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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

One valid sub-chronic oral study on rats is available for reproductive toxicity/develomental screening. There were no detailed clinical signs, signs related to treatment or effects on arena observations, sensory reactivity, grip strength or motor activity at 5 mg/kg/day, nor change in body weight gain or food consumption during the two-week period before mating, or during gestation or lactation and no change in any blood cell or plasma parameter; oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index, litter size, sex ratio or survival and development of the offspring, or macroscopic findings in the adult or offspring.

There were no microscopic changes observed in the reproductive organs at 5, 15 or 45 mg/kg/day, which were considered to be related to treatment. The seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. The slightly low prostrate weight in the 45 mg/kg/day group was thus considered to be of no toxicological significance and of uncertain relationship to treatment.

Short description of key information:

For reproductive/developmental toxicity the no-observed-effect-level (NOEL) was considered to be 15 mg/kg/day.

Justification for selection of Effect on fertility via oral route:

Contemporary study

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 May to 14 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable laboratotory animal supplier
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: Males: 343 to 395 g; Females: 237 to 278 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body (and floor) with a stainless steel mesh lid; changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: certified pelleted diet, ad libitum
- Water (e.g. ad libitum): potable water from the public supply, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23 °C
- Humidity: 40 - 70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light: 12 hours dark.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance TCD was prepared for administration as a series of graded concentrations in the vehicle, corn oil.

Starting with the lowest concentration (2 mg/mL), approximately 50 % of the final volume of vehicle was added to the required amount of pre-weighed test substance and magnetically stirred until dissolved. The solution was made up to final volume with vehicle and mixed using a magnetic stirrer until homogeneous. Higher concentrations (6 and 18 mg/mL) were prepared in ascending order of concentration using the same method.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity - before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix.

Achieved concentration - samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Analysis of the concentration of test substance in the fomulation ws undertaken by gas chromatograhy using external standard calibration. The method was validated prior to use.
Duration of treatment / exposure:
Males: For a minimum of four consecutive weeks, including two weeks prior to pairing.
Females: Two weeks prior to pairing, throughout mating and gestation and until Day 6 of lactation.
F1 animals: No direct treatment.
Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
Not applicable
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the findings of this preliminary study, it is considered that doses to be selected for administration on the follow-up Combined Repeat Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study (OECD 422) should be 5, 15 and 45 mg/kg/day. The doses used in this study (0, 5, 15 and 45 mg/kg/day) were selected in conjunction with the Sponsor.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

BODY WEIGHT: Yes
- Time schedule for examinations: F0 Males, Day that treatment commenced (Week 0), Each week, At necropsy
F0 Females, Day that treatment commenced (Week 0), Each week until mating was detected, Days 0, 6, 13 and 20 after mating, Days 1, 4 and 7 of lactation


Oestrous cyclicity (parental animals):
Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.

Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities,

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: following completion of Week 5 investigations
- Maternal animals: Day 7 of lactation.

GROSS NECROPSY
- All F0 adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Organs were weighed and tissues preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and Table 2 (attached) were prepared for microscopic examination and weighed, respectively. Bilateral organs were weighed together.
Postmortem examinations (offspring):
SACRIFICE
Intraperitoneal injection of sodium pentobarbitone

Time of necropsy; F1 offspring Day 7 of age.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

F1 generation (offspring)

Offspring at scheduled termination
Examined externally; those offspring deemed normal were discarded without further macroscopic examination. Any externally abnormal offspring were examined internally and any abnormal tissues were retained.

Premature deaths (Before Day 7 of age)
Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were subject to fresh macroscopic examination, where possible (external and internal); this also included an assessment for the presence of milk in thestomach, where this was possible.

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

Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including pre-coital interval and mating performance the similarity of the data was such that analyses were not considered to be necessary:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analysed at each timepoint separately:

Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods
Blood chemistry and haematology
Grip strength and motor activity
Oestrous cycles
Gestation length and gestation index
Litter size and survival indices
Organ weights, both absolute and adjusted for terminal body weight
Reproductive indices:
Group Post implantation Live birth
survival index (%) index (%)

1 Mean 90.0 97.6
N 10 10
2 Mean 93.3 98.7
N 10 10
3 Mean 93.5 98.8
N 10 10
4 Mean 96.8 95.0
N 8 8
Offspring viability indices:
Group Viability index %

