RSS-4, DRY SUBSTANCE

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March - 01 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

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

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight range at start of treatment was 335-340 gr (males) or 210-215 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic 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
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle with the light on from 07:00-19:00.

IN-LIFE DATES: From: 09 March - 01 May 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation: 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 specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity of the test substance.

Storage conditions of formulations: At ambient temperature.

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).
- 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 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.
- After successful mating each pregnant female was caged individually in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (06 April 2012), according to a validated method (Project 498508). 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 (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. 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 formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations. No test substance was detected in the Group 1 formulations. The formulations of Group 2 and Group 4 were homogeneous. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 33 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). One control female and one female at 1000 mg/kg bw/day 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 14 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected in consultation with the Sponsor based on the results of the dose range finding study(Project 499082). Since in this dose range finding study no toxicologically relevant clinical signs were noted, it was decided for the main study (Project 498516) to conduct clinical observations at least immediately after dosing.

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 randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected 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

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made for all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. 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.

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: 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: 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

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 (5 animals/sex/group)
- Battery of functions tested: According to test guidelines

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis from 5 males of the control and high dose group and all males suspected to be infertile, if applicable.

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 abnomalities, 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 for 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).

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 (iso-flurane) and subsequently exsanguinated.
Two animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 20 hours and 5 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the macroscopic or microscopic findings.
- Selected 5 animals/sex/group: According to test guidelines
- All remaining females which failed to deliver, the female with total litter loss and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
No thyroid gland organ weight was recorded for one female at 1000 mg/kg bw/day. For another female at 1000 mg/kg bw/day inadvertently the organ weight from the spleen was entered in the computer instead that from the ovaries. The incorrect ovaries organ weight was removed from the tables. Since tissues had been processed for histopathology, it was not possible to complete the missing data. However, sufficient data was available from the remaining selected females from this group for evaluation.
For one female at 1000 mg/kg bw/day, the terminal body weight was recorded at necropsy. It was additional information, that was not required by the protocol.
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY:
According to test guidelines
A few tissues were not available for histopathology because they were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. These are listed in the raw data and pathology report. Sufficient data was available for 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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption (FC; absolute and relative to body weight) was lower for females at 1000 mg/kg bw/day as compared to controls during lactation. This change was most likely caused by the lower FC of the females with smaller litters.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (FC; absolute and relative to body weight) was lower for females at 1000 mg/kg bw/day as compared to controls during lactation. This change was most likely caused by the lower FC of the females with smaller litters.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 1000 mg/kg bw/day had to be sacrificed on Day 1 of lactation since she had a total litter loss. On the same morning, a pale appearance, shallow respiration, piloerection were noted. A few hours thereafter, one nearly entirely cannibalized pup was found in the cage. Gross findings during necropsy included an enlarged placenta in the uterus and part of a pup in her stomach. It was her only pup, since there was only one implantation site. There were no morphological findings in either the reproductive organs or the mammary glands of this female that could be attributed to the test substance.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included scabbing, scaling and alopecia. These findings occurred within the range of background findings to be expected 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 toxicologically relevant.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Locomotor activity as determined by total movements and ambulations was considered to be unaffected by treatment up to 1000 mg/kg bw/day (both sexes). Mean values remained within the normal range of biological variation. All groups showed a comparable habituation profile with high activity in the first interval that decreased over the duration of the test period. For one control male, total movements and ambulations were relatively high, with mean values at the upper end of the normal range. Consequently, the control means for these endpoints were relatively high, but still remained within the normal range of biological variation.


BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. During the entire treatment period there was a trend towards slightly lower body weights and body weight gains for males at 1000 mg/kg bw/day as compared to controls, reaching statistical significance on Day 8 of the mating period only. However, since these changes were only slight, with all values within the normal range of biological variation, this finding was not considered to be toxicologically relevant.

