Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Dec 2016 to 20 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Dec 2016 to 20 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Appearance: yellow powder
- Purity: ≥ 99%
- Date of receipt: 15 July 2016
- Date of expiration: 15 July 2018
- Storage conditions: Ambient temperature (15 - 25°C)
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain selection: The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
- Source: The animals were btained from a colony maintained under specific pathogen-free (SPF) conditions at Charles River Deutschland, Sulzfeld, Germany.
- Age of the males: About 11 weeks at the start of pretreatment period and about 13 weeks at the start of exposure and about 15 weeks at the start of mating.
- Age of the females: About 12 weeks at the start of pretreatment period and about 14 weeks at the start of exposure and about 16 weeks at the start of mating.
- Weight at study initiation: Males: 312.6 - 397.9 g; Females: 206.5 - 242.6 g
- Housing: The animals were housed under conventional conditions in one animal room. No other test systems were housed in the same room during the study. The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a ‘lot’) and by animal number (within each lot the mated females were housed in numerical order). After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet: Food was provided ad libitum from the arrival of the rats until the end of the study, unless precluded by the performance of certain laboratory investigations. From their arrival, the rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of VRF1 (FG) diet is analysed by the supplier for nutrients and contaminants. The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced once per week with fresh portions and topped up when necessary.
- Water: Drinking water was supplied ad libitum from the arrival of the animals until the end of the study. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. The supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.
- Acclimation period: Upon arrival on 30 November 2016, the rats were taken in their unopened shipping containers to a quarantine room and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from 4 randomly selected animals of this study. On 6 December 2016, the results of the serological examinations were received. Based on the serology results the animals were accepted. The animals were subsequently released for experimental use. During the acclimatization period to the laboratory conditions and during the study, the animals were maintained in the same room.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: At least 45% and not exceeding 65% other than during short periods due to room cleaning. At some occasions, relative humidity was slightly below 45% (reaching a minimum of 29.5% on 17 January 2017) for short periods of time.
- Air changes: 10 changes per hour
- Photoperiod: A sequence of 12 hours light and 12 hours dark was maintained.
- Light quality: Lighting was artificial (fluorescent tubes).
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 0.04 - 1 mg/mL
- Amount of vehicle: 5 mL/kg body weight
- Batch no.: A1600985
- Purity: 100 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gavage liquid containing the test substance was destructed in the presence of sulfuric acid and hydrogen peroxide. The samples were subsequently diluted using 1.4 M HNO3 and analysed using inductively coupled plasma - mass spectrometry (ICP-MS). Rhodium was used as internal standard. The test substance was quantified using tellurium (Te) as a marker.
- Validation criteria: The acceptance criterion for linearity was as follows: the correlation coefficient of the calibration curves should be ≥ 0.996.
- Homogeneity: The homogeneity of the test substance was assessed in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. Three samples of each test gavage liquid, taken at different locations in the gavage liquid container, and 1 sample of the control gavage liquid were analysed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the gavage liquid container). The test substance was considered to be homogeneously distributed in the gavage liquid if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
- Content: The content of the test substance was determined in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. The content of the test substance in gavage liquid was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: Animals were caged together until mating occurred.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After the first week of the mating period all females, were successfully mated with their assigned male. Based on this number of mating pairs, the mating period was finalized after one week.
- After successful mating each pregnant female was caged: Caged individually for the birth and rearing of their pups. Dams were allowed to raise their litter until sacrifice, 13 or 14 days after giving birth.
- Any other deviations from standard protocol: Since one animal had to be sacrificed before mating, this animal was replaced by a spare animal until mating occurred.
Duration of treatment / exposure:
The test substance was administered during a pre-mating period of 2 weeks, during mating (1 week) and post-mating up to at least 28 consecutive days for male rats.
The test substance was administered during a pre-mating period of 2 weeks and during mating (1 week), gestation and lactation until (or shortly after) postnatal day 13 (PN 13) for female rats.
Frequency of treatment:
Once daily for 7 days per week
Dose / conc.:
0.2 mg/kg bw/day (actual dose received)
Remarks:
Low-dose
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Remarks:
Mid-dose
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
High-dose
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose rationale: The dose levels were selected in consultation with the sponsor are were based on the results of a 2-weeks doserange finding study with the test substance in rats
Maternal examinations:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study, up to the last week of the gestation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation (erroneously, female animals of lot 1 were weighed on GD 21) and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

NEUROBEHAVIOURAL EXAMINATION
In the week prior to sacrifice, Functional Observational Battery (FOB) tests and spontaneous motor activity were performed in 5 males/group after 28 days of dosing and in 5 females with a litter/group on PN day 13. For females both tests were performed after sacrifice of their pups on PN day 13.

