Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2012 - 12 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Remarks:
Research Toxicology Centre S.p.A., Via Tito Speri, 12/14, 00040 Pomezia (Rome), ITALY
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco Italy
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 176 to 200 g; Females: 151 to 175 g
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least three times a week. During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent
material which was inspected and changed daily. The males were re-caged after mating as they were before mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
Suitable nesting material was provided and was changed at least 3 times a week.
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 4 to 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2°C
- Humidity: 55 ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 July 2012 To: 12 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was suspended in the vehicle (0.5% methylcellulose in purified water) at the final concentrations of 10, 30 and 100 mg/mL. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
Mating was monogamous (one male to one female) with the exception described below. One mid-dose male (no. 54) died during the first mating session. The assigned female (no. 53) was re-mated with a proven male (no. 52) of the same treatment group.
A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred with the exception of female no. 53 as previously described, or 14 days had elapsed. In one occasion of unsuccessful mating with the first assigned male, the female no. 61 (Group 4) was re-mated with a proven male (male no. 74) of the same treatment group. The second pairing combination was monitored for mating until positive signs of copulation were observed.
For three females (nos. 05, 29 and 59) the mating was inadvertently not detected in the first mating session. On the basis of this apparently negative sign, the females were allowed to have the second pairing cohabitation with proven males of the same treatment groups. The second pairing combination was monitored for mating, however due to the increment in body weight, the pregnancy status became evident and the females were
individually caged before completion of the second pairing combination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study no. 93100: BASF Project No.: 05Y0252/12X126) in the range from 10 to 100 mg/mL.
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (check of concentration and homogeneity). The stability was found to be at least 48 hours at room temperature in the concentration range of 10 to 100 mg/mL. Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) were also analysed to check the concentration and homogeneity. The overall results of the analyses were within the limits of acceptance stated in RTC SOPs for concentration (80-120%) and homogeneity (CV <10%).
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing up to the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum (up to Day 26 for non pregnant females) and post partum periods until Day 3 post partum.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CLINICAL SIGNS
Twice before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHTS
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION
Food consumption was recorded at weekly intervals, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 postpartum).

PARTURITION AND GESTATION LENGTH
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
EUTHANASIA
Parental animals that had completed the scheduled test period were killed with carbon dioxide. The males were killed after the mating of all females (i.e. after 38 days of treatment). The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after the positive identification of mating were killed shortly thereafter (Day 26/27 post coitum).

NECROPSY
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding the found dead animal no. 54) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
From all animals completing the scheduled test period the organs indicated in section Table 1 were dissected free of fat and weighed. The ratios of organ weight to terminal body weight were calculated for each animal.

HISTOPATHOLOGY
Samples of all the tissues listed in Table 1 from all animals were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid).
The tissues required for histopathological examination are listed in section 4.4.6. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
a) Tissues specified in section Table 1 from all animals in the control and high dose group killed at term.
b) Tissues specified in section Table 1 from one animal dying during the treatment period.
c) All abnormalities in all groups.
Postmortem examinations (offspring):
EUTHANASIA
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopental.

NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed on Day 4 post partum and examined for external abnormalities and sex confirmation by gonadal inspection.
One pup (dam no. 31 of Group 2) with tail’s abnormalities was retained in fixative (10% neutral buffered formalin).
Statistics:
Group mean values were calculated for all parameters. Data from non-pregnant females were excluded from group mean calculations.
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences among control and treated group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test when “n” was more than 5 (Epididymides, ovaries and testes).
The criterion for statistical significance was p<0.05.
Reproductive indices:
- Copulatory Index (%)
- Fertility Index (%)
- Copulatory Index (%)
- Fertility Index (%)
- Copulatory Interval
- Pre-implantation loss
- Pre-birth loss
Offspring viability indices:
Pup loss at birth, Cumulative pup loss on Day 4, Sex ratios
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One mid-dose male (no. 54) was found dead on Day 5 of the mating phase. The cause of death was attributed to a mis-dosing on the basis of the macroscopic and histopathological findings.
At daily clinical observation, slight to moderate hairloss in different parts of the body surface (muzzle, head, dorsal and ventral regions, and/or lower forelimb) was recorded more frequently in males and females of the high dose group. Additionally slight scab formation was observed at the high dose group.
Corneal opacity and protruding eye were found in a single mid-dose female (no. 59). Cyphosis and piloerection were observed once in the mid-dose group during the mating phase (animal nos. 54 and 41) and post coitum period (animal no. 51).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain were comparable between groups throughout the study for both males and females.
On one occasion, a statistically significantly lower body weight gain was recorded in males of the mid-dose group during the pre-pairing period.
No differences were found in food consumption between the groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No differences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups. Regularity of the oestrous cycle was also found.
The number of implantations, corpora lutea, total litter size, pre-implantation loss and prebirth loss did not differ between groups.
Gestation length was also comparable between groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No differences were found in terminal body weight between groups.
Slightly but significantly higher absolute and relative ovaries weights (up to 14%) were found in the high dose group when compared to controls. In the absence of any histological correlate and since oestrous cycling and reproduction were incospicious, this difference was not considered to be toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Unscheduled death:
A single animal from Group 3 (animal no. 54), treated at 300 mg/kg bw/day, was found dead on Day 5 of the mating phase. Macroscopically, red fluid in the thoracic cavity was observed.
This change suggested that the cause of death was probably due to a mis-dosing and therefore was not treatment related.
Final sacrifice:
The most relevant macroscopic changes detected in the treated animals when compared with controls were the following:
Hairloss and/or scab formation/s in various regions of the body (head, forelimbs, ventral region and neck) in both sexes.
Minimally higher incidence of swollen liver in high dose males.
Pale, firm and raised area in the hepatic left lobe, reduced size of the testis and corresponding epididymidis and prostate in single instances.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The only relevant change observed in treated males and females was minimal scab formation/s in various regions of the body. In the absence of any other finding and since scab formation was minimal and occurs also spontaneously in rats, these findings could be
considered not toxicologically relevant.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The remaining lesions, such as hepatic necrosis, testicular atrophy and consequently reduced sperm in the epididymidis were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
Reproduction/Fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
CLINICAL SIGNS (OFFSPRING)
Mean litter and pup weights were comparable between groups on Days 1 and 4 post partum. No differences were found in sex ratio.
No remarkable differences were noted in the clinical signs of pups between groups. The findings generally recorded were small in size, cold to touch, apparently no food intake, or pale appearance. One low dose pup showed damaged tail.

GROSS PATHOLOGY (OFFSPRING)
No relevant findings were found in the two decedent pups, one in the control group and one in the low dose group.
At termination, one low dose pup (dam no. 31) showed kinked, red and swollen tail. These findings were considered incidental.
No other abnormalities were found in pups sacrificed on Day 4 post partum.
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Reproductive effects observed:
not specified
Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for both males and females.
Executive summary:

Reproduction/developmental toxicity of the test article was investigated in Wistar Hannover rats up to Day 4 post partum following the study design outlined in OECD guideline 421 and in compliance with GLP. Three groups of 10 animals/sex each received the test item at dosages of 100, 300 and 1000 mg/kg bw/day, respectively. The control group of the same number of animals/sex was administered with the vehicle alone (methylcellulose 0.5% in purified water). The test item and vehicle were given by oral gavage at constant volume of 10 mL/kg body weight. Males were treated for approximately 6 weeks including 2 weeks prior to pairing and continuously thereafter up to the day before necropsy. Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3 post partum. The following parameters were evaluated in parental animals: body weight, clinical signs food consumption, oestrous cycle, mating performance, litter data, macroscopic observations and organ weights. The histopathological examination, including the spermatogenic cycle for males, was performed in all animals of the control and high dose groups. Pups were monitored for daily clinical sings and body weight. Post mortem examination of pups was also performed.

One mid-dose male died on Day 5 of the mating phase due to a mis-dosing. Three females were found not pregnant at necropsy: one in each of the control, low and high dose groups. The number of females with live pups on Day 4 post partum was: 9 in each of the control group, low and high dose groups and 10 in the mid-dose group. Hairloss and/or scab formations were mainly recorded in the high dose group. Body weight, body weight gain and food consumption did not differ between groups. Measurements of oestrous cycle, pre-coital interval, copulatory and fertility indices did not show differences between groups. No significant differences were observed in the number of implantation, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups. Litter data including mean litter and pup weights were comparable between groups. No differences were found in sex ratio. No relevant differences were noted in the clinical signs of pups between groups. No relevant findings were found in decedent pups or in pups sacrificed at termination. No differences were found in terminal body weight. Slightly higher ovaries weight was recorded in the high dose group compared to controls. In the absence of any histological correlate, and since oestrous cycling and reproduction were inconspicious, this difference was not considered to be toxicologically relevant. At macroscopic examination, hairloss and/or scab formation/s in various regions of the body (head, forelimbs, ventral region and neck) were found more frequently in both sexes of the high dose group. Microscopically, the only relevant change observed was minimal scab formation/s in various regions of the body of the high dose group. However, these minimal findings could be considered not toxicologically relevant. Evaluation of the spermatogenic cycle did not show differences between the groups. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for both males and females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
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

