Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction and developmental parameters, no effects were noted at any dose level. Thus the NOAEL for reproductive perfomance and offspring development was determined to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-01 to 2008-11-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd., 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approximately 10 weeks old at starting and 12 weeks at mating
- Weight at study initiation: (P) Males: 339 - 433 g; Females: 192 - 271 g
- Fasting period before study:
- Housing: Before mating: 4 animals of the same sex / cage; Mating: 1 male and 1 female / cage; Pregnant females were housed individually

- Diet: ad libitum (ssniff® SM R/M-Z+H "Autoclavable complete feed)
- Water: ad libitum (tap water)
- Acclimation period: 49 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9 – 24.7
- Humidity (%): 34 -70
- Air changes (per hr): 8 -12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in dried peanut oil in concentrations of 3, 30 and 200 mg/mL.

VEHICLE
- Justification for use and choice of vehicle: As this test item is not soluble in water, dried peanut (Arachis) oil was used for dose formulations preparation
- Concentration in vehicle: 3, 30 and 200 mg/mL
- Amount of vehicle: 5 mL/kg
- Lot/batch no.: 127K0016

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 6 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear] referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of test item in the vehicle, peanut oil, was analytically proven. Assessment of test item stability in this vehicle indicated stability at room temperature up to 72 hours at concentrations from approximately 1 to 250 mg/mL in dried peanut oil (LAB study code 08/639-316AN). A separate analytical report provides this information. Analytical control of dosing solutions was conducted during the study from all the concentrations employed; the measured concentrations ranged from 102 to 107 % of nominal concentrations. The analytical results were considered suitable for the study purposes.
Duration of treatment / exposure:
- Males were dosed for 28 days, 14 days pre-mating (PM) and 14 days mating/post- mating period (M).
- Females were dosed for 14 days pre-mating (PM), for up to 6 days mating period (M), through gestation (up to 23 days) (G) and day 3 post-partum (PP/PN) with necropsy the following day, or shortly thereafter.
Frequency of treatment:
Daily
Details on study schedule:
PRE-EXPERIMENTAL PERIOD
Animal arrival: Day [-49]: 13 August 2008
Veterinary control, acclimation: Day [-49] - Day [-1]: 13 August 2008-30 September 2008
Body weight measurement Day [-1]: 30 September 2008
Randomisation: Day [-1]: 30 September 2008
Animal identification: Day [-1]: 30 September 2008
First day of treatment: Day [0] :01 October 2008

TREATMENT PERIOD
Treatment period: M: Daily up to necropsy, 01-28 October 2008 (During the pre-mating and mating periods)
F: Daily up to the necropsy, 01 October–16 November 2008 (During the pre-mating, mating, gestation periods and lactation days)
All animals received the last dose one day before the necropsy.

Body weight measurement: M: Weekly, 01, 08, 15, 22, and 29 October 2008
F: Weekly prior to and during the mating period,
- On gestational days 0, 7, 10, 14, 17 and 20
- On postpartal days 0 and 4 01, 8, and 15 October 2008; from 15 October-up to 17 November 2008 (depending upon mating)

Food consumption measurement:
M: At least weekly prior to and during the mating period, 01, 08, 15, 22, and 29 October 2008
F: Weekly prior to the mating
- On gestational days 0, 7, 14 and 21
- On postpartal days 0 and 4 01, 08, and 15 October 2008; from 15 October up to 17 November 2008 (depending upon mating)

General clinical observation: M: Daily up to the necropsy, 01-28 October 2008 (During the pre-mating and mating periods.)
F: Daily up to the necropsy, 01 October–16 November 2008
(During the pre-mating, mating, gestation and lactation periods.)

