Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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:
17 February 2012 - 18 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 4-Methylthiosemicarbazide (MTSC)
- Physical state: white powder
- Lot/batch No.: 11012441081
- Purity: 99.74%
- Expiry date: 06 January 2013
- Storage conditions: at room temperature and protected from light and humidity.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age and mean body weight at study initiation: at the beginning of the treatment period, the males were approximately 10 weeks old and had a mean body weight of 338 g (range: 306 g to 370 g) and the females were approximately 9 weeks old and had a mean body weight of 222 g (range: 194 g to 248 g). The males and the females were sexually mature and were not siblings. The females were virgin.
- Fasting period before study: no
- Housing: the animals were individually housed, except during pairing and lactation in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen because of software limitations and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litters as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 6 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 29 February 2012 to 25 April 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water obtained by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. It was mixed with the required quantity of vehicle.
The frequency of dose formulation preparation was based on available stability data. The dose formulations were stored at room temperature and protected from light pending delivery to the study room in brown flasks.

VEHICLE
- Concentration in vehicle: 0.1, 0.5 and 2 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs (and for a maximum of 3 weeks)
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: not assessed, dose formulation is a solution
Stability: stable for 9 days at room temperature and protected from light
Duration of treatment / exposure:
In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (i.e. at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period,
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5, 2.5 and 10 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study. In this study, the test item was given to Sprague-Dawley rats at 0.5, 2.5 or 10 mg/kg/day. At 10 mg/kg/day, there were lower hemoglobin concentration, hematocrit and thymus weight in females. At microscopic examination, there were minimal lymphoid atrophy in the thymus and increase in the extramedullar hematopoiesis in the spleen of both sexes, as well as a decrease in adipocyte numbers in the bone marrow of females. At 0.5 and 2.5 mg/kg/day, there were no relevant in-life, organ weight, macroscopic or microscopic findings.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS (mortality):
- Time schedule: at least twice a day during the treatment period.

MORTALITY AND CLINICAL SIGNS: once a day.

BODY WEIGHT (GAIN):
- Time schedule: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coïtum (p.c.) and days 1 and 5 p.p.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 p.p.
During the pairing period, the food consumption was not measured for males or females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females were mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 post-coitum.

GROSS NECROPSY
Macroscopic post-mortem examination of principal thoracic and abdominal organs.

HISTOPATHOLOGY
- ovaries or testes and epididymides of all control and high-dose animals.
- see above sperm parameters
- spleen (in all groups) and thymus (groups 1 and 4).

ORGAN WEIGHTS: epididymides, ovaries, testes, adrenals, heart, liver, kidneys, spleen and thymus.
Postmortem examinations (offspring):
SACRIFICE: on day 5, post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Data are compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
(limited data)
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no in-vivo observation of sperm
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

MORTALITY:
There were no unscheduled deaths.
Three females given 0.5 mg/kg/day were sacrificed on day 25 p.c. for absence of delivery.

CLINICAL SIGNS:
There were no test item-related clinical signs.
Incidental clinical signs included piloerection, area of hair loss, cutaneous lesion and brownish vaginal discharge.

BODY WEIGHT (GAIN):
There were no test item-related effects on mean body weight and mean body weight gain in males.

At 10 mg/kg/day, there was a statistically significant retardation in mean body weight gain throughout the whole gestation period and particularly in the first week of gestation (up to -51% from controls), resulting in a lower mean body weight from day 7 p.c. (up to -11%). These adverse findings were considered to be related to treatment with the test item.
During the pre-mating period, there were a tendency towards a lower mean body weight gain ( 25%) which was considered to be of minor toxicological relevance and non-adverse as there was no statistical significance and no effects on mean body weight.
During the lactation period, the lower mean body weight recorded on days 1 and 5 p.p. when compared with controls and reaching statistical significance on day 1 p.p. was considered to be due to the effects seen on gestation mean body weight. There were no test item-related effects during lactation on mean body weight gain.
There were no test item-related effects on mean body weight and mean body weight gain in females at 0.5 and 2.5 mg/kg/day.

