Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2007 - 08 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
GLP compliance:
yes
Remarks:
NOTOX B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid
- Molecular weight (if other than submission substance): 480.73
- Analytical purity: 99.0%
- Storage condition: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males (5-6 weeks); females (13-14 weeks)
- Weight at study initiation: males (187-218 g); females (235-291 g)
- Housing:
Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (Mill type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/sex/cage. Females were individually housed in Macrolon cages (Mill type, height 18 cm).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 20.1 - 24°C)
- Humidity (%): 30-70 (actual range 30 - 85; Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% CMC (carboxymethyl cellulose) in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. No adjustment was made for specific gravity of the test substance, vehicle and/or formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Amount of vehicle (if gavage): 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
One female was cohabited with one male of the same treatment group. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug (day 0 post-coitum).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations was performed during Week 1 (13 April 2007), Week 2 (23 April 2007), Week 4 (3 May 2007), Week 8 (31 May 2007), Week 16 (26 and 31 July 2007). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 484716).
The accuracies were between 81% and 110% with coefficients of variation < 6.5%.
Duration of treatment / exposure:
males: 103 to 106 days, i.e. 10 weeks before mating, during mating and up to termination (after delivery of litters)
females: 55 to 63 days, i.e. 2 weeks before mating, during mating, during post-coitum, and during 20 to 22 days of lactation
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based among others on a 28-day toxicity study (non-GLP, 1972) with CFY rats by oral gavage. Findings in this study were: mortality of one control male and two 600 mg/kg females, clinical signs at 600 mg/kg, reduced body weight gain at 200 and 600 mg/kg, several affected parameters for haematology at 200 and 600 mg/kg, increased adrenal weight and decreased liver weight at 600 mg/kg and distension of stomach at 200 and 600 mg/kg. The NOAEL in this 28-day toxicity study with CFY rats was set at 50 mg/kg bw.
In addition, a 14-day dose range finding study (NOTOX Project 484928; 2007) was performed. Five Wistar rats/sex/group were treated by gavage at 0, 30, 100 or 300 mg/kg body weight/day. At the high dose level salivation was observed for all animals and two females showed laboured respiration and rales. Body weight and food consumption was slightly affected at this dose level, but recovered during treatment. No findings were observed for clinical biochemistry, macroscopic and histopathological examination. Absolute and relative kidneys weight for males were increased. At all dose levels several haematological parameters were affected for males (increased RDW, decreased haemoglobin, haematocrit, MCV, MCH and MCHC). No clear dose response relationship was apparent (effect was more or less the same for all dose levels).
Based on above results, dose levels of 3, 30 and 300 mg/kg were selected for this one-generation study.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1, 4, 7, 14 and 21

FOOD CONSUMPTION: Yes
Weekly for males and females during the pre-mating treatment period. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on days 1, 4, 7, 14 and 21 post-partum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: yes
Performed during necropsy for males (after delivery of the litters) and females (day 21 post partum or shortly thereafter). Blood samples were collected from 10 rats/sex/group. In cases where the samples were found clotted, other animals were selected at another timepoint for blood collection to ensure for 10 appropriate blood samples per sex and group. The animals were not fasted overnight. Blood samples (0.5 ml) were collected under isoflurane anaesthesia (Abbott B.V. Hoofddorp, The Netherlands). Blood samples were drawn from the aorta before exsanguination and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTA. Blood was stored in a refrigerator prior to analysis on the ADVIA120.
Parameters examined: White blood cells (WBC), Differential leucocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
Litter observations:
STANDARDISATION OF LITTERS
On Day 4 of lactation litters were reduced in size to eight pups by random culling of F1 pups.

PARAMETERS EXAMINED
- Mortality / viability: The numbers of live and dead pups at the First Litter Check (= check at day 1 of lactation) and daily thereafter were determined. If
possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals
- Body weights: Live pups were weighed on lactation days 1, 4, 7, 14 and 21
- Sex: determined for all pups on days 1 and 4
Postmortem examinations (parental animals):
SACRIFICE
All animals surviving to the end of the treatment period and all moribund animals were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. Males were killed as soon as possible after delivery of the litters. Females were killed on day 21 post partum or shortly thereafter. Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period. In case a female was not pregnant, the uterus was stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites.

GROSS NECROPSY
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The Gl tract was looked at thoroughly. Descriptions of all macroscopic abnormalities were recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands): Seminal vesicles, Cervix, Spleen, Coagulation gland, Testes, Epididymides, Uterus,
Ovaries, Vagina, Pituitary gland, All gross lesions, Prostate gland (Testes and epididymis were fixed in modified Davidson's solution).