FOOD CONSUMPTION
Food consumption, calculated as absolute values and relative to body weight, was lower for females at 1000 mg/kg bw/day as compared to controls during lactation. This change was most likely caused by the lower food consumption of the females with smaller litters. Therefore, it was regarded as secondary effect and thus not toxicologically relevant. No toxicologically relevant changes in absolute and relative food consumption were noted for males up to 1000 mg/kg bw/day. During the entire treatment period there was a trend towards slightly lower absolute and relative food consumption for males at 1000 mg/kg bw/day compared to controls. However, since changes were only slight, with all values within the normal range of biological variation, this finding was not considered to be toxicologically relevant. The statistically significantly higher absolute food consumption recorded for females at 1000 mg/kg bw/day compared to controls during Days 0-7 post-coitum was not considered to be toxicologically relevant, since values remained within the range considered normal for rats of this age and strain. Moreover, in case of toxicity, a decrease rather than an increase in food intake would be expected.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.The statistically significantly lower number of platelets noted for females at 1000 mg/kg bw/day as compared to controls was not considered to be toxicologically relevant, since it was a relatively small change with values remaining within the historical range of (1106 ± 217.3) x 10E9/L. The statistically significantly lower red blood cell count (RBC) recorded for males at 300 mg/kg bw/day as compared to controls was considered not to represent a change of biological relevance, since no treatment-related distribution could be established. All values remained within the historical range of (8.60 ± 0.450) x 10E12/L.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated high dose animals (1000 mg/kg bw/day) from control animals:
- Higher alkaline phosphatase (ALP) activities for females.
- Lower cholesterol concentration for males.
- Lower sodium concentrations for males.
- Higher potassium concentrations for both males and females.
- Lower chloride concentrations for males.
The biological relevance of the findings of a higher alkaline phosphatase activity for females at 1000 mg/kg bw/day and a lower cholesterol concentration for males at 1000 mg/kg bw/day is doubted, since there were no findings in other parameters like food consumption, haematology or at the microscopic level that could explain these changes.
Any statistically significant changes at 100 or 300 mg/kg bw/day were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. For one female each at 100 and 300 mg/kg bw/day, the bile acid concentration was above the historical range (i.e. 305.5 and 130.5 µmol/L, respectively, versus a historical range of 39.0 ± 21.94 µmol/L). The reason for these two outliers is not known. Evaluation of in-life and necropsy data did not reveal any sign for an impaired health condition of either female, and a technical error could be excluded. Based on the incidental nature and in the absence of any dose-related trend, this finding was not considered to be biologically relevant.

MACROSCOPIC EXAMINATION
Macroscopic examination at necropsy did not reveal any toxicologically relevant alterations that were related to treatment up to 1000 mg/kg bw/day. Incidental findings among control and treated animals at necropsy included pelvic dilation of the kidneys (unilateral), yellowish, soft cyst or nodule on the tail of the epididymides, tan or dark-red foci on thymus, lungs or clitoral glands (isolated to many), reddish discolouration of the mandibular lymph nodes, enlarged mesenteric lymph nodes or liver, agenesis of the papillary process of the liver, fibrinlike coating of the lateral liver lobe, testes and/or epididymides reduced in size (uni- or bilateral), alopecia and/or scabbing on different parts of the body. The incidence of these findings was within the background range of macroscopic abnormalities that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. Therefore, they were not considered to be toxicologically relevant.