FOOD CONSUMPTION:
There were no test item-related effects on mean food consumption in males.
At 10 mg/kg/day, there was a statistically significant lower mean food consumption in the first week of gestation. Taking into account the amplitude of the change (-12% from controls, p<0.01), the statistical significance and the effects on gestation mean body weight at the same time, this finding was considered to be test item-related. There were no test item-related effects on mean food consumption at this dose-level in pre-mating and lactation periods.
There were no test item-related effects on mean food consumption in females at 0.5 and 2.5 mg/kg/day.

REPRODUCTIVE PERFORMANCE:
Mating and fertility data:
There were no test item-related effects on mating or fertility indexes.
At 2.5 and 10 mg/kg/day, there was a mean pre-coital time slightly prolonged when compared with that of the controls. This was due in each group to one pair that mated 13 days after the start of the pairing period (mid-dose group) or that did not mate within 2 weeks (high-dose group). In both groups, the estrous cycles of the females were blocked in diestrous. The female from the high-dose group was then paired with a proven male and they quickly mated. These delays of mating were considered to be fortuitous as noted in isolated pairs in both groups and as they can also be observed spontaneously in this strain of rat. When removing those outsiders, pre-coital time in both groups remained slightly prolonged than in controls. However, in the absence of any dose-relationship and taking into account that all the other pairs mated in no more than 5 days as it is commonly observed in this strain of rat, an effect of the treatment with the test item was considered unlikely.
All females were pregnant, except two non pregnant females at 0.5 mg/kg/day which were sacrificed on day 25 p.c. for absence of delivery. This was considered to be fortuitous.
Another female of that group did not deliver and was sacrificed on day 25 p.c. but was pregnant. It had brownish vaginal discharge on day 25 p.c. and had brownish content in the dilated uterus and three corpora lutea and implantations sites at necropsy. This was also considered to be fortuitous.

Delivery data:
There were no test item-related effects on the mean duration of gestation, mean number of corpora lutea and implantation sites, mean number of females having delivered or on the mean pre implantation loss at any dose-level.
At 10 mg/kg/day, there were a statistically significantly lower mean number of pups delivered correlating with a higher mean post-implantation loss, when compared with controls. These adverse findings were considered to be test item-related.
At 0.5 and 2.5 mg/kg/day, there were no effects on mean number of pups delivered and mean post implantation loss considered as toxicologically significant (mean data comparable to the control mean and/or including in the historical control data).

ORGAN WEIGHTS:
When compared with controls, there were statistically significant, slight increases (up to +51%) in the mean absolute and relative spleen weights in females given the test item at 10 mg/kg/day. This finding correlated with increased severity and incidence of congestion microscopically. The amount of blood in the spleen is highly variable, depending on the individual reaction to barbiturate euthanasia and the subsequent degree and timing of exsanguination and loss of blood of the isolated spleen. This increased spleen weights was therefore considered to be agonal, most likely related to euthanasia with pentobarbital (Greaves, 2007).
The statistically significant changes in organ weights for adrenals, heart, and liver in females given the test item at 10 mg/kg/day were considered to be a reflection of reductions in body weight (-10%) and not due to test item-related organ toxicity.
There were no significant organ weight changes in males given the test item at any dose-levels.
Other organ weight changes were considered not to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

GROSS PATHOLOGY:
Sacrifices on day 25 p.c.
Only one pregnant female given the test item at 0.5 mg/kg/day, was sacrificed on day 25 p.c. because it had not delivered. At necropsy, both uterine horns were dilated and there was thick brown content in the vagina.
There were no macroscopic findings considered to be test item-related in both non-pregnant females sacrificed on day 25 p.c. One female had focal white discoloration on the gastric wall which correlated microscopically with developmental squamous cyst, and one female showed red discoloration in the thymus correlated with hemorrhages.

Terminal sacrifice
Spleen was enlarged in 2/10 females given the test item at 10 mg/kg/day and a single female at 2.5 mg/kg/day. This correlated with slight congestion in one high-dose female and the mid-dose female. It correlated with moderate hematopoiesis in the other high-dose female.

The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control Sprague-Dawley rats, and were considered to be incidental.