HAEMATOLOGY:
- Time schedule for collection of blood: prior to sacrifice
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: hemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: prior to sacrifice
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4) and bile acids.
Ovaries and uterine content:
The ovaries, uterus (including cervix) and vagina of all female animals were examined. The number of implantation sites and resorptions were counted in the uterus. If necessary the implantation sites were made visible following Salewski, E (1964), Arch. Exp. Path. Pharmacol. 247, 367.
Fetal examinations:
SACRIFICE
- Pups were sacrificed on day 13 of lactation

EXAMINATIONS
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter. At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. The thyroid gland was examined in detail.
- Skeletal examinations: No
- Head examinations: No
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.
- Signs and pathology of pups: Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded.
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**). Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (hematology, clinical chemistry) and T4 hormone data: ‘Generalized Anova/Ancova Test’ (with ‘Automatic’ as data transformation method. This test is an automatic decision tree consisting of: (1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed; (2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test); (3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significancelevels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.
Indices:
For each female, the following was determined:
- number of adult females with normal or abnormal oestrous cycle and cycle duration.
- number of females mated (= placed with males).
- number of females inseminated.
- duration of gestation (= mean number of gestation days).
- number of females surviving delivery.
- number of females with live born and (all) stillborn pups.
- number of implantation sites.
- litter size: number of pups delivered (live- and stillborn) per litter.
- number and sex of live pups at day 0, 4, 7 and 13.
- number of pups lost.

Accordingly, the following reporduction indices were calculated:
- mating days until Day 0 pc (= time between the start of mating and successful copulation)
- duration of gestation (= time between gestation day 0 and day of delivery)
- female mating index (= (number of females inseminated/number of females placed with males) x 100)
- female fertility index (= number of pregnant females*100/number of inseminated females)
- gestation index (= (number of females with live pups / number of females pregnant) x 100)
- prenatal loss (= (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites)
- perinatal loss (= (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered)
- live birth index (= (number of pups born alive/number of pups born) x 100)
- viability index day 0 - 4 (= (number of pup surviving 4 days/number of liveborn on day 0) x100))
- viability index day 4-13 (= (number of pup surviving 13 days/number of liveborn on day 4) x100)
- sex ratio day 0 and 13 (= [(number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13] x 100)
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation period or the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed. On the first day of the mating period, one female of the control group had to be sacrificed in a moribund condition because she had a bad wound due to fighting with here male cage-mate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on body weights and body weight changes of female animals during the pre-mating period.
- On gestation days 7, 14 and 20, body weights of the female animals of the high-dose group were statistically significantly lower than the body weights of the control animals.
- Body weight changes of the female animals of the high-dose group were statistically significantly lower from gestation day 7-14, 14-20 and 0-20.
- During the lactation period, body weights of the females of the high-dose group were statistically significantly lower than the body weights of the control animals whereas no statistically significant effects were observed on body weight changes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on food consumption of female animals during the pre-mating period.
- During the gestation period (GD 7-14 and 14-20), food consumption of the females of the high-dose group was statistically significantly lower than the food consumption of the control animals.
- During the lactation period, in the mid-dose group food consumption was statistically significantly decreased between postnatal days 4 to 7. No other statistically significant effects were observed on food consumption during the lactation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In female animals no differences in red blood cell and coagulation parameters were observed among the control and the treatment groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No differences were observed in clinical chemistry parameters among the control and treatment groups. In female animals, the concentration of cholesterol in the high-dose group was statistically significantly higher than in control animals. In conclusion, except for some incidental findings which were considered chance findings and not related to treatment, no effects on clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects were observed in female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- In female animals, no statistically significant differences were observed in the mean absolute organ weights between the control and treatment groups.
- In female animals of the high-dose group, the relative weights of the brain (15%), liver (12%) and kidneys (12%) were statistically significantly increased as compared to control animals. The effects on organ weights of >10% as observed in female animals of the high-dose group were considered as treatment related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examinations revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
HORMONE ANALYSIS
No statistically significant effects were observed amongst the different groups.
Number of abortions:
no effects observed
Description (incidence and severity):
All females delivered live born pups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites and number of pups delivered were comparable among the groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) accounted 0(0), 0 (0), 1 (3), 1(1) for the control, low-, mid- and high-dose groups, respectively. The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups (the number of still born pups and perinatal loss was highest in the control group).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of gestation was comparable among the groups.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was comparable among the groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy was not affected by treatment, all mated females were pregnant. The gestation index was 100% in all groups
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: develpmental toxicity: no effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4 and 7. On lactation day 13, the weight of the male and female pups of the high-dose group was statistically significantly lower than the weight of the control pups. Body weight changes of the male and female pups of the high-dose group were statistically significantly lower than observed in the control group between lactation days 4-7, 7-13 and 0-13.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of pups delivered per litter and the incidences of live born pups were comparable among the groups. The number of stillborn pups accounted 0, 0, 3 and 1 for the control, low-, mid- and high-dose groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed on sex ratio on day 0 and day 13 among the various groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of pups delivered per litter and the incidences of live born pups were comparable among the groups.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
The number of number of pups that died or were missing between days 0-4 accounted 1, 1, 1 and 4 for the control, low-, mid- and high dose, respectively. After culling on day 4, no pups were lost.
External malformations:
no effects observed
Description (incidence and severity):
- Macroscopic observations: Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects. Obviously, macroscopy could not be performed in the pups that were missing during the lactation period. Macroscopic observations of pups at necropsy on postnatal day 13 revealed no abnormalities.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Microscopic thyroid examinations: Microscopic examinations of the thyroid gland revealed no treatment-related abnormalities.
- Thyroid weight: There were no effects of the absolute and relative weight of the thyroid of male and female pups on post-natal day 13.
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Dec 2016 to 20 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Date of receipt: 15 July 2016
- Date of expiration: 15 July 2018
- Purity: ≥ 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25°C)

