Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 12 July 2000 to 03 April 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to OECD guideline and according to GLP. The analytical investigation at 5mg/ml were carried out as part of a preliminary study which was in compliance with GLP. In the current report no compliance was claimed for this data. However, this did not affect the validity of the report.
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
(1983)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation (P): approximately 5 weeks
- Weight at study initiation (P): 22,4 to 35,1 g for males and 18,2 to 27,1 g for females
- Fasting period before study:
- Housing: Mice were housed singly in MT2 cages (29cm x 11.5cm x 11cm) from North Kent Plastics Limited, Erith, Kent, England throughout the study (except during cohabitation for pairing and when females were with their litter during lactation).
- Diet: commercially available laboratory animal diet (VRF-1), ad libitum. During week 11 of the study, the diet in four food hoppers (group 4 males) was found to be contaminated with mould. Contaminated food hoppers were replaced with food hoppers containing a new batch of diet. Upon completion of the study data from these animals were scrutinized and no effect on the outcome of the study was seen.
- Water: water from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): The animal room had its own supply of filtered air, under positive pressure, which was passed to the atmosphere without recirculation.
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose in 0.1% Tween 80
Details on exposure:
The dosages of the test item were prepared weekly as a mixture in the vehicle. For each concentration the required quantity of test item was weighed, and added to the vehicle and then mixed thoroughly until it was visibly homogeneous. The formulations were re-mixed before and during dosing using a magnetic stirrer.
The animals were dosed daily for 8 weeks prior to pairing until termination at a volume-dosage of 10 mL/kg bw/day. Control animals received the vehicle at the same volume-dosage during the treatment period. The volume administered daily to each animal was based on the last scheduled bodyweight for males throughout the study and for females during the pre-pairing period. For females after mating the volume administered daily was based on the bodyweight recorded before dose administration.
Details on mating procedure:
After eight weeks of treatment, males and females from the same treatment groups were paired on a one-to-one basis. Each morning following pairing the females were examined for the presence of a copulation plug. The day on which positive evidence of mating was found was designated Day 0 of gestation.
One male was killed before pairing and the prospective female of this male was paired with a male that mated successfully within that group. One female at 150 mg/kg/day showed no positive evidence of mating after 14 days of pairing. Therefore, the original male partner was replaced by a male that successfully mated within that group.
Once mating had been detected the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test substance during ambient temperature storage for 2 days and refrigerated storage for 15 days were confirmed at nominal concentrations of 5 and 50 mg/ml in a preliminary study (Huntingdon Life Sciences Ltd. 000054). A copy of this analytical report was attached to the current report.
The homogeneity and stability during ambient temperature storage for 2 days and refrigerated storage for 15 days were confirmed at a nominal concentration of 60 mg/ml as part of the present study. The storage period represented the maximum time from preparation to completion of use. In addition, the stability of discrete (1 ml) samples was confirmed during freezer storage for 8 days.The mean concentrations of test substance analysed during the study (Weeks 1, 9 and 13) were between 1.3% below and 3.6% above nominal concentrations, confirming the accuracy of formulation.
Duration of treatment / exposure:
The animals were dosed daily by the oral route (gavage) for 8 weeks prior to pairing and until termination at a volume-dosage of 10 mL/kg bw/day.
Frequency of treatment:
once daily
Details on study schedule:
After eight weeks of treatment, 13 weeks old males and females from the same treatment groups were paired on a one-to-one basis. Each morning following pairing the females were examined for the presence of a copulation plug. The day on which positive evidence of mating was found was designated Day 0 of gestation. From Day 17 after mating females were inspected three times each day for the onset, progress and completion of parturition. All offspring were examined at approximately 24 hours after birth.
Remarks:
Doses / Concentrations:
50, 150, 600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex and group
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CLINICAL SIGNS AND MORTALITY
All animals were examined at least twice daily throughout the study and any visible signs of reaction to treatment were recorded. Detailed clinical observations were recorded daily during the first week of treatment and weekly thereafter.
Animals sacrificed or found dead were examined macroscopically and specimens of tissues considered abnormal were retained.

BODYWEIGHT
Males were weighed twice weekly for the first two weeks of the study and weekly thereafter. Females were weighed twice weekly for the first two weeks of the study and then weekly until mating was detected. After mating females were weighed on Days 0, 6, 12 and 17 after mating and on Days 1, 4, 7, 14 and 21 of the lactation period.

FOOD CONSUMPTION
Food consumption was recorded weekly for F0 males and females until the animals were paired for mating. Food consumption for females after mating was recorded for Days 0, 6, 12 and 17 of gestation and Days 1, 4, 7 and 14 of the lactation period.

BLOOD SAMPLING FOR PLASMA T3 AND T4
Immediately before termination each animal was bled under isoflurane anaesthesia from the orbital sinus using lithium heparin as an anticoagulant. Approximately 0.5 ml of whole blood was taken from each animal. The samples were then centrifuged and the plasma divided into 2 aliquots, one of which was assayed for total thyroxine (T4) and the other assayed for total tri-iodothyronine (T3) by radioimmunoassay.