Detailed clinical observations: Treatment Day 0, then weekly (M/F), 01, 08, 15, 22, 29 October, and 05, 12 November 2008

Mating period: 15-20 October 2008

Examination of placental sign: On gestational days 13 and 14, 28 October-02 November 2008 (depending upon mating)

Observation of delivery process: From the gestational day 21, 06-11 November 2008 (depending upon mating day)

Body weight measurement of pups: On postnatal days 0 and 4, 06-15 November 2008 (depending upon delivery day)

Necropsy: M: After mating period (after 28 days of treatment), 29 October 2008
F: Dams: on post-partal day 4, or shortly thereafter
Offspring: on post-partal day 4, or shortly thereafter 10-17 November 2008

Analytical control of the dosing solution: First and last weeks of the treatment 06 October, 13 November 2008
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels based on previous studies conducted with this test item in rats (28-Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat SPL Project
Number: 235/217)
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, after treatment approximately at the same time

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of dosing (day 0), at least weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The food consumption was determined at least weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment periods.

OTHER: Mortality and morbidity
- Time schedule: twice daily (at the beginning and end of each day)
Estrous cyclicity (parental animals):
Each morning a vaginal smear was monitored.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis, epididymis (total and cauda), seminal vesicles and prostate weight; histopathology of testes, epididymides, prostate and seminal vesicles
Litter observations:
STANDARDISATION OF LITTERS
- Live pups were counted, sexed, weighed individually with an accuracy of 0.1 g within 24 hours of parturition (on the first day after parturition was complete), and on day 4 post partum.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, behavioural abnormalities, other: runts (pups that were significantly smaller than other pups)

GROSS EXAMINATION OF DEAD PUPS:
Yes, for at least external abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations (the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 post partum.
- These animals were subjected to postmortem examinations (macroscopic) as follows: Pups euthanized at day 4 postpartum, or shortly thereafter (up to day 6 postpartum), were carefully examined for at least gross abnormalities. The found dead, or stillborn pups were subjected to necropsy with macroscopic examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations (found dead pups); internal examinations include autolysis, lungs, stomach, small intestine, large intestine and abdominal cavity

Statistics:
Statistical Evaluation
The statistical evaluation of appropriate data (marked † above) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to assess the significance of inter-group differences. Where significant result
was obtained at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.
Reproductive indices:
Male Mating Index
Male Fertility Index
Female Mating Index
Female Fertility Index
Female Gestation Index
Offspring viability indices:
− Survival index of pups on postnatal days 0 and 4