OTHER SPECIFICS:
- Appearance: yellow powder
Species:
rat
Strain:
Wistar
Remarks:
(Crl:WI(Han))
Details on species / strain selection:
- Species selection: The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
- Strain selection: The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were obtained from a colony maintained under specific pathogen-free (SPF) conditions at Charles River Deutschland, Sulzfeld, Germany.
- Age of the males: About 11 weeks at the start of pretreatment period and about 13 weeks at the start of exposure
- Age of the females: About 12 weeks at the start of pretreatment period and about 14 weeks at the start of exposure
- Weight at study initiation: Males: 312.6 - 397.9 g; Females: 206.5 - 242.6 g
- Housing: The animals were housed under conventional conditions in one animal room. No other test systems were housed in the same room during the study. The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet: Food was provided ad libitum from the arrival of the rats until the end of the study, unless precluded by the performance of certain laboratory investigations. From their arrival, the rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced once per week with fresh portions and topped up when necessary.
- Water: Drinking water was supplied ad libitum from the arrival of the animals until the end of the study. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: Upon arrival on 30 November 2016, the rats were taken in their unopened shipping containers to a quarantine room and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from 4 randomly selected animals of this study. On 6 December 2016, the results of the serological examinations were received. Based on the serology results the animals were accepted. The animals were subsequently released for experimental use. During the acclimatization period to the laboratory conditions and during the study, the animals were maintained in the same room.

DETAILS OF FOOD AND WATER QUALITY:
- Each batch of VRF1 (FG) diet is analysed by the supplier for nutrients and contaminants.
- Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. The supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: At least 45% and not exceeding 65% other than during short periods due to room cleaning. At some occasions, relative humidity was slightly below 45% (reaching a minimum of 29.5% on 17 January 2017) for short periods of time. This deviations were considered not to have affected the validity of the study.
- Air changes: 10 changes per hour
- Photoperiod: A sequence of 12 hours light and 12 hours dark was maintained.
- Light quality: Lighting was artificial (fluorescent tubes).
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was used because this is an anticipated route of human exposure.
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 0.04 - 1 mg/mL
- Amount of vehicle: 5 mL/kg body weight
- Batch no.: A1600985
- Purity: 100 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gavage liquid containing the test substance was destructed in the presence of sulfuric acid and hydrogen peroxide. The samples were subsequently diluted using 1.4 M HNO3 and analysed using inductively coupled plasma - mass spectrometry (ICP-MS). Rhodium was used as internal standard. The test substance was quantified using tellurium (Te) as a marker.
- Validation criteria: The acceptance criterion for linearity was as follows: the correlation coefficient of the calibration curves should be ≥ 0.996.
- Homogeneity: The homogeneity of the test substance was assessed in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. Three samples of each test gavage liquid, taken at different locations in the gavage liquid container, and 1 sample of the control gavage liquid were analysed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the gavage liquid container). The test substance was considered to be homogeneously distributed in the gavage liquid if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
- Content: The content of the test substance was determined in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. The content of the test substance in gavage liquid was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Duration of treatment / exposure:
The test substance was administered during a pre-mating period of 2 weeks, during mating (1 week) and post-mating up to at least 28 consecutive days for male rats.
The test substance was administered during a pre-mating period of 2 weeks and during mating (1 week), gestation and lactation until (or shortly after) postnatal day 13 (PN 13) for female rats.
Frequency of treatment:
Once daily for 7 days per week
Dose / conc.:
0.2 mg/kg bw/day (actual dose received)
Remarks:
Low-dose
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Remarks:
Mid-dose
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
High-dose
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose rationale: The dose levels were selected in consultation with the sponsor are were based on the results of a 2-weeks dose-range finding study with the test substance in rats
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study, up to the last week of the gestation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation (erroneously, female animals of lot 1 were weighed on GD 21) and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