ASSESSMENT OF REPRODUCTIVE PERFORMANCE
The time elapsing between initial pairing and detection of mating was recorded.
From Day 17 after mating females were inspected three times each day for the onset, progress and completion of parturition. The time elapsing between the detection of mating and commencement of parturition was reported. Dams were observed daily for evidence of abnormal maternal behaviour.
Sperm parameters (parental animals):
Immediately after sacrifice the left vas deferens, epididymis and testis of each male was removed and the epididymidis and testis were weighed.
A sample of sperm was expressed from the vas deferens into pre-warmed (37°C) medium Ml99, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). At least 200 sperm per animal were analysed for:
a) The percentages of motile and progressively motile sperm for all groups,
b) Sperm morphology after staining with Nigrosine and Eosin.

The left cauda epididymis of each male was weighed and homogenised in 1 ml of a mixture of 0.9% saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT-mixture) An aliquot of this mixture was assessed for sperm count. The concentration (Million/g) and total number of sperm were reported for Groups 1 and 4 only.
Homogenisation-resistant spermatids
The left testis of each male was weighed and frozen. Prior to analysis, the testis was allowed to thaw, 1 ml of SMT-mixture was added and the testis was homogenised. An aliquot of this mixture assessed for homogenisation-resistant spermatid count. The concentration (Million/g) and total number of spermatids were reported for Groups 1 and 4 only.
Litter observations:
POST-NATAL OBSERVATIONS (Fl OFFSPRING)
Observations at Day 1 of age
All offspring were examined at approximately 24 hours after birth (Day 1 of age) and the a) Number of offspring (live and dead), b) Bodyweights of live offspring - weighed individually on Days 1, 4 (before culling), 7, 14, and 21 of age, c) Sex ratio and d) Observations on individual offspring were recorded for each litter.
Observations after Day 1:
Litters were observed daily for evidence of abnormal appearance or behaviour and mortality of pups. Any offspring found dead were examined externally and internally. Litters containing 11 or more offspring were culled by random selection to 10 (where possible 5 males and 5 females per litter) on Day 4 of age. Culled offspring were killed and discarded without necropsy.
Group mean litter size and litter weights were calculated from the individual litter values. Group mean offspring bodyweight was calculated separately for male and female offspring from the individual litter values.
Postmortem examinations (parental animals):
NECROPSY, F0 GENERATION
The males were sacrificed following successful littering of the females and the females following weaning of their progeny. Immediately before termination each animal was bled under isoflurane anaesthesia from the orbital sinus using lithium heparin as an anticoagulant. Approximately 0.5 ml of whole blood was taken from each animal. The samples were then centrifuged and the plasma divided into 2 aliquots, one of which was assayed for total thyroxine (T4) and the other assayed for total tri-iodothyronine (T3) by radioimmunoassay. After blood sampling the animals were sacrificed by inhaled carbon dioxide and each animal was subjected to a detailed macroscopic examination.

ORGAN WEIGHTS
The following organs, taken from each F0 parental animal were weighed (paired organs were weighed separately): Adrenal glands, Prostate (ventral lobe, males), Brain, Seminal vesicles and Coagulating gland (males), Epididymides (males), Spleen, Kidneys, Testes (males), Liver, Thyroid, Ovaries (with oviduct, females), Uterus with cervix (females), Pituitary.

TISSUES PRESERVED FOR EXAMINATION
Samples of Adrenal glands, Brain, Epididymides (right), Kidneys, Liver, Mammary glands (caudal), Ovaries (with oviduct), Pituitary, Prostate (ventral lobe), Seminal vesicles and Coagulating gland, Spleen, Testis (right), Thyroid, Uterus with cervix, and Vagina, as well as tissues with abnormalities were preserved in 10% neutral buffered formaldehyde (NBF), except for the testes and epididymides, which were preserved in Bouin's fluid for at least 24 hours before transfer to 70% industrial methylated spirit.

HISTOPATHOLOGY
Following tissue samples from parental animals of group 0 and 4 were dehydrated, embedded in paraffin wax, sectioned, stained with Haematoxylin and Eosin and investigated microscopically: Epididymides(right, caput, corpus and cauda), Liver, Ovaries with oviduct(left and right), Prostate (ventral lobe), Seminal vesicles and Coagulating gland, Testis (right), Uterus with cervix, Vagina and tissues with abnormalities.
Testes were stained using a standard Periodic Acid-Schiff (PAS) method. Examination of the ovary of F0 females was limited to a qualitative assessment of the presence of primordial follicles, growing follicles and corpora lutea.
Tissues with abnormalities from all mice should have been subjected to histopathological examination. Since findings were seen across all groups and were clearly not treatment related, microscopic examinations were not performed on these tissues.
Postmortem examinations (offspring):
NECROPSY, F1 OFFSPRING (Day 21 post natal)

ORGAN WEIGHT
Brain, Liver, Spleen and Thymus were weighed from the first male and the first female selected from each litter. In addition, following organs were retained from these animals:
Brain, Epididymides, Liver, Ovaries (with oviduct), Prostate (ventral lobe), Seminal vesicles and coagulating gland, Spleen, Testes, Thymus, Uterus with cervix, Vagina, Tissues with abnormalities.

HISOPATHOLOGY
As no treatment related findings were observed at macroscopic examination in the offspring, tissues from these animals were not examined microscopically.
Reproductive indices:
For each group and sex the Percentage mating, Conception rate, Fertility index, Gestation length and Gestation index were calculated.
Offspring viability indices:
Survival indices for each group were calculated from individual litter values: Post-implantation survival index, Live birth index, Viability index, Lactation index.


CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs were seen at any dose group. No treatment related mortality was observed. However, one male of the high dose group was sacrificed moribund; one female of the high dose group and one females of the control group was found dead. A further female of the control group was sacrificed moribund. None of these mortalities was treatment related.
One total litter loss was seen in each of the treatment groups on or before Day 1 of age. These litter losses were considered to be not treatment related.

BODYWEIGHTS
No effects on body weight were seen in any dosage group.

FOOD CONSUMPTION
The food consumption of males and females remained unaffected by treatment.

REPRODUCTIVE PARAMETER
No effects on mating performance, fertility, gestation length, parturition and gestation indices were seen at any dosage level.
PLASMA T3 AND T4 (details see table 1)
T3 level were slightly increased in animals of both sexes, while T4 level were slightly decreased only in male animals at 600 mg/kg bw/d when compared to control. Only the decreased T4 levels of males at 600 mg/kg bw/day were statistically significant. Since the T3 level was elevated in both sexes and effects on T3 and T4 Level were only seen in the highest dose group these changes in T3/T4 levels could be treatment related.

NECROPSY F0 Parents
No treatment related macroscopic abnormalities were seen at necropsy in males or females.

ORGAN WEIGHTS F0 Parents
Organ weights of female and male sex organs were unaffected by treatment.
At 600 mg/kg bw/day the absolute and relative liver weight (approx. 20% of control) was increased in both sexes. At 150 mg/kg bw/d the absolute and relative liver weight (10% of control) of females was increased, while only relative liver weights for males were increased (5% of control). The increase in liver weights was considered to be an adaptive response to treatment.
Absolute and relative thyroid weights (with parathyroid) of males and females were increased at 600 mg/kg/day (14% and 19% of control in males and 9% and 7%in females, respectively).
Other findings like increased absolute spleen weights in females or increased relative kidney weights in males showed no clear dose response relationship and were considered to be not treatment related.

SPERM ANALYSIS F0 Males
No effects on any parameter examined (motility, progressive motility, epididymal sperm count, homogenisation-resistant spermatids from the testis and sperm morphology) were seen at 600 mg/kg bw/d. Due to the absence of effects at the highest dose group sperm analysis was not conducted at the lower treatment groups.
HISTOPATHOLOGY F0 Parents
An increased incidence of hepatocyte hypertrophy of the centrilobular regions of the liver was seen in 23/32 males and 10/32 females at 600 mg/kg bw/day. The high incidence of centrilobular hepatocytic hypertrophy was considered to be an adaptive response to treatment.
No further treatment related findings were seen upon histopathology. There were no apparent effects on the reproductive organs of males and females.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 150 mg/kg bw/day liver weights were increased in both sexes. Since no histopathological correlates were seen at this dose this effect was considered to be not adverse.
Dose descriptor:
NOAEL
Remarks:
(reproduction performance and fertility)
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Since no effects on mating performance, fertility, gestation length, parturition and gestation indices were seen at the highest dose level tested, the NOAEL was set at 600 mg/kg bw/day.
CLINICAL SIGNS
No clinical signs were seen in the offspring of parental animals from any dose group.
The number of uterine implantation sites (recorded at termination), litter size at birth and survival of offspring to Day 4 showed minor intergroup variation without dose response relationship. No effects on survival to Day 21 were seen. The sex ratio from Day 1 to Day 4 and to Day 21 after birth was comparable throughout the study.

BODYWEIGHT
No differences in bodyweights of offspring at birth and no effects on bodyweight change during lactation were seen.

NECROPSY
No treatment related effects were seen upon macroscopic examination of pups dying before weaning or pups sacrificed at weaning.

ORGAN WEIGHTS
No effects on brain, spleen liver or thymus weights were seen.
Dose descriptor:
NOAEL
Remarks:
(developmental)
Generation:
F1
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Littering, survival and development of the F1 progeny were similarly unaffected at dose levels up to and including 600 mg/kg bw/day.
Reproductive effects observed:
not specified

Table 1: Blood T3 and T4 level in males and females

 

 

Dose (mg/kg bw/d)

0

50

150

600

MALES

Total T3

nmol/l

1.41 +/ -0.16

1.43 +/- 0.17

1.42 +/- 0.17

1.51 +/- 0.14

Total T4

nmol/l

52 +/- 11

53 +/- 12

49 +/- 13

44 +/- 11*

FEMALES

Total T3

nmol/l

1.38 +/- 0.19

1.32 +/- 0.20

1.37 +/- 0.19

1.4 +/- 0.22

Total T4

nmol/l

62 +/- 20

59 +/- 13

57 +/- 18

54 +/- 15

*, p<0,05

Conclusions:
The No-Observed-Effect-Level for reproductive function and survival, growth and development of the offspring to weaning in the context of this study was 600 mg/kg/day.
Executive summary:

The test substance was administered to male and female CD-1 mouse in a one-generation study according to OECD guideline 415 (1983) via gavage in doses of 50, 150 and 600 mg/kg bw/d for a total of 16 weeks.

No treatment-related mortalities, no clinical signs and no effects on bodyweight and food consumption were seen in parental animals of both sexes even at the highest dose group.

At 600 mg/kg bw/d the relative liver weights of males and females were increased and at histopathology centrilobular hepatocytic hypertrophy was seen in these animals. Liver weights were also increased at 150 mg/kg bw/day in both sexes. Since no histopathological correlates were seen at this dose this effect was considered to be not adverse.