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d (high dose group) suriving animals showed soft faeces (cage side observation) were noted in the male animals on days 1-2 (2/28 days of treatment). Female rats showed hunched back position (2/12 females, for 6/40, or 11/43 days), and dry red material (periocular, 1/12 females, for 5/42 days).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Preterminal mortality occurred during the study in 2 females, but was not considered a result of a systemic test item effect. One low dose female was euthanized on day 34 due to pulmonary damage, followed by intrauterine death and poor clinical condition. Collapsed, red discoloured, cystic right lobes of the lungs and small beige liver were also observed in this female. At microscopic examination, the histopathological findings were correlated with gross lesions. Mild diffuse purulent pleuritis was seen in the lungs. Moderate transmural necrosis of the left horn and abscess adhering to the right horn with moderate septic intraluminal haemorrhage were recorded in the uterus. One high dose female was found dead one day after parturition, likely due to post partum haemorrhage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the females, the high dose animals administered 1000 mg/kg bw/day showed slightly lower mean body weight and body weight gain values during the gestation and post-partal (PP) period, attaining statistical significance on PP 4. Following administration of 150, or 15 mg/kg bw/day, the body weight differences were minor, although lower values were also observed occasionally after GD 14 in these animals. A statistically significant lower body weight gain was noted at 15 mg/kg bw/day during the post partum period (PP 0-4).
Food consumption and compound intake (if feeding study):
no effects observed
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic changes were found in preterminal deaths. In the found dead high dose female (4502), mild lymphoid atrophy of the thymus was observed microscopically. The probable cause of death of this female was considered to be the post partum haemorrhage. In the low dose female (2502), the microscopic findings were correlated with gross lesions. Mild diffuse purulent pleuritis was seen in the lungs. Moderate transmural necrosis of the left horn and abscess adhering to the right horn with moderate septic intraluminal haemorrhage were recorded in the uterus.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
MORTALITY AND CLINICAL OBSERVATIONS
- Preterminally dead animals:
Mortality and clinical adverse effects occurred in 2 females at 15 and 1000 mg/kg bw/day, respectively. Female 2502 showed no clinical signs up to and including day 29. From day 29 to days 33/34, hunched back position, piloerection and decreased activity were noted in this animal, with genital bleeding as of day 32. On day 34, paleness, prone position and moribundity occurred, and the animal was euthanized preterminally for humane reasons. At macroscopic examination, pregnancy was shown; however, no foetuses could be identified, but intrauterine haemorrhagic masses, approximately 1x1 cm. Collapsed, red discoloured, cystic right lobes of the lungs and small beige liver were also noted. Based on the lack of similar changes in other animals from this group, or at higher dose levels, the cause of death is not considered a systemic TODI-related effect, but secondary to pulmonary damage followed by intrauterine death and poor clinical condition of this female. Histopathology examination revealed microscopic findings correlated with gross lesions. Mild diffuse purulent pleuritis in the lungs, moderate transmural necrosis of the left horn, abscess adhering to the right horn and moderate septic intraluminal haemorrhage in the uterus were recorded. Female 4502 presented piloerection and genital bleeding after parturition on day 40, followed by death on day 41. Its death was not considered directly related to test item administration, but rather secondary to post-partum haemorrhage. Moreover, it should be noted that all the pups of this female died (4 born, with 16 corpora lutea and 15 implantation sites).

- Surviving animals
1000 mg/kg bw/day (High Dose)
Soft faeces (cage side observation) were noted in the male animals on days 1-2 (2/28 days of treatment). Female rats showed hunched back position (2/12 females, for 6/40, or 11/43 days), and dry red material (periocular, 1/12 females, for 5/42 days).

150 and 15 mg/kg bw/day (Mid and Low Doses)
There were no clinical signs displayed at these dose levels that were considered toxicologically significant, or related to TODI administration.

BODY WEIGHT
Slightly lower body weight and body weight gain values than control animals, with no statistical significance, were observed in the 1000 mg/kg bw/day males throughout the study. In the 150 and 15 mg/kg bw/day males, there were no variations in the body weight, or body weight gain values that were ascribed to test item administration, or were considered toxicologically significant. In the females, the high dose animals administered 1000 mg/kg bw/day showed slightly lower mean body weight and body weight gain values during the gestation and post-partal (PP) period, attaining statistical significance on PP 4. Following administration of 150, or 15 mg/kg bw/day, the body weight differences were minor, although lower values were also observed occasionally after GD 14 in these animals. A statistically significant lower body weight gain was noted at 15 mg/kg bw/day during the post partum period (PP 0-4).

FOOD CONSUMPTION
There were no adverse effects noted in the food consumption during the study, in both the male and female animals, at dose levels of up to and including 1000 mg/kg bw/day.
Minimally higher food consumption was observed in the males administered 150 or 1000 mg/kg bw/day in the mating/post-mating period (days 14 to 21), not correlated with the body weight changes, and in the females at 150 mg/kg bw/day (days 14 to gestation day GD 0).