NEUROBEHAVIOURAL EXAMINATION
In the week prior to sacrifice, Functional Observational Battery (FOB) tests and spontaneous motor activity were performed in 5 males/group after 28 days of dosing and in 5 females with a litter/group on PN day 13. For females both tests were performed after sacrifice of their pups on PN day 13.

HAEMATOLOGY:
- Time schedule for collection of blood: prior to sacrifice
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: hemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: prior to sacrifice
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4) and bile acids.
Sacrifice and pathology:
SACRIFICE, GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
Prior to sacrifice, all adult male and female animals were fasted overnight (water was freely available). All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on 24 January 2017 (after 29 days of dosing). Dams were sacrificed, after overnight fasting, on day 14 of lactation.

GROSS PATHOLOGY
At scheduled necropsy, the organs of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. A detailed scheme of the examined organs is presented in 'Any other information on materials and methods incl. tables'. Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin’s fixative. The reproductive organs and gross lesions of all adult male and female animals were preserved. In addition, a series of other organs/tissues of five adult animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected; same animals as selected for haematology and clinical chemistry) were preserved.

HISTOPATHOLOGY
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination were performed on the preserved organs of all animals of the control and high-dose group. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Other examinations:
BLOOD SAMPLING FOR HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all male and female adult animals for determination of T4 hormone levels in serum samples. In addition, blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). This blood was collected to determine T4 hormone levels in serum samples. Samples were stored in a freezer (≤-18°C) until analysis.
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**).
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (haematology, clinical chemistry) and T4 hormone data: ‘Generalized Anova/Ancova Test’ (with ‘Automatic’ as data transformation method. This test is an automatic decision tree consisting of: (1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed; (2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test); (3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation period or the lactation period.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed. On the first day of the mating period, one female of the control group had to be sacrificed in a moribund condition because she had a bad wound due to fighting with here male cage-mate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on body weights and body weight changes of male and female animals during the pre-mating period and of males during the post-mating period.
- On gestation days 7, 14 and 20, body weights of the female animals of the high-dose group were statistically significantly lower than the body weights of the control animals. Body weight changes of the female animals of the high-dose group were statistically significantly lower from gestation day 7-14, 14-20 and 0-20.
- During the lactation period, body weights of the females of the high-dose group were statistically significantly lower than the body weights of the control animals whereas no statistically significant effects were observed on body weight changes.
- Terminal body weights of male and female animals of the high-dose group were statistically significantly lower than of the male and female animals of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on food consumption of male and female animals during the pre-mating period and of males during the post-mating period.
- During the gestation period (GD 7-14 and 14-20), food consumption of the females of the high-dose group was statistically significantly lower than the food consumption of the control animals.
- During the lactation period, in the mid-dose group food consumption was statistically significantly decreased between postnatal days 4 to 7. No other statistically significant effects were observed on food consumption during the lactation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- In male and female animals no differences in red blood cell and coagulation parameters were observed among the control and the treatment groups.
- As compared to the control group, in male animals of the low-dose group statistically significant lower values were observed for the number of white blood cells, the absolute number of lymphocytes and on the absolute- and relative numbers of basophiles. Since no differences were observed on total and differential white blood cell parameters in male animals of the mid- and high-dose groups nor in the female animals of all treatment groups, the effects observed in the male animals of the low-dose group were considered as chance findings and not related to treatment.
- In conclusion, there were no treatment-related changes in red blood cell and coagulation parameters and in total and differential white blood cell counts.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Except for two incidental findings (statistically significant higher concentrations of ALP in the low-dose group and of potassium in the high-dose group) in male animals, no differences were observed in clinical chemistry parameters among the control and treatment groups.
- In female animals, the concentration of cholesterol in the high-dose group was statistically significantly higher than in control animals.
- In conclusion, except for some incidental findings which were considered chance findings and not related to treatment, no effects on clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- The mean landing footsplay of male animals of the high-dose group was statistically significantly increased in comparison to the control group. Also, the time pattern of activity of males of both the mid-and high-dose groups were significantly different when compared to control males. These findings might reflect a neurotoxic effect of the test substance.
- No other effects were observed in male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- In male and female animals, no statistically significant differences were observed in the mean absolute organ weights between the control and treatment groups.
- The relative liver weight of male animals of the mid- and high-dose groups (8% and 13%, respectively) and the relative kidney weight of the high-dose males (9%) were statistically significantly increased as compared to the control group.
- In female animals of the high-dose group, the relative weights of the brain (15%), liver (12%) and kidneys (12%) were statistically significantly increased as compared to control animals.
- The effects on organ weights of >10% as observed in male- and female animals of the high-dose group were considered as treatment related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examinations revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
HORMONE ANALYSIS
No statistically significant effects were observed amongst the different groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