Thyroid weights were increased in males at 600 mg/kg bw/day. T3 level were slightly increased in animals of both sexes, and T4 level were slightly decreased only in male animals at 600 mg/kg bw/d when compared to control. Although only the decreased T4 levels of males reached statistically significance, these changes in T3/T4 levels could be treatment related.

Due to the increased liver weights with histopathological correlates and the increased thyroid weights which possibly resulted in the changes in T3/T4 levels, the NOAEL for parental toxicity was considered to be 150 mg/kg bw/day.

 No effects on mating performance, fertility, the ability of the females to successfully rear a litter to weaning and the condition, survival and development (to weaning) of the offspring were seen even at the highest dose group. No treatment related macroscopic findings were seen. Thus, the NOAEL for reproduction and developmental toxicity was 600 mg/kg bw/day, respectively.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The mouse model was chosen in agreement with the German Competent Authority to avoid the masking of possible human-relevant effects by massive non-relevant rat-specific effects. Although both rat and mouse do not own thyroxine binding proteins, the effects of phenolic antioxidants on the excretion rate of T3 and T4 in the mouse are known to be slighter than in the rat. It was therefore expected that the mouse model allows the evaluation of human-relevant effects. To gain additional information on the effects on the metabolic and hormonal status, blood samples were taken in the preliminary study (Huntingdon, 2001) to assess the thyroid function (by measuring T3 and T4). Additionally, at termination liver samples were assessed for peroxisomal palmitoyl CoA-oxidase and microsomal lauric acid 12-hydroxylase activity.

Range finding study in mice (Huntington, 2001)

The test substance was administered by gavage at dosages of 50, 150 or 500 mg/kg bw/day to groups of 10 male and 10 female (F0) mice for 15 days prior to pairing and until termination, after weaning, of the filial (F1) generation. Control animals received the vehicle.

No mortality was observed and clinical signs for males and females were unremarkable and are not considered due to treatment. There were no adverse effects on the bodyweight or the food consumption. Mating performance, fertility, gestation and parturition showed no obvious effects of treatment. The size of the F1litters, sex ratios, litter survival, bodyweights and the growth of the offspring to Day 21 of age were not adversely affected by treatment of the F0 parents.

Necropsy of offspring and the F0 adults did not reveal any apparent macroscopic effects of treatment there were no apparent effects on the organs of reproduction. The test substance showed no obvious adverse effects on the general condition, thyroid function, mating performance, fertility, or the progress of gestation or lactation in the treated mice. Furthermore, littering and survival and development of the F1progeny were similarly unaffected. Parental liver weights at 500 mg/kg bw/day were high and hepatic lauric acid 12-hydroxylase activity was increased at this dosage for the males and females and at 150 mg/kg bw/day for the females only. The substance seems to act as a weak inducer of the cytochrome P450 CYP4A subfamily. There was no evidence that the substance was acting as a peroxisome proliferator in the mouse after seven weeks of treatment.The No Observed Adverse Effect Level (NOAEL) was established at 500 mg/kg bw/day.

 

One generation oral toxicity study in mice (OECD 415, GLP) (Huntington, 2001)

The test substance was administered by gavage at dosages of 50, 150 or 600 mg/kg bw/day to groups of 32 male and 32 female (F0) mice for 8 weeks prior to pairing and until termination, after weaning, of the filial (F1) generation. A similarly constituted control group received the vehicle. No mortality was observed and no clinical signs due to treatment have been observed. There were no adverse effects on the bodyweight or the food consumption. Sperm counts, mating performance, fertility, gestation and parturition were not affected. The size of the F1litters, sex ratios, litter survival, bodyweights and the growth of the offspring till weaning were not adversely affected by treatment of the F0 parents. Necropsy of offspring and all adults did not reveal any apparent macroscopic effects of treatment there were no apparent effects on the organs of reproduction. At 150 and 600 mg/kg bw/day liver weight of the females and at 600 mg/kg bw/day of the males was higher in comparison to the Control group. Histopathological examination revealed a centrilobular hepatocytic hypertrophy. This effect is considered an adaptive response to treatment in a high incidence of animals. The thyroid weights of parental males and females (less pronounced) receiving 600 mg/kg bw/day were higher. This effect coincidences with slightly elevated T3levels and slightly reduced T4levels when compared with the Control or other treated groups.

The test substance showed no obvious adverse effects on the general condition, thyroid function, mating performance, fertility, or the progress of gestation or lactation in the treated mice. Furthermore, littering and survival and development of the F1progeny were similarly unaffected.

The No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg bw/day for the F0 generation and 600 mg/kg bw/day for the F1generation.