HISTOPATHOLOGY:
Sacrifices on day 25 p.c.
In one female (0.5 mg/kg/day), sacrificed on day 25 because it had not delivered, there was minimal or slight mixed inflammatory cell infiltrates (with neutrophils) in the mucosa/submucosa of the uterus and vagina. Necrotic material correlated with brown content grossly observed. These inflammatory and necrotic findings were considered to be incidental and not to be related to the test item administration in view of their isolated occurrence at the low dose.
There were no significant microscopic findings in ovaries, spleen and thymus from two non-pregnant females sacrificed on day 25 p.c.

Terminal sacrifice
The following microscopic changes were observed in the spleen:
- there was a trend towards increased germinal center development in males given the test item at 10 mg/kg/day compared to controls,
- severity of hematopoiesis was minimally increased in males given the test item at 10 mg/kg/day,
- there was increased pigment in the spleen in females given the test item at 10 mg/kg/day compared to controls. This was characterized by golden brown pigment in macrophages of the red pulp. Although this finding is commonly observed in control rats, a relationship to treatment could not be excluded,
- incidence and severity of congestion were slightly increased in females given the test item at 10 mg/kg/day compared to controls. This correlated with increased spleen weights at this dose level. These microscopic and organ weight changes related to congestion were considered to be agonal, most likely related to euthanasia with pentobarbital (Greaves, 2007) .

Increased germinal center development and hematopoiesis seen in the spleen from males at 10 mg/kg/day and increased pigment in the spleen from females at 10 mg/kg/day were considered not to be adverse in view of their low magnitude. Germinal centers are indicative of immune stimulation and are generally present in the spleen and lymph nodes (and are recorded in controls from the current study). Extramedullary hematopoiesis is also normally recorded in the spleen from control rats. Pigment is normally present with low severity in the red pulp of the spleen and is related to the erythrocyte metabolism.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental toxicity
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

MORTALITY/VIABILITY/CLINICAL SIGNS:
There were no test item-related deaths or clinical signs in pups.

BODY WEIGHT (GAIN):
At 10 mg/kg/day, there was a statistically significantly higher mean body weight on day 1 p.p. in both sexes when compared with controls. However, mean values were included in the historical control data for males (mean 6.9 [6.6 – 7.4]) and comparable to the historical control data for females (mean 6.6 [6.3-7.0]).
There were no test item-related effects on mean day 1 p.p. body weight at 0.5 and 2.5 mg/kg/day.

Mean body weight gains of pups at the beginning of the lactation period were statistically significantly higher at 10 mg/kg/day in males when compared with controls, but the same trend was observed at this dose-level in females and at 2.5 mg/kg/day in both sexes. This led to higher mean body weights on day 5 p.p. at 2.5 and 10 mg/kg/day with statistical significance for both sexes except for the mid-dose females. These day 5 p.p. pup mean body weights remained in the historical control data for males (mean 10.8 [10.1-11.9]) and females (mean 10.4 [9.6-11.4]).

These findings, which were considered to be mainly related to the lower number of live pups delivered per female when compared with controls, were considered not of toxicological significance. Furthermore, the mean body weights on days 1 and 5 p.p. at 2.5 and 10 mg/kg/day were including in or comparable to the limit of the historical control data range.

SEX RATIO:
No effects.

GROSS PATHOLOGY:
No effects.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for toxic effects on progeny

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weight and mean body weight changes (g)

Sex Male Female
Dose-level (mg/kg/day) 0 0.5 2.5 10 0 0.5 2.5 10
Pre-mating or whole study
Day 1 337 336 338 340 220 222 223 223
Day 15 386 391 395 393 244 249 253 241
Day 36 437 453 456 446  / / / /
Days 1 - 15 +49 +54 +57 +53 +24 +28 +31 +18
Differences from controls / / / / / +17% +29% -25%
Days 1 - 36 100 117 118 106 / / / /
                 
Gestation
Day 0p.c. / / / / 251 259 265 250
Day 7p.c. / / / / 292 298 305 270
Day 14p.c. / / / / 328 333 339 297*
Day 20p.c. / / / / 412 418 419 367**
Differences from controls / / / / / +1% +2% -11%
Days 0 - 7p.c. / / / / +41 +39 +39 +20#
Differences from controls / / / / / -5% -5% -51%
Days 7-14 p.c. / / / / +36 +35 +35 +27#
Days 14-20 p.c. / / / / +83 +85 +80 +70*
Days 0-20 p.c. / / / / +161 +159 +154 +117#
Differences from controls / / / / / -1% -4% -27%
                 