ORGAN WEIGHTS
Higher kidney weights (absolute and relative to body weights) were noted for males at 1000 mg/kg bw/day. In addition, two out of the five selected females at the same high dose level, had kidney weights (absolute: 2.20 and 2.31 gram) that were above the historical control range. As a consequence, the group mean value for females at 1000 mg/kg bw/day was higher as compared to that of control females. Any other statistically significant changes in organ weights of animals treated at the high dose of 1000 mg/kg bw/day as compared to control animals were considered not to be toxicologically relevant since no corroborative findings were noted in clinical laboratory or histopathology parameters, and/or values remained within the normal range of biological variation. These changes consisted of a lower thymus weight for males , and higher liver weights for males and females. For females, the increase in the mean liver weight was caused by the relatively high liver organ weights (absolute: 10.20 and 12.02 gram) for two females at 1000 mg/kg bw/day. A trend towards slightly higher thyroid gland weights (absolute and relative to body weight) was noted for females at 1000 mg/kg bw/day as compared to controls. However, changes were relatively slight, with all values remaining within the normal range of biological variation. Furthermore, there were no pathological findings in the thyroid glands, except for one female, showing follicular diffuse hyperplasia/hypertrophy at a minor degree. Taken this together, no toxicological relevance was attributed to this finding. At the individual level, relatively low testes and epididymides weights were recorded for two males at 100 mg/kg bw/day. This was in line with the macroscopic finding of reduced sizes of testes and epididymides (uni- or bilateral) for these two males. One of these males failed to produce offspring due to massive bilateral seminiferous tubular atrophy in the testes (there was no sperm present). The other male, produced a normal litter, although microscopic examination of the testes and epididymides from this male revealed multifocal seminiferous tubular atrophy in the testes (unilateral) and oligospermia in the epididymides (unilateral), both at a marked degree. At the low incidence and in the absence of a dose-related trend, these findings were not considered toxicologically relevant. Other organ weights and organ to body weight ratios at 1000 mg/kg bw/day were similar to control levels. No changes were noted in organ weights following treatment at 100 or 300 mg/kg bw/day.
For historical control data, see section "Any other information on results incl. tables" further below.

MICROSCOPIC EXAMINATION
Treatment-related microscopic findings were present in high dose females only and consisted of abnormalities in the kidneys and urinary bladder.
Kidneys
- Tubular dilatation was present in 3/5 female (1 minimal, 2 slight) rats treated at 1000 mg/kg bw/day.
- Tubular degeneration and regeneration was present in 5/5 female (1 minimal, 3 slight, 1 moderate) rats treated at 1000 mg/kg bw/day up to moderate degrees. This finding was characterized by variable degrees of basophilic tubules with an increased mitotic index and the presence of apoptotic cells, representing both degeneration and regeneration. The lumen of these tubules often contained eosinophilic granular debris.
Urinary bladder
- Urothelial vacuolation was present at slightly increased incidence and severity in 2/5 female (two slight) rats treated at 1000 mg/kg bw/day compared to 1/5 control, 1/5 100 mg/kg bw/day and 1/5 300 mg/kg bw/day (all at a minimal degree) treated female rats.
- Diffuse hyperplasia of the urothelium was present in 2/5 female (one minimal, one slight) rats treated at 1000 mg/kg bw/day.
At 100 mg/kg bw/day, one female failed to get pregnant because the male with whom she was paired, had massive bilateral seminiferous tubular atrophy in the testes (there was no sperm present). For the other pairs there was no morphological finding that could explain their failure to get pregnant or the total litter loss.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, except for the one male which had no sperm at all. Although rare, this is not an unusual observation. Since there was no sign for necrosis in the testes and in the absence of a doserelated trend, this isolated finding was not considered to be toxicologically relevant. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain and in this type of study.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment up to 1000 mg/kg bw/day. For one female at 1000 mg/kg bw/day with total litter loss, only one implantation site was noted whereas the number of 12 corpora lutea was within normal ranges. Comparable findings are incidentally seen in this type of studies without any relation to treatment. Since for the remaining nine females in the same group the numbers of implantation sites were within normal ranges, no toxicological significance was attributed to this finding. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

GESTATION
The gestation index and duration of gestation were within normal ranges for treated and control females. One female at 1000 mg/kg bw/day lost her only pup before the first litter check. This resulted in a slightly lower gestation index at this highest dose level. The gestation index was 100, 100, 100 and 90% for females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. However, no toxicological significance was attributed to this finding since the gestation index for the high dose group remained within the normal range of biological variation.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction toxicity was noted up to the highest dose level tested (1000 mg/kg bw/day).