Short description of key information:
No adverse effects on fertility were noted in a one-generation study in mice (OECD 415, GLP). The highest tested dose was 600 mg/kg bw and the NOAEL for parental toxicity was 150 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
Study performed according to OECD guideline and according to GLP

Effects on developmental toxicity

Description of key information
In a developmental toxicity study in rabbits (OECD 414, GLP) effects on offspring were noted at doses which were toxic to dams (Harlan 2009). Effects are considered secondary to maternal toxicity.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April to October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1998)
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), 2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany, Niederlassung Kisslegg, Germany
- Age at study initiation: 25 - 36 weeks
- Weight at study initiation: 2265 to 3375 g
- Housing: individually in stainless steel cages equipped with an automatic cleaning system; a piece of wood and a haystick were also provided for environmental enrichment.
- Diet (e.g. ad libitum): pelleted standard Kliba Nafag 3418 (batch no. 70/07) rabbit maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days, under test conditions and after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 - 70%
- Temperature and humidity values outside of the ranges given above occasionally occurred, usually following room cleaning; they were considered not to have any influence on the study.
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs
Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC and 0.1% Tween 80 in highly purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The dose formulations were prepared weekly. The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle (0.5% CMC and highly purified water) were added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle (0.5% CMC and highly purified water) was added. Separate formulations were prepared for each concentration. The correct amount of 0.1% Tween 80 was added each morning to the daily portions of the dose formulations. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. Dose formulations in glass beakers were stored in the refrigerator (2 - 8 °C) where they were stable for 8 days.

VEHICLE
- Identification: 0.5% CMC, 0.1% Tween 80, highly purified water
- Source: Fluka Chemie GmbH, 9471 Buchs / Switzerland
- Batch Number: 1367388 (CMC), 34707017 (Tween 80)
- Storage Conditions: Room temperature (20 ± 5 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical monitoring:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken again as above. The aliquots for analysis of dose formulations were frozen and stored at -20 ± 5 °C until analysis.
The samples were analyzed by gas chromatography coupled to a flame ionization detector following an analytical procedure provided by the Sponsor and adapted at the testing facilities. The test item was used as the analytical standard.

Results of the analytical monitoring:
The identity of the test item was confirmed by its retention time. The test item content of 13 out of 18 samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test item in 0.5% CMC/0.1% Tween 80 was demonstrated for 4 out of 6 groups. It is indicated that homogeneity was easier to achieve for the lower content levels of dose groups 2 and 3 and that correct sampling was difficult due to formulation properties. This had an impact on results of storage stability as well. 2 out of 6 results met the limit of ±10% of nominal only. Deviating results were predominantly above nominal content which is not a sign of test item degradation. Therefore, the application formulations were considered to be stable for at least 8 days when kept at 2-8 °C. In conclusion, the results obtained within this part confirm the correct preparation of application formulations during the conduct of this study.
Details on mating procedure:
After acclimatization, females were placed in cages with sexually mature males (1:1) until copulation had been observed. After mating, the females were removed and placed in individual cages. The day of mating was designated day 0 post coitum.
The male rabbits were from the same source and strain as the females and were used for the mating only; their fertility had been proven and was continuously monitored.
Duration of treatment / exposure:
22 days (from day 6 to 27 post coitum)
Frequency of treatment:
once daily
Duration of test:
27 days (sacrifice was done on day 28)
Remarks:
Doses / Concentrations:
20, 40, 80 mg/kg bw/day
Basis:
actual ingested
(the application volume was 4 mL/kg bw)
No. of animals per sex per dose:
20 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous non-GLP dose range finding toxicity study (RCC B55170, 2008)
Maternal examinations:
MORTALITY AND CLINICAL SIGNS
The dams were examined for mortality twice daily. Cage-side clinical observations were made once daily up to the day of necropsy for assessment of clinical symptoms.

BODY WEIGHTS
Body weights were recorded daily for day 0 to day 28 post coitum.

FOOD CONSUMPTION
Food Consumption was recorded for the following periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24 and 24-28 post-coitum.

BLOOD SAMPLING
Blood samples (approximately 1 mL) were collected from the ear vein from 10 females each in groups 0, 20, 40 and 80 mg/kg bw/day. The samples were taken 24 h before the first administration (day 5 post coitum) and 24 h after the last administration (day 28 post coitum, directly before the necropsy). Plasma samples were prepared from the blood and frozen (-80 °C) for possible examination of the level of T3, T4 and TSH. However, based on the results of this study, these further investigations were not considered necessary.

NECROPSY
At the scheduled necropsy on day 28 post coitum, females were sacrificed by an intravenous injection of sodium pentobarbital and the fetuses removed by Caesarean section. Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents.
Gross macroscopic examination of all internal organs was performed, and particular attention was given to the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 28 post coitum to enable the calculation of the corrected body weight gain.
The following organs from all sacrificed females were weighed: liver, adrenal glands, thyroid gland, pituitary gland.
The above organs (but only half of the liver) were preserved in neutral phosphate buffered 4% formaldehyde solution for possible histopathological examination. The other half of the liver was placed immediately in a bag in pieces of 4 - 5 g and put in liquid nitrogen for storage at -80 °C for possible examination of necessary parameters (for example enzyme induction). However, based on the results of this study, these further investigations were not considered necessary.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. The fetuses were dissected, the organs examined and any abnormal findings were recorded. The sex of each fetus was noted.
2. After the skin had been removed, the cranium was examined for the degree of ossification.
3. From half of the fetuses the heads were separated from the trunks and fixed in Bouin's fixative. They were serially sectioned and examined (evaluation of the internal structures of the heads, including the eyes, brain, nasal passages and tongue) according to the method of Wilson JG (In: Teratology: Principles and Techniques. Eds., J.G. Wilson and J. Warkany, University of Chicago Press, 265-277,1965). Descriptions of any abnormal findings were recorded. After examination, the sections were preserved in a solution of ethyl alcohol and glycerin (one head per container). From the other half of the fetuses the heads were not separated but processed and stained as described in the next paragraph.
4. From all fetuses the skin with the exception of over the paws and the dorsal-cervical fat pads were removed and discarded. The trunks of the fetuses without heads and the fetuses with heads were processed through solutions of ethanol, glacial acetic with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage; the procedure were based on the method of Inouye M (Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and Alizarin red-S. Congenital Anomalies 16, 171-173, 1976). The skeletons were examined and all abnormal findings and variations were recorded
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated
- The Dunnett-test [Dunnett CW, J. Amer. Statist. Assoc. 50, 1096-1121, 1955] (many to one t-test) based on a pooled variance estimate was applied if the variables could not be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test [Miller RG, Simultaneous Statistical Inference, Springer Verlag, New York,1981] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
-Fisher's exact-test [Fisher RA, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1950] was applied for the macroscopical findings.
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study.
One dam of the high dose group (80 mg/kg bw/day) aborted on day 27 post coitum. This female was necropsied on day 28 post coitum, as scheduled. No other clinical signs or symptoms were noted for any dam in any group.

