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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2007 - June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed according to the OECD Consensus document Number 13 The Application of the OECD Principles of GLP to the Organization and Management of Multi-Site studies. The study procedures described in this report were based on the following guidelines: 1) Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996. 2) Environmental Protection Agency (EPA) guideline 40 CFR 799.9365, TSCA combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, December 2000.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA guideline 40 CFR 799.9365
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
pH: ca. 10 at 100 g/l
Stability at higher temperatures: yes, max. 65 °C
Stability in vehicle water: min 96 h
Solubility in vehicle water: yes

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: ca. 10 weeks
• Number of animals: 40 females and 40 males. This resulted in at least 8 pregnant females per group.
• Weight at study initiation: range 290 - 293 g (males), 188-193 g (females)
• Acclimatisation:  5 d
• Accommodation:
- Pre-mating: Animals were housed in groups of 5 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 (MIII type, height 18 cm).
- Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
• Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except the night prior to planned necropsy
• Water: Free access to tap-water.
• Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
• Temperature (°C): 21 ± 3
• Relative humidity (%): 30 – 70
• Air changes (1/h): ca. 15
• Photoperiod (h dark / h light): 12/12 (hours artificial light/ hours darkness per day).

IN-LIFE DATES:
• From August 29 (delivery of animals)to October 26, 2007

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
water
Details on exposure:
Treatment
Parental animals
• Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
• Frequency: once daily for 7 d/w , approximately the same time each day with a maximum of 4 h difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
• Exposure period:
- Males were exposed for 30 d, i.e. 2 w prior to mating, during mating, and up to termination.
- Females were exposed for 42 - 53 d, i.e. 2 w prior to mating, during mating, during post-coitum, and during at least 4 d of lactation.
• Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Offspring
• Offspring was not treated.
Details on mating procedure:
- M/F ratio per cage:
5 animals/sex/cage (Pre-Mating)
1:1 (mating)
5 male animals/cage (post-mating); females were housed individually (post-mating)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations prepared on one day during the treatment period was performed according to the following scheme:
Group Analysis (type of sample)
1 acc (M)
2 acc + hom + stab{t=o} (TMB), stab{t=5, RT} (S)
3 acc (M)
4 acc + hom + stab{t=o} (TMB), stab{t=5, RT} (S)
with: acc=accuracy, hom=homogeneity, stab=stability (hours), T=top, M=middle, B=bottom position of container, S=stability sample taken at middle position of container, RT=room temperature
The analytical method used was based on the results of a separate project for the development and validation of the analytical method (HPLC; NOTOX project 485209)

For each dose group reproduction parameters were expressed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis

Analysis of Dose Preparations:
The pH of the formulations were between 8.31 to 8.68 (Group 1), 10.12 to 10,17 (Group 2), 10.10 to 10.17 (Group 3) and 9.94 to 10.02 (Group 4).
The accuracies of the formulation of Group 2 were between 102 % and 109 %. For Group 3, accuracies of 98 % and 103 % were obtained and for Group 4 the accuracies were between 87 % and 105 %, The value of 87 % in Group 4 was accepted since the accuracy of the duplicate sampie was within the criterion range of 90 - 110 %. Based on the results obtained it was concluded that the formulations were prepared correctly.
No test substance was detected in the Group 1 formulations.
The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation <= 10%).
Formulations at the entire range were stable for at least 5 h when stored at room temperature.
Duration of treatment / exposure:
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day.
Males were exposed for 30 d, i.e. 2 w prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 d (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 d of lactation).
Frequency of treatment:
Once daily (7 d/w), approx. the same time each day with max. 4 h difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
BREEDING PROCEDURES:
Following a minimum of 14 d of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River will supply non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intra-vaginal copulatory plug. This day was designated day 0 post-coitum.
Once mating had occurred, the males and females were separated. A maximum of 14 d was allowed for mating.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mL/kg body weight
Basis:
other: Dose Volume
Remarks:
Doses / Concentrations:
0 mg/kg b.w.
Basis:
nominal in water
control group
Remarks:
Doses / Concentrations:
100 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
Remarks:
Doses / Concentrations:
300 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
Remarks:
Doses / Concentrations:
1,000 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
No. of animals per sex per dose:
10 males: 0/100/300/1000 mg/kg b.w.
10 females: 0/100/300/1000 mg/kg b.w.
Offspring was not treated.
Control animals:
yes, concurrent no treatment
Details on study design:
• Dose selection rationale: range-finding study. Two dose levels (500 and 1,000 mg/kg bw ) were used to set the dose level for the main study. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at these dose levels. A very slight effect on body weight and food consumption was observed for both groups which recovered at the end.
• Rationale for animal assignment (if not random): randomized
• Rationale for selecting satellite groups: not applicable
• Post-exposure recovery period in satellite groups: not applicable
• Section schedule rationale (if not random): not applicable
Positive control:
No positive control used in the study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (parental animals + offspring)
Parental animals:
Mortality: at least twice daily; Clinical signs: at least once daily
The following tests were performed in the first five mated males and females/group with live offspring:
hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent) motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring. The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 min.
Offspring:
Mortality: the numbers of live and dead pups at the First Litter Check (= check at day 1 of lactation) and daily thereafter was determined.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily
Body weight: Yes (parental animals + offspring)
Parental animals: Males and females were weighed on the first day of exposure and weekly there-after. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1 and 4.
Offspring: Live pups were weighed during lactation on days 1 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE:
Weekly, for males and females. 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 postcoitum and after delivery on days 1 and 4 post-partum.