Lactation
Day 1 p.p. / / / / 310 312 321 284*
Differences from controls / / / / / +1% +4% -8%
Days 5 p.p. / / / / 321 333 335 300
Days 1 - 5p.p. / / / / +10 +21 +14 +16
/: not applicable.
Statistically significant: *: p<0.05, #: p<0.001.

Table 2: Mean food consumption (g/animal/day) for females

Sex Female
Dose-level (mg/kg/day) 0 0.5 2.5 10
Pre-mating
Days 1-8 21 22 22 20
Days 8-15 20 20 21 19
Gestation
Days 0 - 7p.c. 25 25 25 22**
Differences from controls / 0% 0% -12%
Days 7-14 p.c. 27 26 25 24
Days 14-20 p.c. 29 28 28 27
Lactation
Days 1 – 5p.p. 42 47 44 40
/: not applicable.
Statistically significant: **: p<0.01

Table 3: The mating and fertility data

Dose-level (mg/kg/day) 0 0.5 2.5 10
Number of animals paired (M + F) 10 + 10 10 + 10 10 + 10 10 + 10
Number of males mated 10 10 10 9
Number of females mated 10 10 10 10 (a)
Mean number of days taken to mate 2.6 2.3 4.5 4.2
(when removing the outsiders)     (3.6) (3)
Number of pregnant females 10 8 10 10
Fertility index 100% 80% 100% 100%
(a): two females were mated with the same male.

Table 4: The delivery data

Dose-level (mg/kg/day) 0 0.5 2.5 10
number of pregnant females 10 8 10 10
number of females which delivered 10 7 10 10
mean duration of gestation (days) 21,2 21,4 21,5 21,8
mean number of corpora lutea 17,9 18,4 18,1 17,4
mean number of implantations 16,9 17 16,5 16,3
mean pre-implantation loss (%) 5,2 7,5 8,1 6,2
mean number of pups delivered 16,5 16,7 15,6 13,3*
mean post-implantation loss (%) # 2,5 1,6 5,2 20,1
Statistically significant: * p< 0,05
#: manually calculated, no statistic performed

Table 5: The mean pup body weight and body weight changes (g)

Sex Male Female
Dose-level (mg/kg/day) 0 0.5 2.5 10 0 0.5 2.5 10
Bodyweight
Day 1 p.p. 6,5 7,1 7,1 7,4** 6,1 6,7 6,6 7,1**
Day 5 p.p. 9,8 10,9 11,3 11,8 9,4 10,3 10,8 11,4**
Bodyweight change
Days 1 - 5 p.p. +3,3 +3,9 +4,2 +4,5** +3,3 +3,6 +4,1 +4,3
Statistically significant: * p< 0,05, ** p<0,01

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 0.5, 2.5 or 10 mg/kg/day. Adverse effects were observed at 10 mg/kg/day. When compared with controls, there was a statistically significantly lower mean food consumption in the first week of gestation associated with a retardation in mean body weight gain over the whole gestation period and particularly in the first week of gestation. There was also a lower mean number of pups delivered correlating with a higher mean post-implantation loss.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 2.5 mg/kg/day (based on the effects seen on mean body weight and mean body weight gain during gestation at 10 mg/kg/day),
- the NOAEL for reproductive performance (mating and fertility) was considered to be 2.5 mg/kg/day (based on increased post-implantation loss at 10 mg/kg/day),
- the NOAEL for toxic effects on progeny was considered to be 2.5 mg/kg/day (based on lower mean number of pups delivered at 10 mg/kg/day).
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, through gestation until day 5 post‑partum (p.p.), according to OECD No. 421, 27 July 1995.

This study provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 5 p.p. The test item was administered as a solution in the vehicle, drinking water treated by reverse osmosis, at dose-levels of 0.5, 2.5 and 10 mg/kg/day.

