Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effect on fertility was noted in a two-generation reproduction toxicity study .

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
The two-generation reproductive toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 May 2013 to 21 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
other: JMAFF, 12 - NohSan No. 8147 (2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Han Wistar rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 5 to 6 weeks and females 4 to 5 weeks; (F1) 20 Day
- Weight at study initiation: (P) Males: 140 - 209 g; Females: 98 - 136 g
- Fasting period before study: No
- Housing: The P generation animals and selected F1 generation animals were housed in groups of four, by sex, until pairing and for males post-pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 23 °C
- Humidity (%): 24 % to 81 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % aqueous carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated for dosing as a suspension in the vehicle, 1 % (w/v) aqueous carboxymethylcellulose at approximately weekly intervals (within the known stability) for
each group.
For use in the first week of dosing, a stock suspension was prepared using the highest concentration (100 mg/mL, Group 4 dose level) and the low and intermediate dose groups were prepared by dilution of this stock; however, the results of analysis of the low and intermediate dose levels were not optimal. After consultation with the Sponsor dosing commenced using the original formulation for Groups 1 and 4 and a re-preparation of Groups 2 and 3 was performed (used from the second day of dosing) by mixing the required amount of test item with the appropriate quantity of vehicle; this method was used for the remainder
of the study.
A weighed quantity of test item was mixed using a pestle with a small quantity of the vehicle in a mortar to form a smooth paste. Via further progressive additions of vehicle and mixing, this was then taken up to near final volume or weight. The mortar was rinsed with vehicle and the rinsings were added to the formulation. Once at final volume/weight (using a density correction of 1.023 for the 100 mg/mL (Group 4)), the formulation was mixed using a laboratory homogeniser, stirred using a magnetic stirring bar and then dispensed into aliquots for dosing.
Formulations were stored frozen (at approximately -18 °C) once made or, if to be used the following day, were stored at room temperature in the dark. Frozen formulations were removed from the freezer and stored overnight at room temperature to defrost. On several occasions throughout the study the bottles cracked and additional formulations had to be prepared. On the day of use, formulations were stirred for at least 30 minutes before and during the dosing procedure.

VEHICLE
1 % (w/v) aqueous carboxymethylcellulose
Details on mating procedure:
- M/F ratio per cage: For pairing, one male and one female from the same group (avoiding sibling mating for F1 animals)
- Length of cohabitation: for up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of test item formulations at concentrations between 0.1 mg/mL and 100 mg/mL in vehicle, spanning those used on this study (5 mg/mL to 100 mg/mL), were examined prior to the start of this study and they were shown to be stable for at least one day at room temperature in the dark and up to eight days when stored frozen (approximately -18 ºC).
Samples were taken from each formulation. The samples from all formulations prepared for use during the first two weeks of treatment (five occasions) were analysed. For the remainder of the first two months of the study, formulation analysis was performed on samples taken from formulations every two weeks; and then once every two months thereafter. All remaining samples were stored frozen (approximately -18 ºC) and will be discarded following the issue of the final report.
Samples from formulations containing the test item were analysed using a previously validated method BFI009LC to determine their homogeneity and achieved concentrations. Samples from the Control formulations were analysed for the absence of the test item.
Duration of treatment / exposure:
All animals were dosed for at least 10 weeks before pairing and then until the day prior to necropsy. Males were dosed before, during and after pairing, and females were dosed before and during pairing, and during gestation and lactation until Day 20 of lactation. Animals that were in mid-parturition at the time of dosing were not dosed on that day. The selected F1 generation was dosed from Day 21 of age.
Frequency of treatment:
once daily
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 males and 24 females per dose level and for each generation.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected on the basis of results from a preliminary study performed in the same laboratory. The test item was administered once daily, via oral gavage, to groups of five male and five female Han Wistar rats at dose levels of 0, 100, 300 or 1000 mg/kg/day for 28 days.
On the basis of this study it was anticipated that there would be minor pathology findings in males and females at 1000 mg/kg/day, which represents the limit dose for studies of this type. Doses of 50 and 250 mg/kg/day were included to determine the dose response of any effects seen at the high dose.