1 Mean 98.7
N 10
2 Mean 98.8
N 10
3 Mean 99.3
N 10
4 Mean 92.3
N 8
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 45 mg/kg/day, one female (No. 48) had a moderate convulsion approximately 2.5 hours after administration on treatment Day 3 and another (No. 50) had a slight convulsion approximately 1-2 hours after administration on Day 4 post-partum.
Two females were killed for welfare reasons on Days 10 or 11 of the pre-mating period.One female (No. 45; 45mg/kg/day) had a slight convulsion on Day 6, and was killed for welfare reasons on Day 11, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical hypertrophy). The factor contributory to death was poor clinical condition.
One female (No. 46; 45mg/kg/day) had a moderate convulsion on Day 6, showed slight tremor on Day 7, and was killed for welfare reasons on Day 10, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical hypertrophy). The factor contributory to death was poor clinical condition.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Overall mean body weight gain (Week 0-5) was slightly high in males treated at 45 mg/kg/day (X 1.3), when compared with Control and was high in females at 15 or 45 mg/kg/day, with relationship to dose (X 1.7 or 1.9, respectively), during the two week pre-mating period.
When compared with Control, body weight gain during gestation was not affected by treatment; however, maternal body weight gain was low in treated females (5, 15 or 45 mg/kg/day) during the first four days of lactation (X 0.36, 0.18 or 0.18, respectively) andoverall lactation body weight gain (Days 1 to 7) was X 0.77, 0.50 or 0.70, respectively.

Overall food consumption of males at 45 mg/kg/day was marginally high in Weeks 4 and 5 (X 1.14), when compared with Control. Overall food consumption of females treated at 15 or 45 mg/kg/day was slightly low during lactation (X 0.88 or 0.84, respectively).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
It was considered that oestrous cycles were unaffected by treatment, during the two week pre-pairing period.
One low dose female (5 mg/kg/day) had an irregular cycle (at least 10 days without oestrous).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Pre-coital interval, mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Mean adjusted liver weight was low (range X 0.87-0.95), without dose-relationship, in males receiving 5, 15 or 45 mg/kg/day and mean prostate weight was low (X 0.78) at 45 mg/kg/day; mean adrenal weight was high in females at 5, 15 or 45 mg/kg/day (X 1.16, 1.19 or 1.27) and
mean brain weight was marginally high, without dose-relationship, in females at 15 or 45 mg/kg/day (both X 1.06), when compared with Control.

Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean offspring body weight at birth (Day 1 of age), following parental treatment at 45 mg/kg/day was low in both sexes (males X 0.90; females 0.88); subsequent body weight gain to Day 7 of age was slightly low (males: X 0.92; females: X 0.90)
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
The mean number of implantations, live litter size on Day 1 and sex ratio showed no adverse effect of parental treatment. The viability index of offspring up to Day 7 of age with maternal treatment at 45 mg/kg/day was lower than Control; this was attributable in part, to six deaths amongst offspring from a single litter (Dam 48).

CLINICAL SIGNS (OFFSPRING)
Clinical signs recorded for the offspring identified occasional pups with findings, but the type of findings and incidence were typical and showed no relationship to treatment.

BODY WEIGHT (OFFSPRING)
Mean offspring body weight at birth (Day 1 of age), following parental treatment at 45 mg/kg/day was low in both sexes (males X 0.90; females 0.88); subsequent body weight gain to Day 7 of age was slightly low (males: X 0.92; females: X 0.90), reflecting slightly low weight gain from Day 4 to Day 7 of age.

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption and compound intake; food efficiency; gross pathology; organ weights; histopathology;
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight;gross pathology; organ weights; histopathology;
Key result
Reproductive effects observed:
no
Conclusions:
Treatment at 15 mg/kg/day did not result in any systemic toxicity. At 45 mg/kg/day, there was no effect of treatment on mating performance, fertility or the number of implantations or
litter size on Day 1 of age but offspring body weights on Day 1 of age and subsequent weight gains from Day 4 to Day 7 of age were low, but there was no conclusive effect on offspring
survival. There was no effect on offspring body weights up to Day 7 of age at 5 or 15 mg/kg/day.

For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg/day and for reproductive/developmental toxicity the no-observed-effect-level (NOEL) was considered to be 15 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Dose descriptor:
NOAEL

Effects on developmental toxicity

Description of key information

For reproductive/developmental toxicity the no-observed-effect-level (NOEL) was considered to be 15 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
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

One valid sub-chronic oral study on rats is available for reproductive toxicity/develomental screening. There were no detailed clinical signs, signs related to treatment or effects on arena observations, sensory reactivity, grip strength or motor activity at 5 mg/kg/day, nor change in body weight gain or food consumption during the two-week period before mating, or during gestation or lactation and no change in any blood cell or plasma parameter; oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index, litter size, sex ratio or survival and development of the offspring, or macroscopic findings in the adult or offspring.

There were no microscopic changes observed in the reproductive organs at 5, 15 or 45 mg/kg/day, which were considered to be related to treatment. The seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. The slightly low prostrate weight in the 45 mg/kg/day group was thus considered to be of no toxicological significance and of uncertain relationship to treatment.

Justification for classification or non-classification

Although general toxicity effects were seen in the study, no effect on the fertlity of parents or on the development of offspring were observed therefore the substance will not be classified at this level.