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

EARLY POSTNATAL PUP DEVELOPMENT
There was a treatment-related effect on the number of dead and living pups at the first litter check and clinical signs at 300 and/or 1000 mg/kg bw/day. Postnatal loss, viability index and sex ratio were unaffected by treatment, and body weight and external macroscopy did not reveal treatment-related findings up to 1000 mg/kg bw/day.

MORTALITY PUPS
Treatment at 1000 mg/kg bw/day resulted in increased mortality of pups. In total 18 pups in six litters were found dead at the first litter check. For one litter of Group 4, there were no living pups at all (only one was born). This altogether resulted in a total number and lower mean number of living pups at first litter check that was 40% lower in the high dose group as compared to the concurrent control group (i.e. 68 versus 115 and 6.8 versus 11.5, respectively). At 300 mg/kg bw/day, there were three litters with in total 6 dead pups at first litter check and one pup missing on Day 2. The mean number of living pups at first litter check at this dose level was only slightly lower than that for controls (i.e. 10.4 versus 11.5) and remained within the normal range of biological variation. However, the number of four dead pups in one litter was higher than is usually encountered, and together with the increased mortality at 1000 mg/kg bw/day, a relationship to treatment could not be excluded. Mortality was within normal ranges in the 100 mg/kg bw/day and control group.

CLINICAL SIGNS PUPS
Four pups from different litters at 1000 mg/kg bw/day had a pale appearance on lactation Days 1 and/or 2, followed by a complete recovery. The finding of pale/lean appearance is not unusual, but the incidence seen in the present study was higher than normal. Taken together with the high mortality in this group it could possibly be an additional indication of a poor condition of some pups during the initial phase of lactation. Incidental clinical symptoms of pups consisted of scabbing, a wound on and/or blue discolouration of the snout, and no 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.

BODY WEIGHT PUPS
Body weights of pups were unaffected by treatment.

MACROSCOPY PUPS
Necropsy did not reveal any toxicologically relevant alterations. Incidental macroscopic findings of pups that were found dead included beginning autolysis, almost entire cannibalization, and/or no milk in the stomach. Incidental macroscopic findings among surviving pups included scabbing of the snout. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

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: Based on increased pup mortality at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day.

Reproductive effects observed:

not specified

Historical control range for female kidneys:

absolute weight of 1.59± 0.170 gram; organ/body weight ratio of 0.69 ± 0.059.

Historical control range for male thymus:

absolute weight of 0.321± 0.0723 gram; organ/body weight ratio of 0.090 ± 0.0197.

Historical control range for male liver:

absolute weight of 8.79± 0.844 gram; organ/body weight ratio of 2.45 ± 0.169.

Historical control range for female liver:

absolute weight of 7.40± 0.832 gram; organ/body weight ratio of 3.24 ± 0.272.

Historical control data for female thyroid glands:

absolute weight of 0.024± 0.0062 gram; organ/body weight ratio of 0.011 ± 0.0028.

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg bw/day, based on findings for the kidneys (both sexes) and urinary bladder (females).
Reproduction NOAEL: At least 1000 mg/kg bw/day.
Developmental NOAEL: 100 mg/kg bw/day, based on increased pup mortality at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has Klimisch score 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Rats were administered orally 0, 100, 300 or 1000 mg/kg bw/d of test substance in a study performed according to OECD 422 (1996). No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment up to 1000 mg/kg bw/day. The fertility NOAEL was determined to be >= 1000 mg/kg bw/d.

Short description of key information:
In an oral OECD 422 study, rats were exposed orally (gavage) to the substance up to 1000 mg/kg bw/day. No reproduction toxicity was observed up to the highest dose level tested, while parental effects were seen at 1000 mg/kg bw/d which included effects on kidneys (both sexes) and urinary bladder (females). The parental NOAEL was determined to be 300 mg/kg bw/d and the fertility NOAEL >= 1000 mg/kg bw/d.