- Rationale for animal assignment:
Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on arrival. The cages were positioned in the battery using a randomised cage allocation procedure. Each animal was uniquely identified by a subcutaneously implanted micro-identification device.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From the start of treatment, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance and each animal was given a detailed clinical examination once each week.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded on the first day of dosing, at weekly intervals throughout the study and then on the day of necropsy.Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (where required for dose volume calculations), 1, 4, 7, 14 and 21 of lactation and on the day of necropsy.
Females that failed to litter were weighed weekly until necropsy to calculate the dose volume to be administered but these data have not been reported. Furthermore, females, where all the pups have died in the litter prior to Day 21 of age, were weighed on Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The amount of food consumed by each cage of males was recorded at weekly intervals during their pre-pairing and post-pairing periods.
Food intake of the females was recorded weekly during the pre-pairing period and over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21 of lactation. Food intake of females, where all the pups have died in the litter prior to Day 21 of age, were recorded on Days 1, 4, 7, 14 and 21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Oestrous cyclicity (parental animals):
For 21 days before the start of the pairing period, vaginal smears were taken daily by lavage. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present.
Sperm parameters (parental animals):
For P and F1 generation: Sperm motility and concentration were assessed for all males killed at scheduled necropsy. The assessment was performed using fluid from one cauda epididymis. The cauda epididymis
was weighed separately. A sample of the epididymal fluid was retained in neutral buffered formalin, a smear was prepared for each Control and high dose male and at least 200 sperm per sample were examined for morphological abnormalities.
After weighing of the testes, the tunica albuginea of one testis was removed and the testis was snap frozen in liquid nitrogen and stored at -20 °C until required. For each Control and high dose male, the testis was thawed, homogenised and the resistant spermatids counted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [P and F1] offspring: The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age.
Pups were weighed individually on Days 1, 4, 7, 14 and 21 of age.
The anogenital distance of the pups in the F1 generation litters was measured on Day 1 of age.


Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All males were killed approximately six weeks after completion of the mating period.
- Maternal animals: Females with litters were killed on Day 21 of lactation at weaning of their litters. The remaining females, including those apparently non-pregnant and those where all the litter had died before Day 21 of age, were also killed at this time.

GROSS NECROPSY
The thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs and uterus were examined. The number of
implantation scars/sites for each female was recorded at necropsy. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred to and retained in neutral buffered formaldehyde. Organs or tissues showing any macroscopic abnormalities were recorded and retained. For each male, one epididymis was processed for sperm evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together):
adrenals prostate & seminal vesicles (incl. coagulating gland), brain, epididymides, spleen, kidneys, testes, liver, thyroids (including parathyroids), ovaries, uterus (incl. uterine cervix and oviducts), pituitary.

For all animals, either whole organs or samples of adrenals prostate & seminal vesicles (incl. coagulating gland), brain, epididymides, spleen, kidneys, testes, liver, thyroids (including parathyroids), ovaries, uterus (incl. uterine cervix and oviducts), pituitary, vagina , with the exception of the eyes, optic nerves, testes and epididymides, were preserved in neutral buffered formaldehyde. The eyes and optic nerves were fixed in Davidson’s fluid and the testes and epididymides were fixed in Bouin’s fixative for 24 hours and then transferred to neutral buffered formaldehyde.
Due to treatment related effects detected in the liver (males and females) and kidneys (males only) in Group 4, the livers of males and females and the kidneys of males only from the remaining dose groups were prepared and examined microscopically.

Postmortem examinations (offspring):
SACRIFICE
With the exception of pups culled on Day 4 of lactation, which were not further examined, a necropsy was conducted on all pups killed or found dead during lactation and all unselected pups on Day 21 of age. The pups were killed by an intraperitoneal injection of sodium pentobarbitone solution (for those up to the age of 14 days) or by exposure to carbon dioxide gas in a rising concentration (for older pups). A dead body weight was recorded for pups where organ weights were to be determined. The thoracic and abdominal cavities were opened by a ventral mid line incision and the major organs were examined.