MATING EVALUATION
- Oestrous Cycle, Reproductive Ability Assessment and Indices
There were no differences considered significant between the control and test item-treated groups with regard to reproductive ability, and in the mating, fertility and gestation indices.
In the male animals, the mating index was 100 % in all groups. A slightly lower male fertility index (92 %, p value<0.01) compared to control was noted at 15 and 1000 mg/kg bw/day. No effects were observed at 150 mg/kg bw/day.
In the female groups, all animals mated. However, at 15 mg/kg bw/day slightly to moderately lower fertility (92 %, p value<0.01) and gestation indices (91 %, p value<0.01) occurred. No effects were noted at 150 mg/kg bw/day. At 1000 mg/kg bw/day, only the fertility index was slightly lower than control (92 %, p value<0.01), while the gestation index was not affected (100 %). As the dose response was not consistent, and no changes were noted in the mid dose group at 150 mg/kg bw/day, these differences were not considered a test item-related effect. Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within 6 days of pairing (cohabitation).

- Evaluation of the Gestation, Parturition and Post-Partal Period
No test item effect on the duration of pregnancy, or abnormalities in the gestation outcome were observed that could be ascribed to the treatment. The mean duration of pregnancy varied from 22.33 days (22 to 23 days) in the controls, 22.40 days (22 to 23 days) in the low dose, 22.17 days (21 to 23 days) in the mid dose to 21.91 days (21 to 22 days) in the high dose group. This variation was not considered to be biologically significant, because it is specific for this rat strain according to LAB Research Ltd. historical control data (gestation length: 22.17 ± 0.44 days, range: 21 to 23 days, n = 41). Females were allowed to litter and rear their offspring. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or post-partal period noted in the treated groups at dose levels up to and including 150 mg/kg bw/day compared to control animals.
At 1000 mg/kg bw/day, the number of pups born was statistically lower than control, and the mean number of live pups on PND 4 was also lower, although not statistically different. However, the numbers of pups in the high dose group were within the normal control range, and therefore these slight changes were not considered to represent a treatment-related effect.
All the parturitions were normal. However, it should be noted that most, or all the foetuses in 2 litters died, or were stillborn (females 2504, administered 15 mg/kg bw/day, and 4502, administered 1000 mg/kg bw/day). The number of corpora lutea and implantations were slightly lower in the 1000 mg/kg bw/day dose group, compared to females in the control or lower dose groups. However, this variation was minor, not statistically significant and was not considered related to test item administration, but to biological variability. Pre-implantation mortality value was lower or comparable in the treated groups (at all dose levels) than in the controls. Intrauterine mortality percentages tended to be above the concurrent control value. However, there were no statistical differences and analysis of individual data suggested that the relatively high values were due to a high intrauterine mortality in individual females, with no consistent dose-response (female 2504, at 15 mg/kg bw/day, females 3509 and 3502, at 150 mg/kg bw/day, and female 4502, found dead, administered 1000 mg/kg bw/day).
All the other individual intrauterine mortality values were low and thus these variations were not considered related to test item administration. A higher, but not statistically significant post-natal mortality was observed at high dose (1000 mg/kg bw/day) due to one female (4502, of which all the pups died). All the other individual data within the high dose group were 0 or low (within 7 %). These minimal differences in post-natal mortality were not considered to be treatment-related.

MACROSCOPIC AND MICROSCOPIC FINDINGS
Preterminal Deaths (Found Dead, or Preterminally Euthanised Animals)

- Macroscopic findings
One high dose female (4502) was found dead on day 41. Red fluid in the perianal area was recorded and dark discolouration/red of the lungs and thymus was found macroscopically in this animal. One low dose female (2502) was preterminally sacrificed on day 34, due to poor clinical condition. No foetuses were noted but intraluminal bilateral haemorrhagic mass in the uterus was present. Collapsed, red discoloured, cystic right lobes of the lungs and small beige liver were observed in this female at necropsy.

- Microscopic findings
In the found dead high dose female (4502), mild lymphoid atrophy of the thymus was observed microscopically. The probable cause of death of this female was considered to be the post partum haemorrhage. In the low dose female (2502), the microscopic findings were correlated with gross lesions. Mild diffuse purulent pleuritis was seen in the lungs. Moderate transmural necrosis of the left horn and abscess adhering to the right horn with moderate septic intraluminal haemorrhage were recorded in the uterus.