ANALYTICAL VERIFICATION OF DOSES OR CONCENTRATIONS

- Linearity: The correlation coefficient was 1.000 during the 6 runs which were performed during this study and therefore the preset criterion was met.

- Homogeneity: The RSD between the mean concentrations at three different locations was < 5% and/or p was ≥ 0.01 for all dose levels of all tested batches. Therefore the test substance was considered to be homogeneously distributed in the gavage liquids. However, the RSD for the low-dose gavage liquid prepared on 17 January 2017 was extremely high (60%), which raises serious questions about the homogeneity of the low-level gavage liquid of this batch.

- Content: The concentration of the test substance was close to intended (90-110%) for all gavage liquids at all dose levels, except for the low-dose level of the gavage liquids prepared on 17 January 2017 (+52%) and 30 January 2017 (-24%) and the mid-dose level of the gavage liquids prepared on 30 January 2017 (-12%). Because the average deviation from the intended concentration was less than 10% for each dose level when all 4 batches are considered, it was assumed that, on average, the animals received the intended test substance dose during the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(diethyldithiocarbamato-S,S')tellurium
EC Number:
244-121-9
EC Name:
Tetrakis(diethyldithiocarbamato-S,S')tellurium
Cas Number:
20941-65-5
Molecular formula:
C20H40N4S8Te
IUPAC Name:
tetrakis(diethyldithiocarbamato-S,S')tellurium
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Date of receipt: 15 July 2016
- Date of expiration: 15 July 2018
- Purity: ≥ 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25°C)

OTHER SPECIFICS:
- Appearance: yellow powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
- Species selection: The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
- Strain selection: The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were btained from a colony maintained under specific pathogen-free (SPF) conditions at Charles River Deutschland, Sulzfeld, Germany.
- Age of the males: About 11 weeks at the start of pretreatment period and about 13 weeks at the start of exposure and about 15 weeks at the start of mating.
- Age of the females: About 12 weeks at the start of pretreatment period and about 14 weeks at the start of exposure and about 16 weeks at the start of mating.
- Weight at study initiation: Males: 312.6 - 397.9 g; Females: 206.5 - 242.6 g
- Housing: The animals were housed under conventional conditions in one animal room. No other test systems were housed in the same room during the study. The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a ‘lot’) and by animal number (within each lot the mated females were housed in numerical order). After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet: Food was provided ad libitum from the arrival of the rats until the end of the study, unless precluded by the performance of certain laboratory investigations. From their arrival, the rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced once per week with fresh portions and topped up when necessary.
- Water: Drinking water was supplied ad libitum from the arrival of the animals until the end of the study. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: Upon arrival on 30 November 2016, the rats were taken in their unopened shipping containers to a quarantine room and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from 4 randomly selected animals of this study. On 6 December 2016, the results of the serological examinations were received. Based on the serology results the animals were accepted. The animals were subsequently released for experimental use. During the acclimatization period to the laboratory conditions and during the study, the animals were maintained in the same room.