REPRODUCTION PROCESSES:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
HAEMATOLOGY: Yes
Time schedule for collection of blood: prior to scheduled postmortem examination, between 7.00 and 10.30 a.m.
Anesthetic used for blood collection: Yes (iso-flurane)
Animals fasted: Yes overnight (with a maximum of 20 hours)
Animal number: the first five mated males/group and the first five females/group

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: prior to scheduled postmortem examination, between 7.00 and 10.30 a.m.
Animals fasted: Yes overnight (with a maximum of 20 hours)
Animal number: the first five mated males/group and the first five females/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: after delivery of offspring
Dose groups that were examined: the first five mated males/group and the first five females/group with live offspring
attery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent) motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring. The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 min.



Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Parameters examined in male parental generations: No sperm parameters were examined.
see also "Parental animals: Observations and examinations"
Litter observations:
The following parameters were examined in offspring:
Mortality/Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter was determined.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed during lactation on Days 1 and 4.
Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS:
All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
• Male animals: Following completion of the mating period (a minimum of 28 d of dose administration), the males were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated, Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs.

• Maternal animals:
- Females which deliver: On lactation Day 5 or shortly thereafter, the females were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated.
- Females which failed to deliver: On post-mating day 24-26, the females which had not delivered were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated.

GROSS NECROPSY:
All animals were fasted overnight (with a maximum of 20 h) prior to planned necropsy, but water was provided.
• Females which deliver: Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs. The number of former implantation sites was counted and recorded. Corpora lutea was also counted and recorded. Gross lesions were saved for possible future histopathological examination.
• Females which failed to deliver:
No organ weights were collected. Tissues were preserved for possible future histopathological examination as described in the following paragraphs, with the following exceptions: Nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using ammonium sulfide solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)). Gross lesions were saved for possible future histopathological examination.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Organ weights: YES
The following organ weights (and terminal body weight) were recorded:
From 5 surviving animals/sex/group (The first 5 mated males per group and the first 5 females with live offspring per group): Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus.
From all remaining males: Epididymides, Testes.
The tissues were prepared for microscopic examination and weighed, respectively.
Statistics:
The following statistical methods were used to analyse the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. The Fisher Exact-test (Fisher, 1950) 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. No statistical analysis was performed on histopathology findings. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each dose group reproduction parameters were expressed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis
Offspring viability indices:
The Viability index was calculated as follows:
[number of live pups on day 4 post partum/number of pups born alive]*100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "details on results"
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see "details on results"
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: see "details on results"

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see "details on results"
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see "details on results"
Reproductive performance:
no effects observed
Description (incidence and severity):
see "details on results"

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period.
One female at 1000 mg/kg (animal no" 75) showed hunched posture and piloerection from Day 20 post-coitum until Day 3 of lactation. In another female at 1000 mg/kg (animal no. 79) piloerection was noted on Day 4 of lactation. Slight salivation was noted incidentally in males and females treated at 1000 mg/kg. This observation was considered related to the taste of the formulation, and was not considered toxicologically relevant.
All other clinical signs (scabs and alopecia) were considered unrelated to treatment

BODY WEIGHT AND WEIGHT GAIN:
A lower mean body weight and body weight gain was recorded for males at 1000 mg/kg during the complete treatment period. For females a lower mean body weight was noted during the complete treatment period (not always statistically significant) and body weight gain was
decreased on Day 8 of pre mating and Day 1 of mating. Females at 100 and 300 mg/kg showed a slightly lower mean absolute body weight on Day 8 of pre mating. As these had recovered on Day 1 of mating, it was not considered toxicologically significant

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption before or after allowance for body weight was reduced in males and females at 1000 mg/kg during the whole observation period (not always statistically significant). Food consumption at 100 and 300 mg/kg was similar to controls.