Terminal Deaths (Scheduled Necropsy)

- Macroscopic Findings
There was no evidence of treatment-related macroscopic changes in any surviving animals administered TODI, that underwent scheduled necropsy at termination. The changes in the jejunum, ileum, caecum, lungs, kidneys, liver, epididymis, seminal vesicles, ovary, thymus, uterus and subcutis were considered to be either incidental or procedure-related.

- Microscopic Findings
The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were of similar histological structure in both control and high dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present. Common background microscopic changes seen across control and high dose males with low severity were noted in a variety of reproductive organs including, testes, epididymides, seminal vesicles and prostate. In the testes, the spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in control and high dose males.

ORGAN WEIGHT
In the male and female animals, there were no toxicologically significant changes in organ weight values noted after administration of TODI during this study, at up to and including 1000 mg/kg bw/day. In the test item-treated males, there were no statistically significant differences when compared with the controls. In the females, the mean terminal body weight was statistically lower at high dose. Brain weights relative to body weight were statistically higher in the low and high dose groups, but this was a consequence of lower body weights and not considered an effect of the treatment on brain weight.





Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
PRETERMINALLY DEAD PUPS
Autolysis was observed on occasion. Red discolouration or pale area of the lungs was found in 15 low dose and 1 high dose pups. The positive floating test of the lungs was recorded in 9/26 pups. Dilatation of the stomach, and yellow discolouration of the stomach and/or small intestine with the presence of yellow gelatinous material were seen in 17 low dose and 1 mid dose animals. In addition, gelatinous red material in the abdominal cavity was noted in animal 2504/12 and empty stomach in animal 3501/15.

Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In the low dose group, 20/144 pups were found dead (6) or were stillborn (14). However, 3 litters only were affected (of female 2504, with a high incidence, and 2507 and 2511, with low incidence of 3/16 and 1/14, respectively). No cannibalized pups were observed in this group. In the mid dose group, 4/155 pups were found dead (with or without cannibalisation) or were stillborn. In the high dose group, a total of 6/131 pups were dead (5, of which 3 cannibalized) or stillborn (1).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
MORTALITY AND CLINICAL OBSERVATIONS
- Preterminally dead pups
No pups died in the control group. In the low dose group, 20/144 pups were found dead (6) or were stillborn (14). However, 3 litters only were affected (of female 2504, with a high incidence, and 2507 and 2511, with low incidence of 3/16 and 1/14, respectively). No cannibalized pups were observed in this group. In the mid dose group, 4/155 pups were found dead (with or without cannibalisation) or were stillborn. In the high dose group, a total of 6/131 pups were dead (5, of which 3 cannibalized) or stillborn (1). There were no test item-related clinical signs noted in these pups. Autolysis was observed on occasion. Red discolouration or pale area of the lungs was found in 15 low dose and 1 high dose pups. The positive floating test of the lungs was recorded in 9/26 pups. Dilatation of the stomach, and yellow discolouration of the stomach and/or small intestine with the presence of yellow gelatinous material were seen in 17 low dose and 1 mid dose animals. In addition, gelatinous red material in the abdominal cavity was noted in animal 2504/12 and empty stomach in animal 3501/15. No deaths or observations in pups were likely attributed to treatment.

- Surviving pups
No clinical or behavioural changes were noted in the surviving pups (F1 generation) following administration of TODI in the parental (P) generation.