Reproduction and developmental toxicity of the test article was investigated in Wistar Hannover rats up to Day 4 post partum following OECD guideline 421 and in compliance with GLP. The study design provided information on gonadal function, mating behaviour, conception, development of the conceptus and parturition. The dosages were: 100, 300 and 1000 mg/kg bw/day. The test item was formulated in methylcellulose (0.5% in purified water) and given at constant volume of 10 mL/kg body weight.

The most relevant changes, recorded during the in vivo phase of the study, were slight/moderate hairloss and/or scab formations in high dose males and females. These findings were also described at macroscopic and/or microscopic evaluation. However, in the absence of any other changes and since these findings were minimal, they could be considered not toxicologically relevant. No remarkable differences were found in body weight/body weight gain or food consumption between groups through the study. Slightly higher ovary weights were noted in the high dose group, however no histological findings were detected. Additionally, oestrous cycling and reproduction were incospicious. Therefore, this difference was not considered to be toxicologically relevant.

The mating performance including the pre-coital interval (i.e. the number of days paired to sperm positive day) and the copulatory evidence (i.e. the presence of sperm and/or copulation plug in situ or in the cage) did not show differences between groups. The resulting copulatory and fertility indices were comparable in all groups. Almost all females were pregnant and had comparable length of gestation. No variations were found in the number of corpora lutea and implantations between groups. All pregnant females gave birth. The litter size at birth and on Day 4 post partum was also similar between groups as well as the mean litter and pup weights. No relevant findings in pups were found at daily clinical observation nor at post mortem examination.

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for males and females.


Short description of key information:
A Reproduction/Developmental Toxicity Screening Test (OECD guideline 421) revealed no potential for reproduction and developmental toxicity up to the highest dose level tested (1000 mg/kg bw).

Justification for selection of Effect on fertility via oral route:
GLP guideline study

Effects on developmental toxicity

Description of key information
A Reproduction/Developmental Toxicity Screening Test (OECD guideline 421) revealed no potential for reproduction and developmental toxicity up to the highest dose level tested (1000 mg/kg bw).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2012 - 12 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Remarks:
Research Toxicology Centre S.p.A., Via Tito Speri, 12/14, 00040 Pomezia (Rome), ITALY
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco Italy
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 176 to 200 g; Females: 151 to 175 g
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least three times a week. During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent
material which was inspected and changed daily. The males were re-caged after mating as they were before mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
Suitable nesting material was provided and was changed at least 3 times a week.
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 4 to 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2°C
- Humidity: 55 ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 July 2012 To: 12 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was suspended in the vehicle (0.5% methylcellulose in purified water) at the final concentrations of 10, 30 and 100 mg/mL. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study no. 93100: BASF Project No.: 05Y0252/12X126) in the range from 10 to 100 mg/mL.
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (check of concentration and homogeneity). The stability was found to be at least 48 hours at room temperature in the concentration range of 10 to 100 mg/mL. Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) were also analysed to check the concentration and homogeneity. The overall results of the analyses were within the limits of acceptance stated in RTC SOPs for concentration (80-120%) and homogeneity (CV <10%).
Details on mating procedure:
Mating was monogamous (one male to one female) with the exception described below. One mid-dose male (no. 54) died during the first mating session. The assigned female (no. 53) was re-mated with a proven male (no. 52) of the same treatment group.
A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred with the exception of female no. 53 as previously described, or 14 days had elapsed. In one occasion of unsuccessful mating with the first assigned male, the female no. 61 (Group 4) was re-mated with a proven male (male no. 74) of the same treatment group. The second pairing combination was monitored for mating until positive signs of copulation were observed.
For three females (nos. 05, 29 and 59) the mating was inadvertently not detected in the first mating session. On the basis of this apparently negative sign, the females were allowed to have the second pairing cohabitation with proven males of the same treatment groups. The second pairing combination was monitored for mating, however due to the increment in body weight, the pregnancy status became evident and the females were
individually caged before completion of the second pairing combination.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing up to the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum (up to Day 26 for non pregnant females) and post partum periods until Day 3 post partum.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
daily
Duration of test:
until day post partum
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Maternal examinations:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CLINICAL SIGNS
Twice before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHTS
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION
Food consumption was recorded at weekly intervals, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 postpartum).