GROSS NECROPSY
For one male and one female pup per litter the following tissues, brain, spleen, kidney, thymus, liver and all gross lesions were retained. All other pups had a gross macroscopic examination and only gross abnormalities were retained.

HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female pup from each litter the following organs were weighed after trimming of fat and other contiguous tissue (left and right kidneys were weighed together): brain, spleen, kidney, thymus and liver.
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis Groups 2, 3 and 4 against Group 1.
All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).
Data were examined for unusually high or low values which could influence the statistical analysis and interpretation (possible outliers). After examining for any outliers, if the variances were clearly heterogeneous, transformations (e.g. log, double arcsine or square root) were used in an attempt to stabilise the variances. If the transformations failed, the data set was examined and a decision taken on further action.
Clinical signs:
no effects observed
Description (incidence and severity):
With the exception of yellow stained bedding seen in the cages of both sexes given 250 or 1000 mg/kg/day (which was considered to be a result of the colour of the test item), there were no clinical signs related to test item administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was only one early decedent, Female 188 given 1000 mg/kg/day, which was killed on Day 23 of gestation due to dystocia. There were no observations in the female reproductive organs that would have caused the dystocia. As there were no other instances of these macroscopic and microscopic findings in other animals this death was considered not to be test item related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From Week 3 for males given 250 mg/kg/day and Week 5 for males given 1000 mg/kg/day, animals gained slightly less weight than the Controls. These differences achieved statistical significance over Weeks 0 to 14 and Weeks 0 to 15 for males given 1000 mg/kg/day (p<0.05). Overall body weight gain (Weeks 0 to 16) was 4 % and 9 % lower than Controls, in the 250 mg/kg/day and 1000 mg/kg/day dose groups respectively, although not statistically significantly different from Controls. Males given 50 mg/kg/day gained weight at a similar rate to, or slightly higher than, the Controls throughout the study. This resulted in an overall body weight gain (Weeks 0 to 16) 3 % higher than Controls.
Females given 50, 250 or 1000 mg/kg/day gained slightly more weight than Controls during the pre-paring period, statistically significantly so for most of this period (p<0.05 and p<0.01), for females given 1000 mg/kg/day.
During the gestation period, females given 1000 mg/kg/day gained slightly more weight than Controls, (p<0.05 over Days 0 to 20 of gestation). Females given 50 or 250 mg/kg/day during gestation and all groups of females given the test item during lactation gained weight at a similar rate to the Controls.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant, dose related increases in group mean body weight adjusted liver weights were apparent in all groups of males (p<0.01) and females given 1000 mg/kg/day
(p<0.01), when compared with Controls.
Statistically significant non-dose related increases in group mean body weight adjusted kidney weights were apparent for all groups of males (p<0.01, p<0.05 and p<0.01, respectively) and females given 1000 mg/kg/day (p<0.01), when compared with Controls.
Group mean adjusted thyroid gland weights for males given 250 mg/kg/day (p<0.01) and both sexes given 1000 mg/kg/day (p<0.01) were statistically significantly higher than Controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic treatment-related findings were seen in the liver in males and females and in the kidneys in males.
Liver:
Minimal or slight hepatocellular hypertrophy was seen in males at all dose levels and in females at the high dose level only. The incidence and severity of this finding were dose related in males.
Kidney:
A treatment-related cortical tubular basophilia was seen in a number of males at all dose levels. Cortical tubular basophilia was accompanied by cortical inflammatory cell infiltrate in some of the affected animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
With the exception of yellow stained bedding seen in the cages of treated animals (which was considered to be a result of the colour of the test item), there were no clinical signs related to administration of the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female 390 (1000 mg/kg/day), was found dead on Day 23 of age; however no findings were seen at necropsy. As this was an isolated occurrence, this death was considered not to be test
item related.
Males 231, 238 and 243 (50 mg/kg/day) were found dead on Days 42, 41 and 40 of age, respectively; findings at necropsy suggested that these deaths were a result of dosing trauma.
