated for up to 14 days
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosage formulations were checked for homogeneity, stability and concentrations.
Check of homogeneity: on dosage forms were prepared at 2, 3 and 5 mg/mL
Check of stability: Each dosage form prepared for homogeneity analysis was sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light)
Check of concentration: in samples taken from each dosage form (including the control) prepared for use in weeks 1, 3 and 6.
Method: validated Gas Liquid Chromatography with Flame Ionisation Detection (GC/FID), Limit of Quantification = 0.02 mg/mL
Duration of treatment / exposure:
In the males:
− 15 days before mating,
− during the mating period (2 weeks),
− until sacrifice (i.e. at least 4 weeks in total),

In the females:
− 15 days before mating,
− during the mating period (2 weeks),
− during pregnancy,
− during lactation until day 5 post-partum inclusive (until sacrifice).

Actual duration: 34 days (males), 42-47 days (females)
Frequency of treatment:
Treatment was administered once daily (7 days per week)
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: [...] weeks: not indicated
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 100, 500 mg/kg/day
Basis:
other: nominal ingested (there was a satisfactory agreement between the nominal and actual concentrations of the test item in the administered dosage forms)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in conjunction with the Sponsor on the basis of the results of a 28-day toxicity study by oral route performed in the same species in which animals received 1, 20, 100 and 500 mg/kg/day.
A significant decrease in the body weight of high-dose females was observed in the last week of treatment in addition to a reduced food consumption.Males from the 500 mg/kg/day group exhibited a significant increase in mean platelet number whereas females exhibited a decreased chloride concentration. A number of alterations were observed in the urinalysis of mid- and high-dose groups. There were variations in organ weights, including the liver and kidney, in animals of both sexes, and adrenals in the 500 mg/kg/day females. Microscopic observations were limited to the kidneys of the mid- and high-dose males, including the appearance of hyaline droplets in the proximal tubular epithelium and regenerative changes of the renal tubules.

- Rationale for animal assignment (if not random):
Animals were allocated to groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and at least once daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment, then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mating (or until sacrifice) and on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was recorded once a week, over a 7-day period, from the first day of treatment until pairing for mating. The quantity of food consumed by each female was recorded once a week, over a 7-day period, from the first day of treatment through
gestation (days 0-7, 7-10, 10-14, 14-17 and 17-20 post-coitum intervals) and lactation (days 1-5 post-partum interval) until sacrifice. During the mating period, the food consumption was not recorded in either males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
No.
Sperm parameters (parental animals):
Parameters examined in all males of parental generation:
[testis weight, epididymis weight,
in control and high dose groups of male parental generation, the tailed and round spermatids, spermatocytes, spermatogonia and the different stages of the spermatogenic cycle were evaluated. In addition, attention was paid to the testicular interstitial cells.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, number of live, dead and cannibalized pups, presence of gross anomalies, weight gain, clinical signs]

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
[yes, for external and internal abnormalities]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the end of the mating period
- Maternal animals: all surviving animals = day 6 post-partum or, for females which had not delivered by day 25 post-coitum, day 25 or day 26 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on the ovaries, testes and epididymides of all males and females of the control and high-dose groups.
The tailed and round spermatids, spermatocytes, spermatogonia and the different stages of the spermatogenic cycle were evaluated. In addition, attention was paid to the testicular interstitial cells.
The testes and epididymides of all the males were weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on day 6 post-partum.
- These animals (and pups found dead) were subjected to postmortem macroscopic examinations.

GROSS NECROPSY
- Performed on all pups (surviving and found dead).

HISTOPATHOLOGY / ORGAN WEIGTHS
There was neither microscopic examination nor organ weighing.
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
hypoactivity, loud breathing, ptyalism, half-closed eyes, piloerection, round back and emaciated appearance
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
biologically significant reduced body weight gain during, at least, the premating period, at all dose-levels
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
biologically significant reduced body weight gain during, at least, the premating period, at all dose-levels
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male given 500 mg/kg/day had hypoactivity and half-closed eyes on day 15 of dosing while another had the same signs on days 12, 13 and 14 of dosing and loud breathing on day 26. All males had ptyalism from day 5 until the end of the treatment period. The deviation of the ptyalism was measured to be 4-5 hours after dosing, and may be related to the taste of the test item.
Two males given 100 mg/kg/day had ptyalism on day 25 of dosing while another had it on days 14, 15 and 16 and again between day 24 and day 27.
There were no clinical signs observed at 20 mg/kg/day.

Premating
Three females given 500 mg/kg/day had hypoactivity and half-closed eyes sporadically towards the end of the second week of dosing. One of these females (H25044) also had piloerection, round back and emaciated appearance on days 15 and 16 of dosing. All females had ptyalism from day 5 of dosing until the end of the premating period.
One female given 100 mg/kg/day had hypoactivity and half-closed eyes on day 12 of dosing. Seven of the ten females at this dose-level had ptyalism towards the end of the second week of dosing and the beginning of the third week.