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Epididymides, Ovaries, Pituitary (weighed when fixed for at least 24 hours), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles, Spleen, Testes, Uterus
Histopathological examination was performed on the spleen (males and females) and uterus (females) for the following animals:
• Group 1 : males 1 -10 and females 97-106.
• Group 4: males 73, 74, 86-93 and females 169, 170, 182-189.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
SACRIFICE
Pups, which were culled, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure. Culling was performed on day 4 of lactation or shortly thereafter. The remaining pups were killed at day 21 post partum or shortly thereafter.

GROSS NECROPSY
Offspring found dead or killed before day 14 of lactation were sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach was examined for the presence of milk. Offspring found dead or killed on or after day 14 of lactation were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs, with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup was preserved in 10% buffered formalin, bouin (Klinipath, Duiven, The Netherlands) or 96% ethanol, as appropriate, for possible further examination. All gross lesions were fixed in 10% buffered formalin.
Statistics:
- if the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex
- the Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution
- the Fisher Exact-test was applied to frequency data
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance
Reproductive indices:
- Percentage mating
- Fertility index
- Conception rate
- Gestation index
- Duration of gestation
- Percentage live males at first litter check
- Percentage live females at first litter check
Offspring viability indices:
- Percentage of postnatal loss days 0-4 post partum
- Percentage of breeding loss day 5 until weaning
- Percentage live males at weaning
- Percentage of live females at weaning
- Viability index
- Weaning index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicological relevant clinical signs were noted. Slight salivation was noted in eight males treated at 3 mg/kg body weight/day for 1-4 days and in all animals treated at 30 and 300 mg/kg from Week 3 onwards (Males Group 3), from Week 2 onwards (Males Group 4 and Females Group 3) and from Week 1 onwards (Females Group 4). On a few occasions moderate salivation was seen (males Group 3 and 4 and Females Group 4). For all animals salivation disappeared within three minutes after dosing (determined on 03 July 2007 which was Week 12 of treatment for the males and Week 4 of treatment for the females). Salivation was considered an adaptive response to the repeated gavage procedure of an alkaline formulation, and was not considered toxicologically relevant. One animal treated at 3 mg/kg (Male 40) showed swelling of the snout. This was also considered to be caused by the alkaline dose formulation and was not considered toxicologically relevant. Incidental findings consisted of alopecia, scabs, swelling of the inguinal region, broken tail apex, bent tail, rales and chromodacryorrhoea. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment related mortality occurred during the study period. One animal treated at 30 mg/kg (Female 162) was sacrificed due to ill health on Day 35 of treatment (Day 19 post-coitum). Prior to sacrifice, this female showed hunched posture, piloerection and pale appearance for two or three days, a slight body weight loss, decreased food consumption and at macroscopic examination several gray-white foci at the kidneys, an enlarged spleen, and eleven resorptions in the uterus.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weights and body weight gain were observed for animals treated at 300 mg/kg body weight/day. Minimal to slight decrease in body weights (up to 8% reduction in group mean values compared to controls) was evident for high dose males as of day 8 of treatment onwards during the premating and postmating periods. Body weight gain was reduced during the first week of treatment for males at 300 mg/kg, and recovered afterwards. For females, decreased body weight gain was observed on Day 20 post-coitum. Body weights and body weight gain of animals treated at 3 and 30 mg/kg remained in the same range as controls over the complete study period. The statistically significantly increased body weight gain observed on a few occasions was not considered toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased food consumption was observed for animals treated at 300 mg/kg body weight/day. This consisted of decreased absolute and relative food consumption for males and females during Week 1 of the treatment period. For females, decreased absolute food consumption was noted on Days 4-7 and 17-20 post-coitum and decreased relative food consumption was seen on Days 4-7 post-coitum. The statistically significantly increased values for relative food consumption noted on several occasions for males of the high dose group were not considered toxicologically relevant as these were due to the lower body weight. The decreased relative food consumption observed during lactation for Group 3 was not considered toxicologically relevant as it was caused by one female (number 158) that showed total litter loss on Day 4 of lactation. Food consumption before or after allowance for body weight was similar between animals treated at 3 and 30 mg/kg and control animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant findings were observed. At the high dose group, increased values for reticulocytes were observed for females (136% of control). However, as this change was very minor and no corroborative findings were noted, it was not considered toxicologically relevant. All other statistical significant changes were not regarded treatment related as no dose response relationship was apparent.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg body weight/day, absolute and relative weights were increased for the spleen (males; 112% of control) and uterus (females; 130% of control). All other statistical significant changes were not regarded treatment related as no dose response relationship was apparent.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. Incidental findings included hernia inguinalis, testes reduced in size, epididymides reduced in size, tail apex bent, thickened nose region, flaccid testes, testes enlarged, dark red discolouration of the testes, stomach grown together with the liver, cerebrum containing fluid, hard nodule at the tail of the epididymides, alopecia at the foreleg or inguinal region, and the uterus containing fluid. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Spleen and uteri were investigated histopathologically. In the spleen, hemapoietic foci and hemosiderin pigments were observed in a similar incidence and severity in either control and high dose animals. These findings were considered to be within the normal range of background pathology encountered in Wistar rats of this age and strain and therefore a treatment relationship can be excluded.