Justification for selection of Effect on fertility via oral route:
Only one study available

Effects on developmental toxicity

Description of key information

In an oral OECD 422 study, rats were exposed orally (gavage) to the substance up to 1000 mg/kg bw/day. Based on increased pup mortality at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day, the developmental NOAEL was determined to be 100 mg/kg bw/d. The parental NOAEL was determined to be 300 mg/kg bw/d, based on findings for the kidneys (both sexes) and urinary bladder (females) at 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March - 01 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (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

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Qualifier:

according to guideline

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight range at start of treatment was 335-340 gr (males) or 210-215 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic 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
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle with the light on from 07:00-19:00.

IN-LIFE DATES: From: 09 March - 01 May 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation: 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 specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity of the test substance.

Storage conditions of formulations: At ambient temperature.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (06 April 2012), according to a validated method (Project 498508). 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 (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. 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 formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations. No test substance was detected in the Group 1 formulations. The formulations of Group 2 and Group 4 were homogeneous. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

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).
- Age at mating of the mated animals in the study: Approximately 14 weeks
- 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 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.
- After successful mating each pregnant female was caged individually in Macrolon plastic cages (MIII type, height 18 cm).
- Any deviations from standard protocol: no

Duration of treatment / exposure:

Males were exposed for 33 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). One female of Group 1 and one of Group 4 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.

Duration of test:

Males: 33 days
Females: 40-53 days

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected in consultation with the Sponsor based on the results of the dose range finding study (Project 499082). Since in this dose range finding study no toxicologically relevant clinical signs were noted, it was decided for the main study (Project 498516) to conduct clinical observations at least immediately after dosing.

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 randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected 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.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made for all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. 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.

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: 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: 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

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 (5 animals/sex/group)
- Battery of functions tested: According to test guidelines

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 (iso-flurane) and subsequently exsanguinated.
Two animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 20 hours and 5 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the macroscopic or microscopic findings.
- Selected 5 animals/sex/group: According to test guidelines
- All remaining females which failed to deliver, the female with total litter loss and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
No thyroid gland organ weight was recorded for one female at 1000 mg/kg bw/day. For another female at 1000 mg/kg bw/day inadvertently the organ weight from the spleen was entered in the computer instead that from the ovaries. The incorrect ovaries organ weight was removed from the tables. Since tissues had been processed for histopathology, it was not possible to complete the missing data. However, sufficient data was available from the remaining selected females from this group for evaluation.
For one female at 1000 mg/kg bw/day, the terminal body weight was recorded at necropsy. It was additional information, that was not required by the protocol.
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY:
According to test guidelines
A few tissues were not available for histopathology because they were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. These are listed in the raw data and pathology report. Sufficient data was available for evaluation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalities, 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 for 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).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

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).

Indices:

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 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

Details on maternal toxic effects:

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 1000 mg/kg bw/day had to be sacrificed on Day 1 of lactation since she had a total litter loss. On the same morning, a pale appearance, shallow respiration, piloerection were noted. A few hours thereafter, one nearly entirely cannibalized pup was found in the cage. Gross findings during necropsy included an enlarged placenta in the uterus and part of a pup in her stomach. It was her only pup, since there was only one implantation site. There were no morphological findings in either the reproductive organs or the mammary glands of this female that could be attributed to the test substance.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included scabbing, scaling and alopecia. These findings occurred within the range of background findings to be expected 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 toxicologically relevant.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Locomotor activity as determined by total movements and ambulations was considered to be unaffected by treatment up to 1000 mg/kg bw/day (both sexes). Mean values remained within the normal range of biological variation. All groups showed a comparable habituation profile with high activity in the first interval that decreased over the duration of the test period. For one control male, total movements and ambulations were relatively high, with mean values at the upper end of the normal range. Consequently, the control means for these endpoints were relatively high, but still remained within the normal range of biological variation.


BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. During the entire treatment period there was a trend towards slightly lower body weights and body weight gains for males at 1000 mg/kg bw/day as compared to controls, reaching statistical significance on Day 8 of the mating period only. However, since these changes were only slight, with all values within the normal range of biological variation, this finding was not considered to be toxicologically relevant.

FOOD CONSUMPTION
Food consumption, calculated as absolute values and relative to body weight, was lower for females at 1000 mg/kg bw/day as compared to controls during lactation. This change was most likely caused by the lower food consumption of the females with smaller litters. Therefore, it was regarded as secondary effect and thus not toxicologically relevant. No toxicologically relevant changes in absolute and relative food consumption were noted for males up to 1000 mg/kg bw/day. During the entire treatment period there was a trend towards slightly lower absolute and relative food consumption for males at 1000 mg/kg bw/day compared to controls. However, since changes were only slight, with all values within the normal range of biological variation, this finding was not considered to be toxicologically relevant. The statistically significantly higher absolute food consumption recorded for females at 1000 mg/kg bw/day compared to controls during Days 0-7 post-coitum was not considered to be toxicologically relevant, since values remained within the range considered normal for rats of this age and strain. Moreover, in case of toxicity, a decrease rather than an increase in food intake would be expected.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.The statistically significantly lower number of platelets noted for females at 1000 mg/kg bw/day as compared to controls was not considered to be toxicologically relevant, since it was a relatively small change with values remaining within the historical range of (1106 ± 217.3) x 10E9/L. The statistically significantly lower red blood cell count (RBC) recorded for males at 300 mg/kg bw/day as compared to controls was considered not to represent a change of biological relevance, since no treatment-related distribution could be established. All values remained within the historical range of (8.60 ± 0.450) x 10E12/L.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated high dose animals (1000 mg/kg bw/day) from control animals:
- Higher alkaline phosphatase (ALP) activities for females.
- Lower cholesterol concentration for males.
- Lower sodium concentrations for males.
- Higher potassium concentrations for both males and females.
- Lower chloride concentrations for males.
The biological relevance of the findings of a higher alkaline phosphatase activity for females at 1000 mg/kg bw/day and a lower cholesterol concentration for males at 1000 mg/kg bw/day is doubted, since there were no findings in other parameters like food consumption, haematology or at the microscopic level that could explain these changes.
Any statistically significant changes at 100 or 300 mg/kg bw/day were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. For one female each at 100 and 300 mg/kg bw/day, the bile acid concentration was above the historical range (i.e. 305.5 and 130.5 µmol/L, respectively, versus a historical range of 39.0 ± 21.94 µmol/L). The reason for these two outliers is not known. Evaluation of in-life and necropsy data did not reveal any sign for an impaired health condition of either female, and a technical error could be excluded. Based on the incidental nature and in the absence of any dose-related trend, this finding was not considered to be biologically relevant.

MACROSCOPIC EXAMINATION
Macroscopic examination at necropsy did not reveal any toxicologically relevant alterations that were related to treatment up to 1000 mg/kg bw/day. Incidental findings among control and treated animals at necropsy included pelvic dilation of the kidneys (unilateral), yellowish, soft cyst or nodule on the tail of the epididymides, tan or dark-red foci on thymus, lungs or clitoral glands (isolated to many), reddish discolouration of the mandibular lymph nodes, enlarged mesenteric lymph nodes or liver, agenesis of the papillary process of the liver, fibrinlike coating of the lateral liver lobe, testes and/or epididymides reduced in size (uni- or bilateral), alopecia and/or scabbing on different parts of the body. The incidence of these findings was within the background range of macroscopic abnormalities that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. Therefore, they were not considered to be toxicologically relevant.