Gestation
During the gestation period at 500 mg/kg/day, three females had hypoactivity and half-closed eyes at the beginning and (for two females) also at the end of gestation. One of the females also had loud breathing on day 8 post-coitum. Female H25044 again had round back and emaciated
appearance for the first four to six days of gestation. All females had ptyalism throughout the gestation period.
At 100 mg/kg/day, seven females had ptyalism, generally during the first half of the gestation period, but also towards the end for some females.

Lactation
At 500 mg/kg/day, two females had hypoactivity and half-closed eyes for up to 5 days and all females had ptyalism until sacrifice on day 6 post-partum.
At 100 mg/kg/day, one female had ptyalism on day 0 post-partum only. Areas of hairloss were observed, also in the control animals, but as this is commonly seen in rats kept under laboratory conditions, it was considered not to be related to treatment.
There were no clinical signs observed at 20 mg/kg/day during the study.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was a dose-related lowering of body weight gain for males and females at all dose-levels during the 2 week premating period, achieving statistical significance at 500 mg/kg/day. During the gestation period, females given 500 mg/kg/day continued to gain less weight than the controls (p<0.01) resulting in statistically significantly low mean body weights on day 20 post-coitum (p<0.01) and days 1 and 5 post-partum (p<0.05), while the other groups gained comparable amounts. During lactation, all female groups gained approximately comparable amounts of weight to the controls.

Male food consumption at all dose-levels was comparable with the controls throughout the study.
During the first week of dosing, females given 500 mg/kg/day consumed statistically significantly less food than the controls (-19%, p<0.001). During the second week, food consumption was still slightly reduced (-14%, not statistically significant). Food consumption remained less than the controls throughout gestation and lactation, achieving statistical significance from day 7 to day 10 and from day 14 to day 20 post-coitum.
There was no effect of treatment on food consumption at 20 or 100 mg/kg/day.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All pairs mated and the mean number of days taken to mate was comparable with the controls for all groups.
There was one non-pregnant female in each of the groups given 20 or 500 mg/kg/day. The remaining females were pregnant, although one female (H25037) in the 500 mg/kg/day dose group did not deliver and was found to have one dead fetus and nine implantation scars in the uterine horns at necropsy. From the poor body weight performance of this female during the gestation period, an unseen delivery was not suspected and therefore it is considered that the scars were implantations resorbed very early during the pregnancy.
The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups at any dose-level or on pup death after birth at 20 and 100 mg/kg/day. One female (H25045) in the 500 mg/kg/day dose group had a dead litter on day 3 post-partum and, as a result, the number of pups dying was statistically significantly higher at 500 mg/kg/day than for the control group.


ORGAN WEIGHTS (PARENTAL ANIMALS)
The differences observed were considered to be of no toxicological importance.


GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related necropsy findings were noted. The few necropsy findings encountered were recognized as those commonly recorded changes in the rat of this strain and age in this laboratory and none were considered to be of toxicological importance.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Morphological examination of the testes: no treatment-related abnormalities were found in the test-treated animals.
Ovaries: the follicular development and the number of corpora lutea were similar in both control and treated animals and showed no disturbance by treatment with the test item.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
at 500 mg/kg/day, one pup was found dead on day 1 post-partum, 11 pups were found dead on day 2 post-partum and the one remaining pup was found dead on day 3 post-partum
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lower body weight gain at all dose-levels
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
From one litter (dose-level of 500 mg/kg/day) one pup was found dead on day 1 post-partum, 11 pups were found dead on day 2 post-partum and the one remaining pup was found dead on day 3 post-partum. No clinical signs had been observed in the pups prior to death.

CLINICAL SIGNS (OFFSPRING)
One pup in the 20 mg/kg/day dose group had no tail. As this was an isolated finding and not also observed in the high-dose group, it was considered to be spontaneous in origin.
All other clinical observations (cold to the touch, anouria, tail necrosed, emaciated appearance, wound on abdomen...) were those commonly seen in pups of this age and as they were often also seen in control pups, they were considered not to be related to treatment.

BODY WEIGHT (OFFSPRING)
Pup weight gain from day 1 to day 5 post-partum was slightly lower than the controls’ at 100 mg/kg/day (-10%) and more markedly lower at 500 mg/kg/day (-22%). Pup body weights were lower than the controls’ on days 1 and 5 post-partum at 500 mg/kg/day and on day 5 post-partum at 100 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING)
There were no treatment-related gross findings.

