Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jan 2012 - 10 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations.

Data source

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

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, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: mean weight at start of treatment was 282 gr (males) or 191 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 23.2°C
- Humidity (%): 34 - 65%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Temporary fluctuations from the light/dark cycle (with a maximum of 1hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered
not to have affected the study integrity.
Temporary deviations from the minimum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 21 February - 10 April 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for the purity of the test substance.
Storage conditions of formulations: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates)).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Mating was overlooked for one female of Group 1, whom later delivered live pups.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted once during the pilot study and were conducted twice during the treatment phase of the main study (Days 1 and 14; 21 February and 5 March 2012) using the HPLC-UV method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined during the first analysis (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the Day 1 and Day 14 formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and no test substance was detected in the Group 1 formulations. The formulations from Days 1 and 14 of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room under normal laboratory light conditions for at least 6 hours (stability was only conducted for Day 1 formulations).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One females of Group 1 and one of Group 3 were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the dose range finding study (NOTOX Project 498685) where 500, 1000, 125 and 250 mg/kg were tested. Since the relevant effects at 125 and 250 mg/kg completely resolved after a few days of treatment, the dose levels for the main study were: 30, 100 and 300 mg/kg body weight.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were conducted for all animals, and were started between immediately after up to 1 hour (e.g. 0-1 hour) after dosing (on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period (between immediately after up to 1 hour (e.g. 0-1 hour) after dosing) this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4. In order to monitor the health status Group 4 males were weighed more often. This was documented in the study raw data.

FOOD CONSUMPTION:
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines

* 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group and animals suspected to be infertile).
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on 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 Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

One or more pups from 4 litters (2 from Group 1, 1 from Group 2 and 1 from Group 3) were not observed on individual occasions. Sufficient data is available for a thorough evaluation.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY
- All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines (+ deviations)
- All remaining animals and females which failed to deliver: According to test guidelines.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 21 hours. The fasting period was only slightly longer and was considered not to have adversely affected the macroscopic or microscopic findings.

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines.
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY
According to test guidelines.