PARTURITION AND GESTATION LENGTH
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

ESTROUS CYCLE
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

EUTHANASIA
Parental animals that had completed the scheduled test period were killed with carbon dioxide. The males were killed after the mating of all females (i.e. after 38 days of treatment). The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after the positive identification of mating were killed shortly thereafter (Day 26/27 post coitum).

NECROPSY
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding the found dead animal no. 54) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
From all animals completing the scheduled test period the organs indicated in section Table 1 were dissected free of fat and weighed. The ratios of organ weight to terminal body weight were calculated for each animal.

HISTOPATHOLOGY
Samples of all the tissues listed in Table 1 from all animals were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid).
The tissues required for histopathological examination are listed in section 4.4.6. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
a) Tissues specified in section Table 1 from all animals in the control and high dose group killed at term.
b) Tissues specified in section Table 1 from one animal dying during the treatment period.
c) All abnormalities in all groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

EUTHANASIA
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopental.

NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed on Day 4 post partum and examined for external abnormalities and sex confirmation by gonadal inspection.
One pup (dam no. 31 of Group 2) with tail’s abnormalities was retained in fixative (10% neutral buffered formalin).
Statistics:
Group mean values were calculated for all parameters. Data from non-pregnant females were excluded from group mean calculations.
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences among control and treated group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test when “n” was more than 5 (Epididymides, ovaries and testes).
The criterion for statistical significance was p<0.05.
Indices:
Copulatory Index, Fertility Index, Copulatory Index, Fertility Index, Copulatory Interval, Pre-implantation loss, Pre-birth loss, Pup loss at birth, Cumulative pup loss on Day 4, Sex ratios
Details on maternal toxic effects:
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One mid-dose male (no. 54) was found dead on Day 5 of the mating phase. The cause of death was attributed to a mis-dosing on the basis of the macroscopic and histopathological findings.
At daily clinical observation, slight to moderate hairloss in different parts of the body surface (muzzle, head, dorsal and ventral regions, and/or lower forelimb) was recorded more frequently in males and females of the high dose group. Additionally slight scab formation was observed at the high dose group.
Corneal opacity and protruding eye were found in a single mid-dose female (no. 59). Cyphosis and piloerection were observed once in the mid-dose group during the mating phase (animal nos. 54 and 41) and post coitum period (animal no. 51).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain were comparable between groups throughout the study for both males and females.
On one occasion, a statistically significantly lower body weight gain was recorded in males of the mid-dose group during the pre-pairing period.
No differences were found in food consumption between the groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No differences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups. Regularity of the oestrous cycle was also found.
The number of implantations, corpora lutea, total litter size, pre-implantation loss and prebirth loss did not differ between groups.
Gestation length was also comparable between groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No differences were found in terminal body weight between groups.
Slightly but significantly higher absolute and relative ovaries weights (up to 14%) were found in the high dose group when compared to controls. In the absence of any histological correlate and since oestrous cycling and reproduction were incospicious, this difference was not considered to be toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Unscheduled death:
A single animal from Group 3 (animal no. 54), treated at 300 mg/kg bw/day, was found dead on Day 5 of the mating phase. Macroscopically, red fluid in the thoracic cavity was observed.
This change suggested that the cause of death was probably due to a mis-dosing and therefore was not treatment related.
Final sacrifice:
The most relevant macroscopic changes detected in the treated animals when compared with controls were the following:
Hairloss and/or scab formation/s in various regions of the body (head, forelimbs, ventral region and neck) in both sexes.
Minimally higher incidence of swollen liver in high dose males.
Pale, firm and raised area in the hepatic left lobe, reduced size of the testis and corresponding epididymidis and prostate in single instances.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The only relevant change observed in treated males and females was minimal scab formation/s in various regions of the body. In the absence of any other finding and since scab formation was minimal and occurs also spontaneously in rats, these findings could be
considered not toxicologically relevant.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The remaining lesions, such as hepatic necrosis, testicular atrophy and consequently reduced sperm in the epididymidis were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
CLINICAL SIGNS (OFFSPRING)
Mean litter and pup weights were comparable between groups on Days 1 and 4 post partum. No differences were found in sex ratio.
No remarkable differences were noted in the clinical signs of pups between groups. The findings generally recorded were small in size, cold to touch, apparently no food intake, or pale appearance. One low dose pup showed damaged tail.