BODY WEIGHT
There were no statistical differences between the group mean litter weights on day 0 or 4. The mean pup weight-gain-per-litter at 15 mg/kg bw/day, was higher than control, and the mean pup weight-per-litter was lower than control in the 1000 mg/kg bw/day dose level. These differences were small and within expected variations in the population of Wistar rats at this age (litter means: body weight, PN0: 5.9-8.0 g, PN4: 8.7-14.6 g, body weight gain, 2.5-6.6 g). When evaluated as all individual pups/group, statistically significant lower mean body weights (days 0) were recorded at 15, 150 and 1000 mg/kg bw/day, with a difference of up to -8 %. On day 4, statistically higher values were found at 15 mg/kg bw/day and statistically lower values at 150 and 1000 mg/kg bw/day. Body weight gains at 0 - 4 days were statistically higher than control at 15 mg/kg bw/day. However, pup weights and weight gains in all groups were within the expected range based on contemporaneous study data (mean values/all pups: body weight, PN0: 4.3- 8.6 g, PN4: 5.9-15.4 g, body weight gain, 1-7.1g), thus the statistical differences were not considered to be biologically significant.

MASCROSCOPIC AND MICROSCOPIC FINDINGS
- Preterminal deaths (found death or preterminally euthanized animals)
A total of 30 pups was found dead and/or cannibalized post partum between days 0 - 1. Necropsy was performed on 20, 3 and 3 animals at dose levels of 15, 150 and 1000 mg/kg bw/day, respectively. Autolytic changes were found in 20, 1 and 2 pups from the low, mid and high dose, respectively. Macroscopic findings were noted in the stomach and/or small intestine, lungs, or abdominal cavity.

OTHER
No external abnormalities ascribed to test item administration were detected at the clinical or macroscopic examinations of the pups. The number of pups
and pup survival were lower in the treated groups than in the control group. However, the values were within the historical range, and no dose response was present. Thus, these variations were not considered toxicologically significant, or related to TODI administration. The sex ratios were similar in the control and treated groups. No statistically significant variations were observed on PN4 compared to PN0. There were no changes that were ascribed to test item administration in the survival index values at PN0 or PN4 for either sex.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction and developmental parameters, no effects were noted at any dose level. Thus the NOAEL for reproductive perfomance and offspring development was determined to be 1000 mg/kg bw/day.
Executive summary:

The objective of this study was the Reproduction/Developmental Toxicity Screening Test with the test item TODI in the rat according to OECD 421 and the draft OECD guidance document 43. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 15, 150 or 1000 mg/kg bw/day compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses.

Clinical observations for signs of ill health or reaction to treatment were made once daily. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly. Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of selected organs and tissues were determined. A histopathological examination was performed on the selected preserved organs and tissues of the animals of the control and high dose groups, and on abnormal tissues from low and mid dose groups.

 

TODI administered daily by oral gavage in Wistar rats, for 28 days in the male animals, and up to 46 days (females) led to minor clinical signs at 1000 mg/kg bw/day. These consisted of transient changes such as soft faeces (cage side observation) in the males on 2/28 days of treatment, and occasional hunched back position and dry red material, periocular, in the females. There were no toxicologically significant clinical signs at dose levels below and including 150 mg/kg bw/day. Preterminal mortality occurred during the study in 2 females, but was not considered a result of a systemic test item effect. One low dose female was euthanized on day 34 due to pulmonary damage, followed by intrauterine death and poor clinical condition. The female was pregnant (corpora lutea and implantation sites present). However, at necropsy no foetuses could be identified, but intraluminal bilateral haemorrhagic masses in the uterus were present. Collapsed, red discoloured, cystic right lobes of the lungs and small beige liver were also observed in this female. At microscopic examination, the histopathological findings were correlated with gross lesions. Mild diffuse purulent pleuritis was seen in the lungs. Moderate transmural necrosis of the left horn and abscess adhering to the right horn with moderate septic intraluminal haemorrhage were recorded in the uterus.

One high dose female was found dead one day after parturition, likely due to post partum haemorrhage. Slightly lower body weight and body weight gain values than control animals, with no statistical significance, were observed in high dose males (1000 mg/kg bw/day) throughout the study. High dose females showed slightly lower mean body weight and body weight gain values during the gestation and post-partal period, attaining statistical significance on PND 4. There were no adverse effects noted in the food consumption during the study, in either the male or female animals, at dose levels of up to and including 1000 mg/kg bw/day. There were no significant differences between the control and test item treated groups with regard to reproductive ability, or in the mating, fertility or gestation indices. Test item administration did not impact the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation). No test item effect on the duration of pregnancy was observed. All females littered in 21 to 23 days. There were no abnormalities in pups that were ascribed to the treatment. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or post-partal period noted in the treated groups at dose levels up to and including 1000 mg/kg bw/day compared to control animals.