BODY WEIGHTS
In the high dose group (80 mg/kg bw/day), mean percentage body weight gain was generally statistically significantly reduced from day 9 post coitum onwards. Over days 6-28, mean body weight increased by 3.3% compared to 6.3% in the control group. Although mean body weight also became reduced in comparison to the control group as the study progressed, it was at no time statistically significantly reduced. Mean corrected body weight gain was slightly reduced (-5.9% compared to -4.7% in the control group). This was considered to be a sign of maternal toxicity.
In the mid and low dose groups (40 and 20 mg/kg bw/day) mean body weight, body weight gain and corrected body weight gain were not affected by treatment with the test item.

FOOD CONSUMPTION
In the high dose group (80 mg/kg bw/day), mean food consumption was reduced, at times statistically significantly, throughout the treatment period (-24.9% over days 6-28 post coitum, compared to the control group). This was considered to be a result of treatment with the test item. In the 40 mg/kg bw/day group, there was a slight transient reduction in food consumption over days 9-12 (-9.2%) and 21-24 post coitum (-21.6%) but this was not statistically significant and did not have an effect on the mean body weight in this dose group. In the low dose group treated with 20 mg/kg bw/day, mean food consumption was not affected by treatment.

REPRODUCTION PARAMETERS (for details, see table below)
All females in all groups were mated.
One female each in the control and the mid dose groups (0 and 40 mg/kg bw/day) were not pregnant as well as 2 in the high dose group (80 mg/kg bw/day). These occurrences were considered to be incidental. Post-implantation loss was statistically significantly increased in the high dose group (27.5% in 12 out of 17 dams compared to 10.0% in 9 out of 19 dams in the control group). As a result, the total number of fetuses was statistically significantly reduced (4.6 fetuses per dam compared to 6.2 in the control group). This was considered to be a test item-related effect. One female in the high dose group aborted on day 27 post coitum. This was considered likely to be a further post-implantation loss and therefore may also be test item-related. In the low and mid dose groups, no test item-related effects were noted in the reproduction data.

NECROPSY
Gross examination of the dams after necropsy revealed several abnormalities in the high dose group (80 mg/kg bw/day) when compared to the remaining groups; the findings consisted of raised crateriform area in the stomach containing hair, pale kidneys, and enlarged gall bladder. Due to the increase in the incidence of these findings in this
dose group, they were considered likely to be related to treatment with the test item.
Findings in the stomach were noted in one dam each in the low (20 mg/kg bw/day) and mid (40 mg/kg bw/day) dose groups. The low dose dam had a raised area on the pylorus and the mid dose dam had a red area in the stomach. Since these were the only findings in the stomach in these dose groups, they were considered likely to be incidental.
Referring to the organ weights, the weights of the left and right thyroid and adrenal glands were increased in the high dose group. These increases were considered to be probably test item-related but since they were not statistically significant, they were not considered to be adverse. In the low and mid dose groups, the organ weights were similar to those of the control group.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at the same dose as maternal toxicity

Details on embryotoxic / teratogenic effects:
SEX RATIO
No test item-related effects on the sex ratio of the fetuses were noted in any group.

FETAL WEIGHTS
In the high dose group (80 mg/kg bw/day), mean fetal weights were statistically significantly reduced (30.5 g versus 32.7 g in the control group; p<0.01). This was considered to be an effect of the observed maternal toxicity in this dose group.
Mean fetal weights in the low and mid dose groups (20 and 40 mg/kg bw/day) were within control range (31.7 and 32.4 g, respectively) and thus were not affected by treatment with the test item. The statistically significant reduction of body weight of the males on an individual basis in the low dose group (31.7 g versus 33.4 g in the control group; p>0.05; see table below) was within the range of the historical control data for litters of this size and therefore was incidental.

EXAMINATION OF SECTIONED HEADS
Examination of fixed sectioned fetal heads did not reveal any test item-related effects. In fact, in the high dose, one fetuse of 41 (i.e. 2% of the fetuses) examined showed dilation of the lateral brain ventricles. The finding was incidental.