DETAILS OF FOOD AND WATER QUALITY:
- Each batch of VRF1 (FG) diet is analysed by the supplier for nutrients and contaminants.
- Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. The supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: At least 45% and not exceeding 65% other than during short periods due to room cleaning. At some occasions, relative humidity was slightly below 45% (reaching a minimum of 29.5% on 17 January 2017) for short periods of time.
- Air changes: 10 changes per hour
- Photoperiod: A sequence of 12 hours light and 12 hours dark was maintained.
- Light quality: Lighting was artificial (fluorescent tubes).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 0.04 - 1 mg/mL
- Amount of vehicle: 5 mL/kg body weight
- Batch no.: A1600985
- Purity: 100 %
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: Animals were caged together until mating occurred.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After the first week of the mating period all females, were successfully mated with their assigned male. Based on this number of mating pairs, the mating period was finalized after one week.
- After successful mating each pregnant female was caged: Caged individually for the birth and rearing of their pups. Dams were allowed to raise their litter until sacrifice, 13 or 14 days after giving birth.
- Any other deviations from standard protocol: Since one animal had to be sacrificed before mating, this animal was replaced by a spare animal until mating occurred.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gavage liquid containing the test substance was destructed in the presence of sulfuric acid and hydrogen peroxide. The samples were subsequently diluted using 1.4 M HNO3 and analysed using inductively coupled plasma - mass spectrometry (ICP-MS). Rhodium was used as internal standard. The test substance was quantified using tellurium (Te) as a marker.
- Validation criteria: The acceptance criterion for linearity was as follows: the correlation coefficient of the calibration curves should be ≥ 0.996.
- Homogeneity: The homogeneity of the test substance was assessed in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. Three samples of each test gavage liquid, taken at different locations in the gavage liquid container, and 1 sample of the control gavage liquid were analysed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the gavage liquid container). The test substance was considered to be homogeneously distributed in the gavage liquid if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
- Content: The content of the test substance was determined in the batches of gavage liquids, prepared on 28 December 2016, 17 January 2017, 30 January 2017 and 13 February 2017. The content of the test substance in gavage liquid was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Duration of treatment / exposure:
The test substance was administered during a pre-mating period of 2 weeks, during mating (1 week) and post-mating up to at least 28 consecutive days for male rats.
The test substance was administered during a pre-mating period of 2 weeks and during mating (1 week), gestation and lactation until (or shortly after) postnatal day 13 (PN 13) for female rats.
Frequency of treatment:
Once daily for 7 days per week
Details on study schedule:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
0.2 mg/kg bw/day (actual dose received)
Remarks:
Low-dose
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Remarks:
Mid-dose
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
High-dose
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose rationale: The dose levels were selected in consultation with the sponsor are were based on the results of a 2-weeks doserange finding study with the test substance in rats
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study, up to the last week of the gestation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation (erroneously, female animals of lot 1 were weighed on GD 21) and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

NEUROBEHAVIOURAL EXAMINATION
In the week prior to sacrifice, Functional Observational Battery (FOB) tests and spontaneous motor activity were performed in 5 males/group after 28 days of dosing and in 5 females with a litter/group on PN day 13. For females both tests were performed after sacrifice of their pups on PN day 13.

HAEMATOLOGY:
- Time schedule for collection of blood: prior to sacrifice
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: hemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: prior to sacrifice
- Animals fasted: Yes (water was freely available)
- How many animals: all animals
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4) and bile acids.
Oestrous cyclicity (parental animals):
Vaginal smears to evaluate the estrus cycle length and normality were made daily for the 14 days during the pre-treatment period, during the pre-mating period and until confirmation of mating. Smears were stained and evaluated for estrus cyclicity in all females. An additional vaginal smear was made at the day of sacrifice but the results of the microscopic examination of the uterus and vagina did not give any indication for further examination of these smears.
Sperm parameters (parental animals):
A detailed scheme including examined male reproductive organs is presented in 'Any other information on materials and methods incl. tables'.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes/no
- If yes, maximum of 4 pups/sex/litter as nearly as possible. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Parturition and litter evaluation: At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.
- Anogenital distance in pups: At lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as mean per litter and corrected by the cube root of body weight.
- Culling and blood collection for hormone analysis: Blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). This blood was collected to determine T4 hormone levels in serum samples. Samples were stored in a freezer (≤-18°C) until analysis. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method based on the manufacturer’s protocol.
- On postnatal day 13 all surviving male pups were examined for the presence of nipples and/or areolas.
- Signs and pathology of pups: Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded.
Postmortem examinations (parental animals):
SACRIFICE, GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
Prior to sacrifice, all adult male and female animals were fasted overnight (water was freely available). All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on 24 January 2017 (after 29 days of dosing). Dams were sacrificed, after overnight fasting, on day 14 of lactation.