There were no test item-related alterations in the delivery data of TODI treated dams as compared to the control value. The number of corpora lutea and implantations were slightly lower in the high dose group (1000 mg/kg bw/day), compared to females in the control or lower dose groups. However, this variation was minor, not statistically significant and was not considered related to test item administration, but to biological variability. Pre-implantation mortality value was lower or comparable in the treated groups (at all the dose levels up to and including 1000 mg/kg bw/day) than in the controls. Relative intrauterine mortality tended to be above the concurrent control value. However, there were no statistical differences and analysis of individual data suggested that the relatively high values were due to a high intrauterine mortality in individual females, with no consistent dose-response (female 2504, at 15 mg/kg bw/day, females 3509 and 3502, at 150 mg/kg bw/day, and female 4502, found dead, administered 1000 mg/kg bw/day). All the other individual intrauterine mortality values were low and thus these variations were not considered related to test item administration. A higher, but not statistically significant post-natal mortality was observed at 1000 mg/kg bw/day due to one female (4502, of which all the pups died). All the other individual mortality data within the group were 0 or low (within 7 %). This was not considered to be treatment-related. Litter examination did not reveal any clinical test item-related effects compared to observations noted in the control group. In the F1 generation, there were no pups with adverse clinical changes, or gross abnormalities. Gross necropsy of dead pups showed no effects of treatment. At necropsy examination, dilatation of the stomach, yellow discolouration of the stomach and/or small intestine with the presence of the yellow gelatinous, autolytic changes and red discolouration or pale area of the lungs were found in these pups.

The number of pups and pup survival were lower in the treated groups than in the control animals, however, all values were within the historical range, and no dose response was noted. Thus, these variations were not considered toxicologically significant, or related to TODI administration. The sex ratio was similar in the control and treated groups. No significant variations were observed on PN4 compared to PN0. In the parent animals surviving to scheduled termination, the administration of TODI at dose levels of 15, 150 or 1000 mg/kg bw/day was not associated with any test item-related macroscopic or microscopic findings. There were no treatment-related organ weight changes.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction parameters, no effects were noted at any dose level, resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate and adverse effects, the NOAEL was 1000 mg/kg bw/day.

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
Quality of whole database:
Guideline and GLP compliant study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction and development:

TODI was examined for its effect on reproduction in a reproduction toxicity screening test according to OECD Guideline 421. TODI administered daily by oral gavage to Wistar rats, for 28 days (males) and up to 46 days (females) at dose levels of 15, 150 or 1000 mg/kg bw/day led to minor clinical signs in the high dose group. The few transient clinical findings noted at high dose consisted of soft faeces in males, and occasional hunched back position and dry red material, periocular, in single females. There were no toxicologically significant clinical signs at dose levels below and including 150 mg/kg bw/day. Preterminal mortality occurred during treatment in 2 females, but was not considered a result of a systemic test item effect. One low dose female was euthanised on day 34 due to pulmonary damage, followed by intrauterine death and poor clinical condition. One high dose female presented post-partum haemorrhage and was found dead one day after parturition. Slightly lower body weight and body weight gain values than control animals, with no statistical significance, were observed in high dose males throughout the study. In the high dose females, slightly lower mean body weight and body weight gain values were noted during the gestation and post-partal (PP) periods, attaining statistical significance on PP 4. There were no adverse effects noted in food consumption during treatment, in either the male or female animals, at dose levels of up to and including 1000 mg/kg bw/day. The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, gestation, parturition or post-partal period. There were no adverse findings at macroscopic or microscopic examination at up to and including 1000 mg/kg bw/day. There were no treatment-related organ weight changes. In the F1 generation, there were no pups with adverse clinical changes, or gross abnormalities. No toxicologically significant effect on pup body weight, or pup survival was noted. Gross necropsy of dead pups showed no effects of treatment with the test item. In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction and developmental parameters, no effects were noted at any dose level. Thus the NOAEL for reproductive perfomance and offspring development was determined to be 1000 mg/kg bw/day.