OTHER FINDINGS (OFFSPRING)
The percentage of male pups was slightly low at 500 mg/kg/day, when compared with the controls.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
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

Conclusions:
Systemic toxicity was observed at all dose-levels, as evidenced by reduced body weight gain during, at least, the premating period (at least 13% less weight gained than the controls during the first 2 weeks of treatment) (statistically significant at 500 mg/kg/day). As body weight gains
which are reduced by more than 10% are considered biologically significant, all groups had biologically significantly reduced body weight gains. Clinical signs of hypoactivity and half-closed eyes were also observed at 100 and 500 mg/kg/day and food consumption was lower than controls’ for females given 500 mg/kg/day.
There were no effects of treatment on pairing but a relationship between development of the fetuses and pups and treatment cannot be excluded because there was one female with only implantation scars and a dead fetus in the uterine horns and another female which delivered a litter of small pups which consequently died on day 3 post-partum.
Mean pup body weight at 500 mg/kg/day was less than that of the controls on day 1 post-partum and pup body weight gain was reduced at 100 and 500 mg/kg/day.
There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day, and the NOAEL for toxic effect on reproductive performance and on progeny was 100 mg/kg/day.
Executive summary:

Methods:

Three groups of ten male and ten female Sprague-Dawley rats received the test item, 2-EHN (CAS No. 27247-96-7), daily, by oral (gavage) administration, before mating and through mating and, for the females, through gestation until day 5 post-partum, at dose-levels of 20, 100 or 500 mg/kg/day. Another group of ten males and ten females received the vehicle, 0.5% tween 80 in purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 10 mL/kg.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals. The dams were allowed to litter and rear their progeny until day 6 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post-partum.

The males were sacrificed after completion of the mating period. The body weight, testes and epididymides weights were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. A microscopic examination was performed, for the control and high-dose group, on the testes and epididymides with special emphasis on the stages of spermatogenesis and histopathology of interstitial testicular structure.

The dams were sacrificed day 6 post-partum (or from day 25 post-coitum for females which did not deliver) and a macroscopic examination of the principal thoracic and abdominal organs was performed. In the females which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. A microscopic

examination was performed on the ovaries of the control and high-dose group females.

The pups were sacrificed on day 6 post-partum and were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

Results:

There were no animals prematurely sacrificed for reasons of poor clinical condition during the study.

All animals given 500 mg/kg/day had ptyalism throughout the study which, when measured, lasted 4-5 hours after dosing and may be related to the taste of the test item formulations. Hypoactivity, half-closed eyes and loud breathing were also observed sporadically in a few males and females. One female also had emaciated appearance, round back and piloerection for some days during the study. The majority of the females given 100 mg/kg/day had ptyalism during the premating and gestation periods and a few males had ptyalism during the premating period, which may be related to the taste of the test item formulations. One female at 100 mg/kg/day had hypoactivity and half-closed eyes on day 12 of dosing. There were no clinical signs at 20 mg/kg/day.

Male and female groups at all dose-levels gained less weight than the controls during the premating period in a dose-related manner achieving statistical significance at 500 mg/kg/day. Females given 500 mg/kg/day continued to gain less weight during gestation, however all groups had approximately comparable weight gains to the controls during lactation. Females given 500 mg/kg/day consumed less food than the controls throughout the study. This was not also observed in the males and there were no effects of treatment at the other dose-levels.

All pairs mated and the mean number of days taken to mate was comparable with the controls for all groups.

There were no effects on the numbers of corpora lutea or implantations. The mean numbers of pups born per litter were comparable with the controls at all dose-levels.

At 500 mg/kg/day, there was one female with one dead fetus and nine implantation scars in the uterine horns and one female which delivered a litter of small pups, all of which were dead by day 3 post-partum. As pup survival was higher in the control group, a relationship to treatment cannot be excluded.

Mean pup body weight at 500 mg/kg/day on day 1 post-partum was non-statistically significantly lower than that of the controls. Pup weight gain from day 1 to day 5 post-partum was non-statistically significantly lower than the controls’ at 100 and 500 mg/kg/day in a

dose-related manner, and mean pup body weights were non-statistically significantly lower than the controls’ on day 5 post-partum at both dose-levels.

There were no treatment-related pup clinical signs or necropsy findings.

There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Conclusion:

The test item, 2-EHN (CAS No. 27247-96-7), was administered daily by oral gavage to male and female Sprague-Dawley rats, from before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 20, 100 or 500 mg/kg/day.

Systemic toxicity was observed at all dose-levels, as evidenced by reduced body weight gain during, at least, the premating period (at least 13% less weight gained than the controls during the first 2 weeks of treatment) (statistically significant at 500 mg/kg/day). As body weight gains which are reduced by more than 10% are considered biologically significant, all groups had biologically significantly reduced body weight gains. Clinical signs of hypoactivity and half-closed eyes were also observed at 100 and 500 mg/kg/day and food consumption was lower than controls’ for females given 500 mg/kg/day.

There were no effects of treatment on pairing but a relationship between development of the fetuses and pups and treatment cannot be excluded because there was one female with only implantation scars and a dead fetus in the uterine horns and another female which delivered a litter of small pups which consequently died on day 3 post-partum.

Mean pup body weight at 500 mg/kg/day was less than that of the controls on day 1 post-partum and pup body weight gain was reduced at 100 and 500 mg/kg/day.

There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day, and the NOAEL for toxic effect on reproductive performance and on progeny was 100 mg/kg/day.