HAEMATOLOGY:
A slightly higher white blood cell count was noted in males and females at 1000 mg/kg (not statistically significant for males). In addition, relative numbers of reticulocytes were increased in males at 1000 mg/kg. Other findings reaching statistical significance (increased basophils in males at 100 and 300 mg/kg, increased mean corpuscular volume (MCV) in females at 300 and 1000 mg/kg, increased mean corpuscular haemoglobin (MCH) in females at 300 mg/kg, decreased mean corpuscular haemoglobin concentration (MCHC) in females at 1000 mg/kg) were considered to
be of no toxicological relevance as these findings either remained within the range considered normal for rats of this age and strain or showed no dose response relationship.

CLINICAL BIOCHEMISTRY:
Increased activity of alanine aminotransferase was noted in males at 1000 mg/kg and for two females at 1000 mg/kg. One male at 1000 mg/kg showed increased activity of aspartate aminotransferase. Other findings reaching statistical significance (decreased levels of total bilirubin in males at 100
and 300 mg/kg, increased level of total bilirubin in males at 1000 mg/kg, slightly decreased sodium in males at 1000 mg/kg, and slightly decreased albumin in females at 300 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

ORGAN WEIGHTS:
Males at 1000 mg/kg showed a lower mean terminal body weight as compared to that of controls. Organ/body weight ratios were increased for liver, spleen, testes and epididymides in males and for liver, spleen, kidneys and adrenals in females at 1000 mg/kg. The increased organ/body weight ratio noted for brain in males at 1000 mg/kg was considered to be caused by the relatively low terminal body weight rather than being a direct effect of
treatment. Absolute brain weight was slightly decreased in females at 1000 mg/kg. However, as this was very slight and the brain weight ratio was unaffected, this finding was not considered toxicologically relevant. No organ weight changes were noted at 100 and 300 mg/kg body weightlday.

GROSS PATHOLOGY:
There were no treatment-related findings. There were no significant changes in the reproductive organs of those animals that had failed to
mate; the ovaries of one rat in Group 3 had apparently not ovulated regularly based on the observation that the corpora lutea were hypercellular suggesting ageing. There was a usual distribution of stage types in the testes stained with PAS. There were occasional observations of commonplace changes in the tissues of young laboratory rats. These were all of small degree and variable incidence. None were associated with treatment.

HISTOPATHOLOGY:
macroscopic findings: no treatment-related findings. Those few observations that were present were those that are commonplace in the tissues of young laboratory rats
microscopic findings: no treatment-related findings.

REPRODUCTION DATA:
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weightlday. No treatment related findings were observed for mating performance, fertility parameters, duration of gestation and number of dead and living pups at first litter check.

BREEDING DATA:
Breeding parameters were unaffected by treatment up to 1000 mg/kg body weight/day. Postnatal loss and viability index were similar for the control and treated groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: Generation: definitive reproduction, breeding, development (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
see "details on results"
Mortality / viability:
no mortality observed
Description (incidence and severity):
see "details on results"
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "details on results"
Sexual maturation:
no effects observed
Description (incidence and severity):
see "details on results"
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see "details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "details on results"
Histopathological findings:
no effects observed
Description (incidence and severity):
see "details on results"

Details on results (F1)

VIABILITY (OFFSPRING):
Development of pups was unaffected by treatment up to 1,000 mg/kg bw/day.

CLINICAL SIGNS (OFFSPRING):
Incidental clinical symptoms consisted of scabs, discolouration tail apex, and blue spot neck. Incidental macroscopic findings consisted of wound at the head, scabs at the neck, and dark red discolouration of the tail apex. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

BODY WEIGHT (OFFSPRING):
(Mean) body weights were similar for the control and treated groups.

GROSS PATHOLOGY (OFFSPRING):
All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with the test substance by oral gavage in male and female rats at dose levels of 100, 300 and 1,000 mg/kg bw./d revealed parental toxicity at 1,000 mg/kg bw/d. No reproduction, breeding and developmental toxicity was observed for treatment up to 1,000 mg/kg bw/d. Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg. The definitive reproduction, breeding and developmental NOAEL was established as being 1,000 mg/kg.