EXTERNAL AND VISCERAL EXAMINATIONS (for details, see the attached tables)
The main findings were summarized in a table (see below). During visceral examination of the fetuses, findings were noted in:
63% examined fetuses (in 100% litters) in the control group;
58% examined fetuses (in 100% litters) in the low dose group (20 mg/kg bw/day);
59% examined fetuses (in 94% litters) in the mid dose group (40 mg/kg bw/day);
66% examined fetuses (in 94% litters) in the high dose group (80 mg/kg bw/day).
Abnormalities were noted in 2% of fetuses in the control group, 3% in the low and mid dose groups, and 9% in the high dose group. Whereas the findings in the low and mid dose groups were considered to be incidental, in the high dose group, the higher incidence of abnormalities was considered to be test item-related, since there was also a higher incidence of post-implantation loss at the same dose level. Since maternal toxicity was noted in this dose group and there was no clear pattern in the nature of the findings, a direct effect of the test item on the fetuses is not suspected. None of the variations noted were considered to be test item-related. Although there was an increased incidence of an additional artery arising from the aortic arch, this was within the range of the historical control data.

SKELETAL EXAMINATIONS (for details, see the attached tables)
In the high dose group 4, the number of supernumerary ribs was marginally increased on a fetus basis compared to the concurrent control, but was not statistically significant on a litter basis. There was a statistically significant increase in incompletely ossified limbs/digits in all 3 treated groups (20, 40 and 80 mg/kg bw/day). The level in the low dose group was within the range of the historical control data and was therefore considered unlikely to be of toxicological relevance. The level in the mid dose group was within the historical control data on a litter level but outside on a fetus level. This was considered to be a transient and non-adverse effect of the test item. The level in the high dose group was outside the historical control range and was considered to be related to the maternal toxicity at this dose level.
During cartilage examination of the fetuses, abnormal findings were noted in:
6% examined fetuses (in 16% litters) in group 1
4% examined fetuses (in 20% litters) in group 2
7% examined fetuses (in 33% litters) in group 3
10% examined fetuses (in 41% litters) in group 4
In the high dose group, there was a decreased incidence of short costal cartilage 10 as well as a decreased incidence of costal cartilage 7 not reaching the sternum. These findings, together with the increased incidence of supernumerary costal cartilages, suggest a possible minor disturbance in the development of the axial skeleton. This would not be unexpected in the presence of the observed maternal toxicity at this dose level. In the mid dose group, these effects only attained statistical significance on a fetal level and therefore the toxicological relevance is unclear.
Abnormalities:
not specified
Developmental effects observed:
not specified

Summary of reproduction data:

 

Reproduction Parameters

 

Dose groups (mg/kg bw/day)

0

20

40

80

NUMBER OF DAMS

19

20

18

17

CORPORA LUTEA

 

 

 

 

 

Total

154

148

135

130

 

Mean/Dam

8.1

7.4

7.5

7.6

PRE-IMPLANTATION LOSS

24

24

30

21

 

% OF CORP. LUTEA

15.6

16.2

22.2

16.2

 

MEAN

1.3

1.2

1.7

1.2

 

NUMBER OF DAMS AFFECTED

11

12

11

10

IMPLANTATION SITES

130

124

105

109

 

% OF CORP. LUTEA

84.4

83.8

77.8

83.8

 

MEAN

6.8

6.2

5.8

6.4

POST-IMPLANTATION LOSS

13

5

11

30

 

% OF IMPLANT. SITES

10

4

10.5

27.5**

 

MEAN

0.7

0.3

0.6

1.8

 

NUMBER OF DAMS AFFECTED

9

4

6

12

EMBRYONIC/FETAL DEATHS TOTAL

13

5

11

30

EMBRYONIC RESORPTIONS

12

5

10

23

 

% OF IMPLANT. SITES

9.2

4

9.5

21.1**

 

MEAN

0.6

0.3

0.6

1.4

 

NUMBER OF DAMS AFFECTED

9

4

6

8

FETAL RESORPTIONS

1

0

1

7

 

% OF IMPLANT. SITES

0.8

 

1

6.4*

 

MEAN

0.1

 

0.1

0.4

 

NUMBER OF DAMS AFFECTED

1

 

1

4

FETUSES

 

 

 

 

 

TOTAL

117

119

94

79

 

% OF IMPLANT. SITES

90

96

89.5

72.5**

 

MEAN

6.2

6

5.2

4.6

 

LIVE FETUSES

117

119

94

79

 

DEAD FETUSES

0

0

0

0

 

ABNORMAL FETUSES

0

0

0

0

SEX OF THE FETUSES

 

 

 

 

TOTAL MALES

57

53

46

33

 

% OF FETUSES

48.7

44.5

48.9

41.8

 

MEAN

3

2.7

2.6

1.9

TOTAL FEMALES

60

66

48

46

 

% OF FETUSES

51.3

55.5

51.1

58.2

 

MEAN

3.2

3.3

2.7

2.7

LIVE MALES

57

53

46

33

LIVE FEMALES

60

66

48

46

WEIGHTS OF LIVE FETUSES (g)

(LITTER BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

19

20

18

17

 

MEAN

33.3

32.4

33.3

30.4*

MALES

 

 

 

 

 

N (LITTERS)

19

19

17

16

 

MEAN

34

32.5

33.6

31.2*

FEMALES

 

 

 

 

 

N (LITTERS)

17

20

16

16

 

MEAN

32.6

32.3

32.1

30.8

WEIGHTS OF LIVE FETUSES (g)