ORGAN WEIGHTS
Higher kidney weights (absolute and relative to body weights) were noted for males at 1000 mg/kg bw/day. In addition, two out of the five selected females at the same high dose level, had kidney weights (absolute: 2.20 and 2.31 gram) that were above the historical control range. As a consequence, the group mean value for females at 1000 mg/kg bw/day was higher as compared to that of control females. Any other statistically significant changes in organ weights of animals treated at the high dose of 1000 mg/kg bw/day as compared to control animals were considered not to be toxicologically relevant since no corroborative findings were noted in clinical laboratory or histopathology parameters, and/or values remained within the normal range of biological variation. These changes consisted of a lower thymus weight for males , and higher liver weights for males and females. For females, the increase in the mean liver weight was caused by the relatively high liver organ weights (absolute: 10.20 and 12.02 gram) for two females at 1000 mg/kg bw/day. A trend towards slightly higher thyroid gland weights (absolute and relative to body weight) was noted for females at 1000 mg/kg bw/day as compared to controls. However, changes were relatively slight, with all values remaining within the normal range of biological variation. Furthermore, there were no pathological findings in the thyroid glands, except for one female, showing follicular diffuse hyperplasia/hypertrophy at a minor degree. Taken this together, no toxicological relevance was attributed to this finding. At the individual level, relatively low testes and epididymides weights were recorded for two males at 100 mg/kg bw/day. This was in line with the macroscopic finding of reduced sizes of testes and epididymides (uni- or bilateral) for these two males. One of these males failed to produce offspring due to massive bilateral seminiferous tubular atrophy in the testes (there was no sperm present). The other male, produced a normal litter, although microscopic examination of the testes and epididymides from this male revealed multifocal seminiferous tubular atrophy in the testes (unilateral) and oligospermia in the epididymides (unilateral), both at a marked degree. At the low incidence and in the absence of a dose-related trend, these findings were not considered toxicologically relevant. Other organ weights and organ to body weight ratios at 1000 mg/kg bw/day were similar to control levels. No changes were noted in organ weights following treatment at 100 or 300 mg/kg bw/day.
For historical control data, see section "Overall remarks, attachments" further below.

MICROSCOPIC EXAMINATION
Treatment-related microscopic findings were present in high dose females only and consisted of abnormalities in the kidneys and urinary bladder.
Kidneys
- Tubular dilatation was present in 3/5 female (1 minimal, 2 slight) rats treated at 1000 mg/kg bw/day.
- Tubular degeneration and regeneration was present in 5/5 female (1 minimal, 3 slight, 1 moderate) rats treated at 1000 mg/kg bw/day up to moderate degrees. This finding was characterized by variable degrees of basophilic tubules with an increased mitotic index and the presence of apoptotic cells, representing both degeneration and regeneration. The lumen of these tubules often contained eosinophilic granular debris.
Urinary bladder
- Urothelial vacuolation was present at slightly increased incidence and severity in 2/5 female (two slight) rats treated at 1000 mg/kg bw/day compared to 1/5 control, 1/5 100 mg/kg bw/day and 1/5 300 mg/kg bw/day (all at a minimal degree) treated female rats.
- Diffuse hyperplasia of the urothelium was present in 2/5 female (one minimal, one slight) rats treated at 1000 mg/kg bw/day.
At 100 mg/kg bw/day, one female failed to get pregnant because the male with whom she was paired, had massive bilateral seminiferous tubular atrophy in the testes (there was no sperm present). For the other pairs there was no morphological finding that could explain their failure to get pregnant or the total litter loss.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, except for the one male which had no sperm at all. Although rare, this is not an unusual observation. Since there was no sign for necrosis in the testes and in the absence of a doserelated trend, this isolated finding was not considered to be toxicologically relevant. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain and in this type of study.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: (Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.)