GROSS PATHOLOGY
At the day of necropsy, a vaginal smear was taken from each female. At scheduled necropsy, the organs of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. A detailed scheme of the examined organs is presented in 'Any other information on materials and methods incl. tables'. Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin’s fixative. The reproductive organs and gross lesions of all adult male and female animals were preserved. In addition, a series of other organs/tissues of five adult animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected; same animals as selected for haematology and clinical chemistry) were preserved.

HISTOPATHOLOGY
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination were performed on the preserved organs of all animals of the control and high-dose group. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

BLOOD SAMPLING FOR HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all male and female adult animals for determination of T4 hormone levels in serum samples. In addition, blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). This blood was collected to determine T4 hormone levels in serum samples. Samples were stored in a freezer (≤-18°C) until analysis.
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on day 13 of lactation

GROSS NECROPSY
At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. A detailed scheme of the examined organs is presented in 'Any other information on materials and methods incl. tables'.
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**). Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (hematology, clinical chemistry) and T4 hormone data: ‘Generalized Anova/Ancova Test’ (with ‘Automatic’ as data transformation method. This test is an automatic decision tree consisting of: (1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed; (2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test); (3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significancelevels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.
Reproductive indices:
For each mating, the following were determined:
- number of adult females with normal or abnormal oestrous cycle and cycle duration.
- number of females mated (= placed with males).
- number of females inseminated.
- number of males with pregnant females.
- duration of gestation (= mean number of gestation days).
- number of females surviving delivery.
- number of females with live born and (all) stillborn pups.
- number of implantation sites.
- litter size: number of pups delivered (live- and stillborn) per litter.
- number and sex of live pups at day 0, 4, 7 and 13.
- number of pups lost.