GROSS PATHOLOGY (OFFSPRING)
No relevant findings were found in the two decedent pups, one in the control group and one in the low dose group.
At termination, one low dose pup (dam no. 31) showed kinked, red and swollen tail. These findings were considered incidental.
No other abnormalities were found in pups sacrificed on Day 4 post partum.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for both males and females.
Executive summary:

Reproduction/developmental toxicity of the test article was investigated in Wistar Hannover rats up to Day 4 post partum following the study design outlined in OECD guideline 421 and in compliance with GLP. Three groups of 10 animals/sex each received the test item at dosages of 100, 300 and 1000 mg/kg bw/day, respectively. The control group of the same number of animals/sex was administered with the vehicle alone (methylcellulose 0.5% in purified water). The test item and vehicle were given by oral gavage at constant volume of 10 mL/kg body weight. Males were treated for approximately 6 weeks including 2 weeks prior to pairing and continuously thereafter up to the day before necropsy. Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3 post partum. The following parameters were evaluated in parental animals: body weight, clinical signs food consumption, oestrous cycle, mating performance, litter data, macroscopic observations and organ weights. The histopathological examination, including the spermatogenic cycle for males, was performed in all animals of the control and high dose groups. Pups were monitored for daily clinical sings and body weight. Post mortem examination of pups was also performed.

One mid-dose male died on Day 5 of the mating phase due to a mis-dosing. Three females were found not pregnant at necropsy: one in each of the control, low and high dose groups. The number of females with live pups on Day 4 post partum was: 9 in each of the control group, low and high dose groups and 10 in the mid-dose group. Hairloss and/or scab formations were mainly recorded in the high dose group. Body weight, body weight gain and food consumption did not differ between groups. Measurements of oestrous cycle, pre-coital interval, copulatory and fertility indices did not show differences between groups. No significant differences were observed in the number of implantation, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups. Litter data including mean litter and pup weights were comparable between groups. No differences were found in sex ratio. No relevant differences were noted in the clinical signs of pups between groups. No relevant findings were found in decedent pups or in pups sacrificed at termination. No differences were found in terminal body weight. Slightly higher ovaries weight was recorded in the high dose group compared to controls. In the absence of any histological correlate, and since oestrous cycling and reproduction were inconspicious, this difference was not considered to be toxicologically relevant. At macroscopic examination, hairloss and/or scab formation/s in various regions of the body (head, forelimbs, ventral region and neck) were found more frequently in both sexes of the high dose group. Microscopically, the only relevant change observed was minimal scab formation/s in various regions of the body of the high dose group. However, these minimal findings could be considered not toxicologically relevant. Evaluation of the spermatogenic cycle did not show differences between the groups. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for both males and females.

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

Reproduction and developmental toxicity of the test article was investigated in Wistar Hannover rats up to Day 4 post partum following OECD guideline 421 and in compliance with GLP. The study design provided information on gonadal function, mating behaviour, conception, development of the conceptus and parturition. The dosages were: 100, 300 and 1000 mg/kg bw/day. The test item was formulated in methylcellulose (0.5% in purified water) and given at constant volume of 10 mL/kg body weight.

The most relevant changes, recorded during the in vivo phase of the study, were slight/moderate hairloss and/or scab formations in high dose males and females. These findings were also described at macroscopic and/or microscopic evaluation. However, in the absence of any other changes and since these findings were minimal, they could be considered not toxicologically relevant. No remarkable differences were found in body weight/body weight gain or food consumption between groups through the study. Slightly higher ovary weights were noted in the high dose group, however no histological findings were detected. Additionally, oestrous cycling and reproduction were incospicious. Therefore, this difference was not considered to be toxicologically relevant.

The mating performance including the pre-coital interval (i.e. the number of days paired to sperm positive day) and the copulatory evidence (i.e. the presence of sperm and/or copulation plug in situ or in the cage) did not show differences between groups. The resulting copulatory and fertility indices were comparable in all groups. Almost all females were pregnant and had comparable length of gestation. No variations were found in the number of corpora lutea and implantations between groups. All pregnant females gave birth. The litter size at birth and on Day 4 post partum was also similar between groups as well as the mean litter and pup weights. No relevant findings in pups were found at daily clinical observation nor at post mortem examination.

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered 1000 mg/kg bw/day for males and females.


Justification for selection of Effect on developmental toxicity: via oral route:
GLP guideline study

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the data, classification for reproduction/developmental toxicity is not warranted under Directive 67/548/EEC.

 

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for reproduction/developmental toxicity is not warranted under Regulation (EC) No.1272/2008.