Another group of 10 males and 10 females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage‑volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked prior to administration of the dose formulation in study weeks 1, 3 and 6.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recordedonce a week. The animals were paired for mating after two weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 p.p.

The males were sacrificed after completion of the pairing period and the dams on day 6 p.p. Final body weights and selected organs weights (epididymides, testes, heart, adrenals, liver, spleen, ovaries, thymus, kidneys) were recorded and a macroscopic post‑mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (thymus, epididymides, testes, ovaries) from the control- and high-dose groups, on all macroscopic lesions and on spleen from all animals.

The pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.


Results 

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within the acceptance criteria (± 10% of the nominal concentration).

 

There were no test item-related deaths or clinical signs.There were no test item-related effects on mean body weight and on mean food consumption in males at any dose-level and in females at 0.5 and 2.5 mg/kg/day. There were no toxicologically relevant effects on mating or fertility indexes at any dose-level and on the other reproductive parameters up to 2.5 mg/kg/day.

 

At 10 mg/kg/day and when compared with controls, there was a lower mean food consumption in the first week of gestation (-12%, p<0.01) associated with a retardation in mean body weight gain over the whole gestation period (-27%, p<0.001) and particularly in the first week of gestation (‑51%, p<0.001), leading to lower mean body weights (-11% at the end of gestation, p<0.01). There were a tendency towards a lower mean body weight gain over the pre-mating period (-25%) which was considered to be of minor toxicological relevance. There were no test item‑related effects on reproductive parameters, except a lower mean number of pups delivered (13.3,vs.controls, p<0.05) correlating with a higher mean post-implantation loss (20.1%, vs. 2.5% in controls). All these effects were considered to be adverse, except the effects on mean body weight gain during the pre-mating period.

At pathology and when compared with controls, there were slight statistically significant increases (up to +51%) in the mean spleen weights in females treated at 10 mg/kg/day along with increased golden brown pigment in macrophages of the splenic red pulp and congestion microscopically. At this dose-level, there were in males a trend towards increased germinal center development (six males vs. one in controls) and increased severity of hematopoiesis (eight males vs. three in controls) in the spleen. All these findings in the spleen were considered not as adverse in view of their low magnitude. At 0.5 and 2.5 mg/kg/day, there were no test item-related organ weight, macroscopic or microscopic changes.

In pups, there were no test item-related deaths, clinical signs or necropsy findings and no test item-related effects on percentage of male pups per litter at any dose-level.

Mean body weight on day 1 p.p. in both sexes was higher at 10 mg/kg/day (both sexes mixed up: vs. in controls, p<0.01). There were no test item-related effects on mean day 1 p.p. body weight at 0.5 and 2.5 mg/kg/day. Mean body weight gains of pups at the beginning of the lactation period were higher at 2.5 and 10 mg/kg/day (both sexes mixed up: +4.2 g and +4.4 g (p<0.05) in ascending group order, vs. +3.3 g in controls), leading to higher dose-related mean body weights on day 5 p.p. (both sexes mixed up:11.0 g (p<0.05) and 11.7 g (p<0.01) in ascending group order, vs. in controls). These findings were considered to be mainly related to the slightly lower number of pups born when compared with controls and were included or comparable to historical control data; they were thus considered not of toxicological significance.


Conclusion

The test item was administered daily by oral gavage to male and female Sprague‑Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 0.5, 2.5 or 10 mg/kg/day.

 

Adverse effects were observed at 10 mg/kg/day. When compared with controls, there was a statistically significantly lower mean food consumption in the first week of gestation associated with a retardation in mean body weight gain over the whole gestation period and particularly in the first week of gestation. There was also alower mean number of pups delivered correlating with a higher mean post-implantation loss.

 

Based on the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 2.5 mg/kg/day (based on the effects seen on mean body weight and mean body weight gain during gestation at 10 mg/kg/day),

.         the NOAEL for reproductive performance (mating and fertility) was considered to be 2.5 mg/kg/day (based on increased post-implantation loss at 10 mg/kg/day),

.         the NOAEL for toxic effects on progeny was considered to be 2.5 mg/kg/day (based on lower mean number of pups deliveredat 10 mg/kg/day).