(INDIV. BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

117

119

94

79

 

MEAN

32.7

31.7

32.4

30.5**

MALES

 

 

 

 

 

N (LITTERS)

57

53

46

33

 

MEAN

33.4

31.7*

33.3

30.9**

FEMALES

 

 

 

 

 

N (LITTERS)

60

66

48

46

 

MEAN

32

31.7

31.6

30.2*

*, p<0.05; **, p<0.01

Summary of fetal data: see attached document.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Range finding study in non-pregnant rabbits (RCC, 2008)

The test substance was administered by gavage at dosages of 100, 200 or 500 mg/kg bw/day to groups of 2 non-pregnant rabbits for 22 consecutive days. The females at 500 mg/kg were sacrificed on day 5 of the treatment period and the females at 200 mg/kg were sacrificed on day 4 of the treatment period due to very low or absent food consumption. The females at 100 mg/kg survived until the scheduled necropsy. No clinical signs were noted in any of the females in any group during the study. At 500 mg/kg, mean food consumption decreased by 88.2% during the treatment period as compared to the pre-treatment period, at 200 mg/kg by 88.5% and at 100 mg/kg by 41.9%. As a result, body weights decreased slightly in all dose groups.

At necropsy, test item-related findings noted included reddish adipose tissue, a distended bladder filled with fluid and yellow material, and foci on the pylorus. Based on these data, doses of 10, 30 and 100 mg/kg are considered appropriate for a subsequent dose range-finding study on embryo-fetal development in rabbits.

Range finding prenatal developmental study in rabbit (RCC 2008)

The test substance was administered by gavage at dosages of 10, 30 or 100 mg/kg bw/day to groups of 5 mated female rabbits from day 6 through day 27 post coitum. Control animals were dosed with the vehicle alone. All females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section.

No test item-related maternal deaths occurred. No clinical signs were noted which were considered to be treatment-related.

At 100 mg/kg bw, mean food consumption was reduced throughout the whole of the treatment period. Mean body weight gain was very slightly reduced during the treatment period. As a result, mean body weights became increasingly reduced as the treatment period progressed.

Post-implantation loss was statistically significantly increased in high dosed females. No maternal, macroscopical findings were noted which were considered to be related to treatment with the test item.

At 100 mg/kg bw, the weights of the fetuses on a litter and individual basis were reduced, but not statistically significantly. Sex ratio was not considered to have been affected. No abnormal findings were noted, which were considered to be test item-related.

Based on these data, dose levels of 20, 40 and 80 mg/kg body weight/day were considered appropriate for a subsequent main study on embryo-fetal development in rabbits.

Prenatal developmental study in rabbits (OECD 414, GLP) (Harlan 2009)

The test substance was administered by gavage at dosages of 20, 40 or 80 mg/kg bw/day mated female rabbits from day 6 through day 27 post coitum. Control animals were dosed with the vehicle alone. All females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section.

All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study. At 80 mg/kg bw, mean food consumption was reduced throughout the treatment period, partially showing statistical significance,. Mean body weight gain was statistically significantly reduced from day 9 post coitum onwards. Mean body weight became reduced as a result in comparison to the control group as the study progressed. Corrected body weight gain was slightly reduced. This was considered to be a result of treatment with the test item.

Post-implantation loss was statistically significantly increased in high dosed females and as a result, the total number of fetuses was statistically significantly reduced. One dam aborted on day 27 post coitum. During macroscopic examination, a higher number of findings was noted in group 4 than in the other dose groups, especially in the stomach, kidneys and gall bladder. These findings were considered to be related to treatment with the test item.

No test item-related effects on the sex ratio of the fetuses were noted in any group. At 80 mg/kg bw, mean fetal weights were statistically significantly reduced. This was considered to be an effect of the observed maternal toxicity in this dose group.Examination of fixed sectioned fetal heads did not reveal any test item-related effects.

At 80 mg/kg bw, there was a higher incidence of abnormalities, which was considered to be a result of the maternal toxicity observed in this dose group. Since maternal toxicity was noted in this dose group and there was no clear pattern in the nature of the findings, a direct effect of the test item on the fetuses is not suspected.

In high dose animals, the number of supernumerary ribs was marginally increased on a fetus basis compared to the concurrent control, but was not statistically significant on a litter basis. There was reduced cranial ossification and a statistically significant increase in incompletely ossified limbs/digits. There was a decreased incidence of short costal cartilage 10 as well as an increased incidence of costal cartilage 7 not reaching the sternum. These findings suggest a possible minor disturbance in the development of the axial skeleton.

Based on the results of this study, the maternal NOEL was considered to be 40 mg/kg body weight/day. The NOAEL for fetal organisms was considered to be 40 mg/kg body weight/day.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available one-generation study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for fertility under Directive 67/548/EEC.

The developmental toxicity study is considered reliable and suitable for classification purposes under 67/548/EEC. Developmental effects were considered to be secondary to maternal toxicity. As a result the substance is not considered to be classified for developmental toxicity under Directive 67/548/EEC.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available one-generation study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for fertility under Regulation (EC) No. 1272/2008.

The available developmental study is reliable and suitable for classification purposes under Regulation 1272/2008. Developmental effects were considered to be secondary to maternal toxicity. As a result the substance is not considered to be classified for fertility under Regulation (EC) No. 1272/2008.

In the one generation study, no effects via lactation were observed.