The following reproduction indices were calculated:
- mating days until Day 0 pc (= time between the start of mating and successful copulation)
- duration of gestation (= time between gestation day 0 and day of delivery)
- female mating index (= (number of females inseminated/number of females placed with males) x 100)
- male mating index (= (number of males placed with females /number of females inseminated) x 100)
- female fertility index (= number of pregnant females*100/number of inseminated females)
- male fertility index (= number of males with pregnant females*100/number of males placed with females)
- gestation index (= (number of females with live pups / number of females pregnant) x 100)
- prenatal loss (= (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites)
- perinatal loss (= (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered)
- live birth index (= (number of pups born alive/number of pups born) x 100)
- sex ratio day 0 and 13 (= [(number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13] x 100)
Offspring viability indices:
- viability index day 0 - 4 (= (number of pup surviving 4 days/number of liveborn on day 0) x100))
- viability index day 4-13 (= (number of pup surviving 13 days/number of liveborn on day 4) x100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation period or the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed. On the first day of the mating period, one female of the control group had to be sacrificed in a moribund condition because she had a bad wound due to fighting with here male cage-mate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on body weights and body weight changes of male and female animals during the pre-mating period and of males during the post-mating period.
- On gestation days 7, 14 and 20, body weights of the female animals of the high-dose group were statistically significantly lower than the body weights of the control animals. Body weight changes of the female animals of the high-dose group were statistically significantly lower from gestation day 7-14, 14-20 and 0-20.
- During the lactation period, body weights of the females of the high-dose group were statistically significantly lower than the body weights of the control animals whereas no statistically significant effects were observed on body weight changes.
- Terminal body weights of male and female animals of the high-dose group were statistically significantly lower than of the male and female animals of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related effects on food consumption of male and female animals during the pre-mating period and of males during the post-mating period.
- During the gestation period (GD 7-14 and 14-20), food consumption of the females of the high-dose group was statistically significantly lower than the food consumption of the control animals.
- During the lactation period, in the mid-dose group food consumption was statistically significantly decreased between postnatal days 4 to 7. No other statistically significant effects were observed on food consumption during the lactation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- In male and female animals no differences in red blood cell and coagulation parameters were observed among the control and the treatment groups.
- As compared to the control group, in male animals of the low-dose group statistically significant lower values were observed for the number of white blood cells, the absolute number of lymphocytes and on the absolute- and relative numbers of basophiles. Since no differences were observed on total and differential white blood cell parameters in male animals of the mid- and high-dose groups nor in the female animals of all treatment groups, the effects observed in the male animals of the low-dose group were considered as chance findings and not related to treatment.
- In conclusion, there were no treatment-related changes in red blood cell and coagulation parameters and in total and differential white blood cell counts.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Except for two incidental findings (statistically significant higher concentrations of ALP in the low-dose group and of potassium in the high-dose group) in male animals, no differences were observed in clinical chemistry parameters among the control and treatment groups.
- In female animals, the concentration of cholesterol in the high-dose group was statistically significantly higher than in control animals.
- In conclusion, except for some incidental findings which were considered chance findings and not related to treatment, no effects on clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- The mean landing footsplay of male animals of the high-dose group was statistically significantly increased in comparison to the control group. Also, the time pattern of activity of males of both the mid-and high-dose groups were significantly different when compared to control males. These findings might reflect a neurotoxic effect of the test substance.
- No other effects were observed in male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examinations revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
HORMONE ANALYSIS
No statistically significant effects were observed amongst the different groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- During the pre-treatment phase, four animals showed an irregular estrous cycle and, consequently, these animals were excluded for allocation to the different dosing groups.
- No statistically significant effects were observed on the estrous cycle of the remaining animals during the 2 weeks pre-treatment period and during the pre-mating phase, up to and including the day the animals were mated.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- All females that were placed with males were mated within 5 days . The mean time to mating was comparable among the groups.
- Except for the control group, male and female mating and fertility indices were 100% for all groups. In the control group, male mating index was 100%, but the female mating index 91.7% since one was sacrificed before mating occurred.
- The male fertility index was 91.7% since pregnancy was not confirmed in one (spare) animal.
- Pregnancy was not affected by treatment, all mated females were pregnant, the mean duration of gestation was comparable among the groups and the gestation index was 100% in all groups.
- All females delivered live born pups, there were no females with only stillborn pups.
- The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) accounted 0(0), 0 (0), 1 (3), 1(1) for the control, low-, mid- and high-dose groups, respectively. In the mid-dose group, one dam had 3 stillborn pups and in the high-dose group one dam had one stillborn pup.
- The number of implantation sites and number of pups delivered were comparable among the groups. Consequently, no statistically significant differences were observed on prenatal loss between the control and treatment groups. The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups (the number of still born pups and perinatal loss was highest in the control group).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
other: behaviour (functional finding)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related signs in pups during the lactation period
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- The mean number of pups delivered per litter and the incidences of live born pups were comparable among the groups.
- The number of stillborn pups accounted 0, 0, 3 and 1 for the control, low-, mid- and high-dose groups, respectively whereas the number of number of pups that died or were missing between days 0-4 accounted 1, 1, 1 and 4 for the control, low-, mid- and high dose, respectively.
- After culling on day 4, no pups were lost.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4 and 7.
- On lactation day 13, the weight of the male and female pups of the high-dose group was statistically significantly lower than the weight of the control pups.
- Body weight changes of the male and female pups of the high-dose group were statistically significantly lower than observed in the control group between lactation days 4-7, 7-13 and 0-13.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weight: There were no effects of the absolute and relative weight of the thyroid of male and female pups on post-natal day 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects. Obviously, macroscopy could not be performed in the pups that were missing during the lactation period.
- Macroscopic observations of pups at necropsy on postnatal day 13 revealed no abnormalities.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic examinations of the thyroid gland revealed no treatment-related abnormalities.
Other effects:
no effects observed
Description (incidence and severity):
DEVELOPMENTAL PARAMETERS
- Sex ratio: No statistically significant differences were observed on sex ratio on day 0 and day 13 among the various groups.
- Pup anogenital distance: There were no treatment related effects on the absolute (expressed in mm) and corrected (expressed in mm/g) anogenital distance of male and female pups.
- Nipple retention: There were no effects on nipple retention of male pups on post-natal day 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

ANALYTICAL VERIFICATION OF DOSES OR CONCENTRATIONS

- Linearity: The correlation coefficient was 1.000 during the 6 runs which were performed during this study and therefore the preset criterion was met.

- Homogeneity: The RSD between the mean concentrations at three different locations was < 5% and/or p was ≥ 0.01 for all dose levels of all tested batches. Therefore the test substance was considered to be homogeneously distributed in the gavage liquids. However, the RSD for the low-dose gavage liquid prepared on 17 January 2017 was extremely high (60%), which raises serious questions about the homogeneity of the low-level gavage liquid of this batch.

- Content: The concentration of the test substance was close to intended (90-110%) for all gavage liquids at all dose levels, except for the low-dose level of the gavage liquids prepared on 17 January 2017 (+52%) and 30 January 2017 (-24%) and the mid-dose level of the gavage liquids prepared on 30 January 2017 (-12%). Because the average deviation from the intended concentration was less than 10% for each dose level when all 4 batches are considered, it was assumed that, on average, the animals received the intended test substance dose during the study.

Applicant's summary and conclusion