The partial list of organs was taken from one male of Group 4, where the full list should have been taken. A few other organs were not available from individual animals for histopathology. Missing tissues are listed in raw data and pathology report. There is sufficient data for a thorough evaluation.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.
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. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Ref. 5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg hunched posture and salivation were noted for all animals. Piloerection, swelling of the abdominal area and flat gait were also noted for a few animals of both sexes, though at a much lower frequency than hunched posture and salivation. Lastly, lethargy, pale appearance and diarrhoea were noted for individual animals at this dose level. Salivation was also seen for all animals at 100 mg/kg, and for one female and four males at 30 mg/kg. In all cases, including the animals at 300 mg/kg, this was likely a physiological response due to the taste of the test substance instead of a sign of systemic toxicity. Piloerection and hunched posture were noted on two occasions for one animal at 30 mg/kg. No toxicological relevance was attributed to these findings as they occurred only on two occasions for a single animal at this dose level. Incidental findings that were noted for control and/or treated animals included rales, scabs and alopecia. These findings occurred within the range of background findings encountered for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, they were not considered to be toxicologically relevant..
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males and one female died spontaneously and two additional females were killed in extremis at 300 mg/kg. The males died after 12 and 13 days of treatment, respectively, and the female died after 29 days on test. The females killed in extremis were euthanized after 30 and 41 days on test, respectively. These animals had only slight weight loss in the time before their deaths. Clinical signs including hunched posture, salivation, piloerection, flat gait, abdominal swelling and pale appearance were noted for some or all animals before their deaths. One female at 100 mg/kg was euthanized because she had a total litter loss (her litter consisted of a single pup). One female that was euthanized in extremis also had a total litter loss where her litter of three pups was found dead at the first litter check.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gains were significantly lower beginning on Day 8 of the premating period until the end of the treatment period for males at 300 mg/kg. While the differences from controls were not statistically significant, females at this dose level also had lower body weights and gains for most of the post coitum and lactation periods (body weight gains were significantly lower for females on Days 4 and 11 during the post coitum period only). One female of Group 1, one female of Group 3 and one of Group 4 had lower body weight gains throughout the post-coitum period, which was attributable to their pregnancy status. These females were all pregnant but did not produce litters with live pups. The female of Group 1 had implantation sites only, the female of Group 3 was found with a single fetus in the uterus (she never delivered), and the female of Group 4 gave birth to three pups that were all dead at the first litter check. No other toxicologically relevant changes in body weights and body weight gain were noted up to 100 mg/kg. Absolute body weights were lower for males at 30 mg/kg during Day 8 of the premating period up to Day 1 of the mating period. This was not considered to be toxicologically relevant because the difference from controls was only slight and occurred in the absence of a dose response effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg absolute and relative food consumption was significantly lower than controls on Days 7-11 of the post coitum period. Slightly lower absolute and relative values than control values were noted over Days 1-8 of the premating period for both sexes (Days 1-15 for males, absolute food consumption only), and for females on Days 0-4 and 1-4 of the post coitum and lactation periods, respectively. These effects were seen at the beginning of the treatment period and were mostly resolved over time, though they were treatment related. There were no other effects on absolute or relative food consumption noted.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematology parameters were affected for females at 300 mg/kg only. Compared to controls, significantly lower values were obtained for eosinophils, haemoglobin, red blood cells and mean corpuscular haemoglobin concentration (MCHC) and significantly higher mean corpuscular volume (MCV) values were seen. When taken together with slightly increased (not statistically significant) reticulocytes and red blood cell distribution width (RDW), these findings are suggestive of slight anemia. There were no other toxicologically relevant effects on haematology parameters up to 100 mg/kg.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At 300 mg/kg, a significant decrease in alkaline phosphatase (ALP) and total protein were noted for males, along with significantly lower creatinine and inorganic phosphate levels for females only. Females at 300 mg/kg also had significantly higher alanine aminotransferase (ALAT; also for females at 100 mg/kg) and aspartate aminotransferase (ASAT) compared to controls. These effects were treatment related but in the absence of any effects on the liver or kidneys, the toxicological relevance of these changes are doubted. The higher ALAT and bile acid means for males at 300 mg/kg were both attributable to very high values obtained for one male of Group 4 and thus these differences were not considered to be toxicologically relevant. No other toxicologically relevant changes in clinical biochemistry parameters were seen.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Both total movements and ambulatory counts were reduced for males and females at 300 mg/kg compared to control animals (only significant for male total movements). While lethargy and flat gait were only noted for a few animals in this group, a relationship to treatment could not be excluded. All groups had a habituation profile with very high activity in the first interval that decreased over the duration of the test period. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal for all animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related microscopic findings in the 300 mg/kg treated rats in the following organs/tissues:
Thymus: Lymphoid atrophy was noted in 5/5 unscheduled deaths (one minimal, four moderate) and in the surviving rats at an increased incidence (5/9) and/or severity (up to moderate).
Stomach:
- Hyperplasia of the forestomach was noted in 5/5 unscheduled deaths (three slight, two moderate) and in the surviving rats at an increased incidence (5/9) and/or severity (up to slight).
- Ulceration of the forestomach was noted in 2/5 unscheduled deaths (up to slight).
- Hyperplasia of the limiting ridge was noted in 1/5 unscheduled deaths (slight) and in the surviving rats at an increased incidence (6/9) and/or severity (up to slight).
- Inflammation of the forestomach was noted in 1/5 unscheduled deaths (slight).
- Edema of the forestomach was noted in 1/5 unscheduled deaths (slight).
- Hyperkeratosis of the forestomach was noted in 1/10 of the surviving rats (minimal).
Skeletal muscle:
- Myofiber degeneration/regeneration was noted in 3/5 unscheduled deaths (one minimal, two slight, females only) and in the surviving females at an increased incidence (5/5) and/or severity (up to moderate).
Mesenterial lymph node:
- Congestion/erythrophagocytosis was noted in 5/5 unscheduled deaths (two slight, three moderate) and in the surviving female rats at an increased incidence (5/9) and/or severity (up to moderate).
The recorded microscopic findings in the 0 mg/kg, 30 mg/kg and 100 mg/kg treated animals were within the normal range of background alterations encountered in Wistar (Han) rats of this age and strain and therefore not regarded as test item related. There were no morphological findings in the reproductive organs of either sex in the 0 mg/kg (Group 1), 30 mg/kg (Group 2), 100 mg/kg (Group 3) and 300 mg/kg (Group 4) treated rats which account for their infertility, failure to deliver healthy pups or reduction in litter size. Furthermore, the spermatogenic staging profiles were normal for all males evaluated.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE DATA
The mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites were unaffected by treatment up to 300 mg/kg. There were 8, 10, 9 and 6 litters available for evaluation in the control, 30, 100 and 300 mg/kg groups, respectively. In the control group, 1 female was not pregnant, and one female had implantation sites only. At 100 mg/kg, one female had a total litter loss and another female did not deliver any live pups. At 300 mg/kg two female were both pregnant but were killed in extremis or died before the scheduled necropsy on gestation Days 10 and 12, respectively. One female in this group was not pregnant, and one had a litter of three pups who were all found dead at the first litter check. Additional details are given in the attached document that indicates pages 55, and 57-58 from the study report.