Female 377 (1000 mg/kg/day) was killed on Day 18 of gestation due to an abnormal gait (at necropsy this animal was found to have suffered a dislocated hip).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Some statistically significant differences in food intake were noted between animals given the test item and Controls, however, these were considered to be incidental and not test item related.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, there were dose related, statistically significant increases in group mean body weight adjusted liver (p<0.01), kidney (p<0.05 and p<0.01 for males given 250 or 1000 mg/kg/day, respectively) and thyroid weights (p<0.01) for males given the test item, when compared with Controls. There was also a dose related increase in body weight adjusted liver weight for all groups of females given the test item (p<0.05 and p<0.01 for females given 250 or 10 mg/kg/day, respectively) and increases in body weight adjusted kidney (p<0.01) and thyroid weights (p<0.01) for females given 1000 mg/kg/day, when compared with Controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic treatment-related findings were seen in the liver in males and females and in the kidneys in males.
Liver:
Minimal or slight hepatocellular hypertrophy was seen in males and females given 250 or 1000 mg/kg/day.
Kidney:
A treatment-related increase in the incidence of cortical tubular basophilia was seen in a number of males at all dose levels.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
With the exception of yellow stained bedding seen in the cages of treated animals (which was considered to be a result of the colour of the test item), there were no clinical signs related to administration of the test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Female 390 (1000 mg/kg/day), was found dead on Day 23 of age; however no findings were seen at necropsy. As this was an isolated occurrence, this death was considered not to be test item related.
Males 231, 238 and 243 (50 mg/kg/day) were found dead on Days 42, 41 and 40 of age, respectively; findings at necropsy suggested that these deaths were a result of dosing trauma. Female 377 (1000 mg/kg/day) was killed on Day 18 of gestation due to an abnormal gait (at necropsy this animal was found to have suffered a dislocated hip).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There was no effect of the test item on the age of balano-preputial separation.
For females there was a statistically significant (p<0.01) delay in vaginal opening at 1000 mg/kg/day. For the Control animals the group mean was 31.3 days and for females given 1000 mg/kg/day the group mean was 32.5 days. As the mean difference between these groups was only just over one day, this finding was considered not to be of biological significance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, there were dose related, statistically significant increases in group mean body weight adjusted liver (p<0.01), kidney (p<0.05 and p<0.01 for males given 250 or 1000 mg/kg/day, respectively) and thyroid weights (p<0.01) for males given the test item, when compared with Controls. There was also a dose related increase in body weight adjusted liver weight for all groups of females given CA4920 (p<0.05 and p<0.01 for females given 250 or 1000 mg/kg/day, respectively) and increases in body weight adjusted kidney (p<0.01) and thyroid weights (p<0.01) for females given 1000 mg/kg/day, when compared with Controls.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Microscopic treatment-related findings were seen in the liver in males and females and in the kidneys in males.
Liver:
Minimal or slight hepatocellular hypertrophy was seen in males and females given 250 or 1000 mg/kg/day.
Kidney:
A treatment-related increase in the incidence of cortical tubular basophilia was seen in a number of males at all dose levels
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on sperm parameters.
The number of follicles in the ovaries of females was similar across all the groups, including Controls. Occasional differences were considered a likely consequence of individual animal variability and were considered not to be related to treatment.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adjusted liver (male) and kidney (female) weights were significantly increased in animals given 1000 mg/kg/day, when compared with Controls.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
kidney
Reproductive effects observed:
no
Conclusions:
No effect on reproductive performance, mating behaviour, conception or pup development in either generation was noted during the course of the study and therefore, for effects on
reproduction, the No Observed Adverse Effect Level was considered to be 1000 mg/kg/day.
In terms of general toxicity, a marginal decrease in male body weight gain was seen at 250 or 1000 mg/kg/day. Liver, kidney and thyroid gland weights were increased in animals given the test item when compared with Controls and were associated, in the parental animals of both generations, with microscopic findings of hepatocellular hypertrophy and increased renal cortical tubular basophilia.
Executive summary:

Effects of the test item on the integrity and performance of the male and female reproductive systems, including gonadal function, the oestrous cycle, mating behaviour, conception, gestation, parturition, lactation and weaning, and the growth and development of the offspring over two successive generations in the rat, when administered orally by gavage.