Details on embryotoxic / teratogenic effects:
Treatment at 1000 mg/kg bw/day resulted in increased mortality of pups. In total 18 pups in six litters were found dead at the first litter check. For one litter there were no living pups at all (only one was born). This altogether resulted in a total number and lower mean number of living pups at first litter check that was 40% lower in the high dose group as compared to the concurrent control group (i.e. 68 versus 115 and 6.8 versus 11.5, respectively).
At 300 mg/kg bw/day, there were three litters with in total 6 dead pups at first litter check and one pup missing on Day 2. The mean number of living pups at first litter check at this dose level was only slightly lower than that for controls (i.e. 10.4 versus 11.5) and remained within the normal range of biological variation. However, the number of four dead pups in one litter was higher than is usually encounted together with the increased mortality at 1000 mg/kg bw/day, a relationship to treatment could not be excluded.
Pup mortality was within normal ranges in the 100 mg/kg bw/day and control group.
Four pups from different litters at 1000 mg/kg bw/day had a pale appearance on lactation Days 1 and/or 2, followed by a complete recovery. The finding of pale/lean appearance is not unusual, but the incidence seen in the present study was higher than normal. Taken together with the high mortality in this group it could possibly be an additional indication of a poor condition of some pups during the initial phase of lactation.
Body weights of pups and gross pathology at necropsy were unaffected up to the highest dose level tested (1000 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

other: clinical findings for pups at 1000 mg/kg bw/day.

Abnormalities:

not specified

Developmental effects observed:

not specified

REPRODUCTIVE DATA

No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment up to 1000 mg/kg bw/day. For one female at 1000 mg/kg bw/day with total litter loss, only one implantation site was noted whereas the number of 12 corpora lutea was within normal ranges. Comparable findings are incidentally seen in this type of studies without any relation to treatment. Since for the remaining nine females in the same group the numbers of implantation sites were within normal ranges, no toxicological significance was attributed to this finding. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

GESTATION

The gestation index and duration of gestation were within normal ranges for treated and control females. One female at 1000 mg/kg bw/day lost her only pup before the first litter check. This resulted in a slightly lower gestation index at this highest dose level. The gestation index was 100, 100, 100 and 90% for females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. However, no toxicological significance was attributed to this finding since the gestation index for the high dose group remained within the normal range of biological variation.

PARTURITION/MATERNAL CARE

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg bw/day, based on findings for the kidneys (both sexes) and urinary bladder (females).
Reproduction NOAEL: At least 1000 mg/kg bw/day.
Developmental NOAEL: 100 mg/kg bw/day, based on increased pup mortality at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has Klimisch score 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Rats (10/sex/dose) were administered orally 0, 100, 300 or 1000 mg/kg bw/d of test substance in a study performed according to OECD 422 (1996). An increased incidence in pup mortality was observed at 300 and 1000 mg/kg bw/day. In the high dose group of 1000 mg/kg bw/day also clinical signs were noted for pups such as pale/lean appearance, considered as indicative for poor condition of the pups. No substance-related effects were observed on gestation index and duration, parturition, maternal care or body weights of pups. Based on increased pup mortality at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day, the developmental NOAEL was determined to be 100 mg/kg bw/d.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available

Justification for classification or non-classification

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats by oral gavage, an increased incidence in pup mortality was observed at 300 and 1000 mg/kg bw/day and clinical findings for pups at 1000 mg/kg bw/day. The NOAEL for maternal toxicity in this study is 300 mg/kg bw/day, based on effects on kidneys and urinary bladder observed at 1000 mg/kg bw/day. Although the incidence of pup mortality at 300 mg/kg bw/day was only slightly different from the normal range observed, together with the increased incidence in mortality observed at 1000 mg/kg bw/day, a relationship to treatment cannot be excluded. Consequently, occurrence of developmental toxicity/teratogenicity in the absence of maternal toxicity cannot be excluded. Together with the severity of the developmental effect observed, it is suggested to classify Magnesium bis(2 -ethylbutanoate) as suspected human reproductive toxicant Cat 2.