GESTATION
The gestation index was much lower at 300 mg/kg (66.7%) compared to controls (88.9%). At 300 mg/kg the low gestational index percentage was attributable to the two pregnant females that did not survive long enough to deliver their litters, the single non-pregnant female, and the total litter loss at first litter check for one female of group 4, who was also euthanized in extremis. Aside from these animals, all other females of the group produced viable, but relatively small litters. However, a relationship to treatment cannot be excluded.

PARTURITION/MATERNAL CARE
No pups were found for one female of Group 3, though she was observed when starting delivery. At the macroscopic examination one fetus was found in the right uterine horn, and she had only one implantation site. A treatment related cause could not be determined for this. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Incidental clinical symptoms of pups consisted of scabs on the tail apex or abdomen, blue or black discoloration of the tail apex, cold, pale appearance and insufficient milk in the stomach. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of dead pups at first litter check and the postnatal loss was higher for females at 300 mg/kg. No pups of the control group, and five, one and eight pups of the 30, 100 and 300 mg/kg groups, respectively, were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalised. The single pup from 100 mg/kg was the only pup from the female with a total litter loss. Additionally, three of the eight pups were from the female of Group 4 wat was killed in extremis and were all found dead at the first litter check. Thus it is possible the pup mortality was secondary to maternal toxicity. However, the data collectively suggest a higher mortality for animals at 300 mg/kg. Additional details are given in the attached document that indicates pages 55 and 57-58 from the study report.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment up to 300 mg/kg. Additional details are given in the attached document that indicates page 59 from the study report.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Incidental macroscopic findings of pups that were found dead/and or the single pup that was euthanized in extremis included the absence of milk in the stomach and cannibalism of the right front leg. Incidental macroscopic findings noted for surviving pups included black discoloration of the tail
apex, blue tail and endorotation of the right hindleg. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Histopathological findings:
not examined

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
The mean number of living pups at the first litter check was significantly lower for females at 300 mg/kg with 7.3 pups per litter compared to 11.4 pups per litter for control animals. This may be in part attributable to the total litter loss for one female of group 4 and to the low number of litters for evaluation (with two females not surviving to give birth). As this female was euthanized in extremis, it is possible that her total litter loss was secondary to maternal toxicity. However, taken together with the higher number of dead pups at first litter check, the increased percentage of pups lost after birth (3.9% at 300 mg/kg compared to none lost for controls) and the relatively small litter sizes (6-10 pups) of the remaining litters, this was considered treatment related. At 300 mg/kg the male to female ratio (39/61) was lower than for controls (54/46), though this difference was not statistically significant. This is likely due to the fewer number of litters available and the relatively smaller size of these litters, however, an effect of treatment could not be excluded. The viability index was unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity, including a lower gestation index, fewer living pups and more dead pups at first litter check, and increased postnatal loss was observed at 300 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg body weight/day revealed parental and developmental toxicity at 300 mg/kg body weight/day. Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived: Parental NOAEL: 100 mg/kg/day, Reproduction NOAEL: at least 300 mg/kg/day, Developmental NOAEL: 100 mg/kg/day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of the test material was conducted in rats by oral gavage following OECD TG 422 and under GLP conditions.

The test material was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day (10 animals/sex/dose; dose levels based on a dose range finding study). Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-49 days). Formulation analysis showed that the formulations were prepared accurately, were homogenous and were stable for at least 6 hours at room temperature.

No reproductive toxicity was observed up to the highest level tested (300 mg/kg).

Developmental toxicity, including a lower gestation index, fewer living pups and more dead pups at first litter check, and increased postnatal loss was observed at 300 mg/kg. No treatment -related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. duration of gestation, parturition, maternal care and clinical signs, body weights, and macroscopic findings of pups).

In conclusion, treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg body weight/day revealed parental toxicity and developmental toxicity at 300 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was 100 mg/kg/day for both parental toxicity and developmental toxicity. No reproductive toxicity was observed up to the highest level tested (300 mg/kg). Therefore, the NOAEL for reproduction was >= 300 mg/kg.