Four groups of 24 male and 24 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (1 % aqueous carboxymethylcellulose), 50, 250 or 1000 mg/kg/day of the test item for 10 weeks before pairing, during pairing, gestation, lactation and until necropsy. Four groups of 24 male and 24 female F1 generation animals were selected from the weaned P generation litters; these animals were dosed once daily by oral gavage at dose levels of 0, 50, 250 or 1000 mg/kg/day from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy. All animals were examined for effects on general condition, body weight and food consumption. The stage of the oestrous cycle was recorded for at least 21 days prior to pairing for P and F1 females, and during the pairing period vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to weaning. The day of sexual development was recorded for all selected F1 animals. Anogenital distance was recorded for all F1 generation pups on Day 1 of age. The P and F1 parental males were subjected to macroscopic necropsy once successful littering was completed. The testes and epididymides were removed and weighed and sperm evaluation was conducted. The P and F1 parental females were killed and subject to necropsy on Day 21 of lactation. A macroscopic necropsy was performed and the number of implantation scars was recorded. For the P and F1 parental males and females a selection of organs were weighed, fixed and microscopically examined. Unselected P generation pups and all F1 generation pups were killed on Day 21 of age. A gross macroscopic necropsy was performed on all pups and, for one male and one female pup per litter from the P and F1 generations, brain, liver, kidney, spleen and thymus weights were recorded. Homogenisation resistant testicular spermatid counts were performed for the P and F1 parental males and ovarian follicle evaluation for the F1 generation parental females.

There were no deaths or adverse clinical observations considered to be related to the test item administration.

A marginal decrease in group mean body weight gain was evident in males given 250 or 1000 mg/kg/day over the duration of the dosing period, when compared with Controls; however, all other groups given the test item across both generations gained weight at a similar rate to, or slightly higher than, Controls throughout the study. There was no effect on food intake for any dose group across either generation, when compared with Control animals. Oestrous cycles, fertility and mating data in both generations were unaffected by treatment with the test item.

There were no test item related effects on the number of pups born or cumulative pup survival for the P or F1 generation litters, when compared with Controls.

There was no biologically significant effect on the age of sexual maturity for males and females and there was also no effect of treatment on the anogenital distance for F1 generation pups.

There was a dose-related increase in group mean body weight adjusted liver weights for all groups of P and F1 generation parental males, P Generation parental females given 1000 mg/kg/day, F1 parental females given 250 or 1000 mg/kg/day, Day 21 of age P

generation female pups and F1 generation male pups given 1000 mg/kg/day. There was a dose-related increase in group mean body weight adjusted kidney weights for all test item treated P generation parental males, P generation parental females given 1000 mg/kg/day, F1 generation parental males given 250 mg/kg/day, F1 generation parental males and females given 1000 mg/kg/day and for Day 21 of age P and F1 generation female pups given 1000 mg/kg/day. Group mean adjusted thyroid gland weights were higher than Controls for P and F1 generation parental males and females given 1000 mg/kg/day P generation parental males given 250 mg/kg/day and F1 generation parental males given 50 or 250 mg/kg/day. There was no effect of treatment on sperm parameters or spermatid counts in the P or F1 generation males or ovarian follicles of F1 generation females.

Treatment-related microscopic findings in the P generation were found in the liver (hepatocellular hypertrophy) in males at all dose levels and in females given 1000 mg/kg/day only and in the kidneys (cortical tubular basophilia) in males at all dose levels. Treatmentrelated microscopic findings in the F1 generation were similar to those seen in the P generation with hepatocellular hypertrophy in males and females given 250 or 1000 mg/kg/day and an increased incidence of cortical tubular basophilia in the kidneys in males at all dose levels.

No effect on reproductive performance, mating behaviour, conception or pup development in either generation and therefore, for effects on reproduction were observed, the No Observed Adverse Effect Level was considered to be 1000 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1
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 two-generation reproductive toxicity study was conducted solely to comply with non-EU national registration requirements.