In respect of the performance of a extended-one-generation study it has to be considered that in practice exposition can be excluded and that the Reproduction/ Developmental Toxicity Screening Test with TODI in the Rat according to OECD 421 revealed no effects at the limit dose (1000 mg/kg bw) in F1 although in the high dose females, slightly lower mean body weight and body weight gain values were noted during the gestation and post-partal periods. Thus, the cited study (OECD 421) can be regarded as sufficient to evaluate this endpoint.In addition, due to the very limited bioavailability of TODI after any relevant route of exposure, further studies are scientifically not meaningful and therefore not in line with animal welfare ideas.

Effects on developmental toxicity

Description of key information

TODI was examined in a reproduction toxicity screening test. Under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction and developmental parameters, no effects were noted at any dose level. Thus the NOAEL for reproductive performance and offspring development was determined to be 1000 mg/kg bw/day.

 

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
Quality of whole database:
Guideline and GLP compliant study
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

Developmental toxicity / teratogenicity

In respect of a Developmental toxicity / teratogenicity study it has to be considered that in practice exposition can be excluded and that the Reproduction/ Developmental Toxicity Screening Test with TODI in the Rat according to OECD 421 revealed no effects at the limit dose (1000 mg/kg bw) in F1 although in the high dose females, slightly lower mean body weight and body weight gain values were noted during the gestation and post-partal periods. Thus, the cited study (OECD 421) can be regarded as sufficient to evaluate this endpoint.

TODI was examined for its effect on reproduction in a reproduction toxicity screening test according to OECD Guideline 421. TODI administered daily by oral gavage to Wistar rats, for 28 days (males) and up to 46 days (females) at dose levels of 15, 150 or 1000 mg/kg bw/day led to minor clinical signs in the high dose group. The few transient clinical findings noted at high dose consisted of soft faeces in males, and occasional hunched back position and dry red material, periocular, in single females. There were no toxicologically significant clinical signs at dose levels below and including 150 mg/kg bw/day. Preterminal mortality occurred during treatment in 2 females, but was not considered a result of a systemic test item effect. One low dose female was euthanised on day 34 due to pulmonary damage, followed by intrauterine death and poor clinical condition. One high dose female presented post-partum haemorrhage and was found dead one day after parturition. Slightly lower body weight and body weight gain values than control animals, with no statistical significance, were observed in high dose males throughout the study. In the high dose females, slightly lower mean body weight and body weight gain values were noted during the gestation and post-partal (PP) periods, attaining statistical significance on PP 4. There were no adverse effects noted in food consumption during treatment, in either the male or female animals, at dose levels of up to and including 1000 mg/kg bw/day. The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, gestation, parturition or post-partal period. There were no adverse findings at macroscopic or microscopic examination at up to and including 1000 mg/kg bw/day. There were no treatment-related organ weight changes. In the F1 generation, there were no pups with adverse clinical changes, or gross abnormalities. No toxicologically significant effect on pup body weight, or pup survival was noted. Gross necropsy of dead pups showed no effects of treatment with the test item. In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for TODI for parental effects was 150 mg/kg bw/day. For reproduction and developmental parameters, no effects were noted at any dose level. Thus the NOAEL for reproductive perfomance and offspring development was determined to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the results of the Reproduction/Developmental Toxicity Screening Test the test item TODI is not classified and labelled according to Regulation 1272/2008/EC (CLP).