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
Reproduction parameters were unaffected by treatment up to 300 mg/kg body weight/day. Mating performance, fertility parameters, duration of gestation and number of dead and living pups at first litter check were similar for the control and treated groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproduction peformance was not affected.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental clinical symptoms consisted of small appearance, wound, scabs, red or blue discolouration, tail apex missing, little or no milk, cold, pale appearance and alopecia. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No toxicological relevant findings were observed for postnatal loss, living pups on Day 4 post partum, breeding loss between Days 5-21 post partum, living pups on Day 21 post partum, viability index and weaning index. The decreased value for postnatal loss and increased value for viability index observed for the low dose group was not regarded toxicologically relevant. The increased value for breeding loss and decreased value for weaning index observed for the low dose group was due to the loss of one litter (number 134) on Day 7 of lactation. Additionally, total litter loss was observed for one dam (number 158) of the mid dose group on Day 4 of lactation. These two total litter losses were not regarded toxicologically significant at this low incidence.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg body weight/day, decreased body weights were observed for male and female pups that revealed statistical significance on Days 14 (male and female: 90% of control) and 21 (male and female: 89% of control) of lactation. Body weights of the low and mid dose groups remained in the same range as controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination of the pups revealed small appearance, no milk, spleen reduced in size and bilobed, tail apex missing, pelvic dilation of the kidneys, and alopecia. One pup of Group 3 (male 5 of litter 150) showed thickened anus and yellow fluid coming from the anus; most probably due to an inflammation. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results, the parental No Observed Adverse Effect Levels (NOAEL) were established at 30 mg/kg/day based on decreased body weights, body weight gain and food consumption of males and females.
The NOAEL for developmental toxicity was set at 30 mg/kg bw based on reduced pup weight during lactation, but not associated with any other developmental adverse effect.
The NOAEL for reproductive toxicity was set at >= 300 mg/kg/day based on the absence of effects.
Executive summary:

A one-generation reproduction toxicity study rats by oral gavage following OECD guideline was performed. The purpose of this study was to investigate effect of the test item on male and female reproductive performance and preliminary information on developmental effects after oral (gavage) administration to male and female Wistar rats during one complete reproductive cycle. After acclimatisation, 24 rats per dose and sex (F0-generation) were exposed to daily doses of 0 (vehicle, 1% aqueous CMC), 3, 30 and 300 mg/kg body weight/day at a dose volume of 5 ml/kg. Males were treated for 103 to 106 days, i.e. 10 weeks prior to mating, during mating, and up to termination (after delivery of litters). Females were treated for 55 to 63 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 20 to 22 days of lactation. F0-females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation litters were reduced in size to eight pups by random culling of F1 pups. Evaluated parameters Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity. All animals were subjected to daily clinical observation. Body weight and food consumption were measured on several occasions over the treatment period. At necropsy, macroscopic observations and organ weights were recorded and blood was collected for haematology investigations. Histopathology of the spleen and uterus was performed. Reproduction parameters, breeding data and pup development were assessed. Accuracy and homogeneity of formulations were demonstrated by analyses. There were no toxicological relevant changes for mortality, clinical signs, haematology investigations, macroscopic examination, organ weights, microscopic examination, reproduction and breeding data that were considered to be an effect of treatment. Salivation was observed for animals treated at 30 and 300 mg/kg; this was considered an adaptive response to the repeated gavage procedure of an alkaline formulation, and was not considered toxicologically relevant. At 300 mg/kg body weight/day, minimal to slight decrease in body weights, body weight gain and food consumption (notably Week 1) were observed for the parental animals and lower body weights were observed for male and female pups. Based on the results, the parental and developmental No Observed Adverse Effect Levels (NOAEL) for the test item were established at 30 mg/kg/day. The reproduction NOEL was established at 300 mg/kg/day.