Effects on developmental toxicity

Description of key information

No developmental effects were noted during a two-generation reproduction toxicity study and in a prenatal/developmental toxicity study.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Remarks:
The developmental toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 March 2015 to 07 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: JMAFF, 12 - NohSan No. 8147 (2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 180 g to 246 g
- Fasting period before study: No
- Housing: Females were housed individually in grid-floor cages over paper lined trays.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 40% to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % aqueous carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at approximately weekly intervals (within the known stability period), for each group separately, as a suspension in 1 % aqueous carboxymethylcellulose.
A weighed quantity of test item was added to a mortar, wetted with a small quantity of vehicle and initially made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a tared beaker on a balance.
The mortar was thoroughly rinsed out with vehicle and these rinsings were added to the suspension which was then made up to final weight with vehicle and mixed with a laboratory homogeniser.
Formulations were divided into daily aliquots and were stored frozen (approximately -20 °C) or, if to be used the following day, formulations were stored at room temperature, protected from light. Frozen aliquots were removed from storage on the afternoon before dosing and stored at room temperature overnight to defrost. Formulations were stirred from 30 minutes before the start of dosing, until the completion of their use for dosing, to ensure thorough resuspension and homogeneity.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first preparation occasion, test item formulations were analysed to assess homogeneity and achieved concentration of test item, using a validated method (BFI009LC (4)). Once homogeneity had been established, subsequent formulations for use on the last day of dosing were analysed to determine achieved concentrations only. Vehicle (for Controls) from the first preparation and that for use on the last day of dosing was analysed to confirm the absence of the test item.
All remaining samples were retained frozen (approximately -18 ºC) and discarded once the final formulation analysis results were accepted.
Details on mating procedure:
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Day 6 of gestation to Day 19 of gestation
Frequency of treatment:
once daily
Duration of test:
From Day 0 of gestation to Day 20 of gestation (necropsy)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in consultation with the Sponsor on the basis of results from a reproduction / developmental toxicity screening test and a two-generation reproduction toxicity study in the rat performed in the same laboratory.
- Rationale for animal assignment (if not random): Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on Day 0 of gestation, making sure that females mated with the same male were spread across the
groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily from the start of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 5 to 20 of gestation, inclusive.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food intake was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The major organs were examined. Gravid uterus weight and placenta weights were recorded and organs or tissues showing macroscopic abnormalities were removed and retained in neutral buffered formaldehyde. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to
confirm pregnancy status. The uterus was retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in neutral buffered formaldehyde.

Ovaries and uterine content:
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses.
The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
Fetal examinations:
- External examinations: Yes: All
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Approximately 50 % of the live foetuses were fixed in Bouin’s fluid for subsequent examination for visceral abnormalities using a combined sectioning/dissection technique.
The remaining foetuses were briefly fixed in 70 % alcohol and subjected to micro-dissection. The viscera were examined and the foetuses were eviscerated. The carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined for skeletal variants and abnormalities.
Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Other defects were classified as minor abnormalities or variations.
- Head examinations: No data
Statistics:
All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. The litter, rather than the foetus, was considered as the experimental unit. Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).

Dunnett’s test: For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags are presented in the tables of results in the final report.

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female given 1000 mg/kg/day (Animal 78) had piloerection on Day 8 of gestation; as this female also had marked initial body weight loss, an association with treatment would seem likely, however, since this was an isolated occasion it was considered not to be adverse. There were no other clinical signs during the study at any dose level.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Over Days 6 to 8 of gestation, there was a slight mean body weight loss in the group given 1000 mg/kg/day, with notable intra animal variation. From Day 9 of gestation onwards, group mean body weight gain was comparable with Controls; therefore, the initial body weight effect at 1000 mg/kg/day was considered not to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the group given 1000 mg/kg/day, animals ate statistically significantly less over Days 6 to 9 of gestation (p<0.01); thereafter, food intake was similar to Controls.
At 50 or 250 mg/kg/day, there was no effect of CA4920 on food intake.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Two females (Control Female 14 and Female 74 given 1000 mg/kg/day) had total early intrauterine deaths and two females were not pregnant (Female 63 given 250 mg/kg/day and Female 73 given 1000 mg/kg/day). Consequently, there were 21, 22, 21 and 20 females in the groups given 0, 50, 250 or 1000 mg/kg/day respectively, with live foetuses on Day 20 of gestation. There were no effects of the test item on pregnancy parameters, the numbers of implantations, incidence of pre- and post-implantation loss and numbers of live foetuses were similar in all groups.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Only one major foetal abnormality was present in one foetus given 250 mg/kg/day (the hind limb fibula(e) was not ossified) and there were no statistically significant differences from Control in the overall incidences of minor or variant foetal abnormalities in the treated groups.
In the groups given 250 or 1000 mg/kg/day, there was a significant (p<0.05) increase in the number of litters with foetuses showing the variant finding of increased renal pelvic cavitation. At 1000 mg/kg/day, there was also a significant increase (p<0.05) in the numbers of litters with foetuses having an extra or vestigial 14th rib (with costal cartilage), one or more of the 11th to 13th ribs with long costal cartilage and incomplete cartilaginous styloid process(es) in the skull. The number of foetuses showing these minor or variant abnormalities was either very small, lacked any dose response or were within or just marginally above the background data ranges.
These findings do not indicate an effect of the test item on foetal development.
Visceral malformations:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull
Description (incidence and severity):
Only one major foetal abnormality was present in one foetus given 250 mg/kg/day (the hind limb fibula(e) was not ossified) and there were no statistically significant differences from Control in the overall incidences of minor or variant foetal abnormalities in the treated groups.
In the groups given 250 or 1000 mg/kg/day, there was a significant (p<0.05) increase in the number of litters with foetuses showing the variant finding of increased renal pelvic cavitation. At 1000 mg/kg/day, there was also a significant increase (p<0.05) in the numbers of litters with foetuses having an extra or vestigial 14th rib (with costal cartilage), one or more of the 11th to 13th ribs with long costal cartilage and incomplete cartilaginous styloid process(es) in the skull. The number of foetuses showing these minor or variant abnormalities was either very small, lacked any dose response or were within or just marginally above the background data ranges.
Developmental effects observed:
no
Conclusions:
Maternal changes seen at 1000 mg/kg/day were considered to be transient and of no adverse outcome.There was no adverse effect on pregnancy or embryonic or foetal development. On this basis, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and No Observed Effect Level (NOEL) for embryo-foetal development was considered to be 1000 mg/kg/day.
Executive summary:

Four groups of 22 sexually mature timed-mated female Crl:WI(Han) rats were dosed with 0 (vehicle), 50, 250 or 1000 mg/kg/day of the test item, once daily by oral gavage, from Days 6 to 19 of gestation, inclusive.

All animals were observed daily from the start of treatment and body weights and food intake were recorded throughout. Animals were killed on Day 20 of gestation, a necropsy performed and the internal organs examined for gross abnormalities. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal abnormalities. Placental and gravid uterus weights were also recorded.

Slight mean body weight loss was evident after the first two days of dosing at 1000 mg/kg/day and less food was eaten over this period, from Day 9 of gestation onwards, body weight gain and food intake were similar to controls.

There were 21, 22, 21 and 20 females in the groups given 0, 50, 250 or 1000 mg/kg/day, respectively, with live foetuses on Day 20 of gestation. There were no effects on pregnancy parameters; values in all groups were similar. At all dose levels, mean foetal and placental weights were comparable with those of the Control group and there was no effect on foetal sex ratio.

There were no maternal macroscopic abnormalities related to the test item. There was no adverse effect of the test item on the incidence of external, visceral, skeletal or cartilaginous major or minor foetal abnormalities or variants.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1
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

The developmental toxicity study was conducted solely to comply with non-EU national registration requirements.

Justification for classification or non-classification

Based on the above mentioned studies, the substance does not have to be classified as reprotoxicant according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.