Ethoxytrimethylsilane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May - 06 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethoxytrimethylsilane

EC Number:

217-370-6

EC Name:

Ethoxytrimethylsilane

Cas Number:

1825-62-3

Molecular formula:

C5H14OSi

IUPAC Name:

ethoxytrimethylsilane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Trimethyl ethoxy silane
- Physical state: Colourless liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approximately 9 - 10 weeks old at start of the study (11 - 12 weeks old at start of treatment and 13 - 14 weeks old at mating)
- Weight at study initiation: Males: 373 - 484 g, females: 244 – 316 g at the start of the treatment
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed (with pups), respectively
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" provided ad libitum
- Water: tap water from the municipal supply provided ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 23.4
- Humidity (%): 30 – 71
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (as a visibly stable homogenous suspension). Formulations were prepared at appropriate concentrations according to the dose level and treatment volume selected.
Formulations were prepared at given intervals (formulations were stored at room temperature in tightly closed glass containers for not more than 5 days before use) as follows. The calculated volume of the vehicle and the calculated amount of test item were added into a beaker; they were mixed vigorously by a vortex or repeated manual inversion to make a homogenous formulation. Glass containers were closed immediately after preparation. Based on the
analytical method validation study formulations in the 10 - 250 mg/mL concentration range were found to be stable after 7 days of storage at room temperature.

VEHICLE
- Concentration in vehicle: 12.5, 37.5, 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: A0381515 / A0384372

Details on mating procedure:

- M/F ratio per cage: one female and one male from the same dose group
- Length of cohabitation: Females remained with the same male until copulation occurred.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Females remained with the same male until copulation occurred.
- After successful mating each pregnant female was caged (how): Sperm positive females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and/or homogeneity was performed at the Analytical Department of the Test Facility using a validated GC-FID (Gas Chromatography – Flame Ionization Detector) method. Duplicate samples were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and approximately last weeks of treatment and approximately midway during the treatment period), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Formulation samples were kept at room temperature in a closed glass or plastic container until analysis.
The measured test item concentrations of the individual test item containing dose formulations varied between 93% and 101% of their nominal concentrations. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.
All test item formulation samples were found to be homogeneous. Acceptance criteria of the homogeneity was that the coefficient of variation (CV%) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
Formulations were considered to be adequately stable under the study conditions (stability period of 7 days).

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, throughout gestation and until the day before the necropsy (13-day post-partum dosing).

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data including the results of an OECD TG 414 study with trimethyl silanol (CAS 1066-40-6) in rats and a 14-day (repeated dose) Dose Range Finding (DRF) study in the rat performed at the Test Facility. For the DRF, the test material was administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 100, 250 and 600 mg/kg bw/day. At 600 mg/kg bw/day, test item-related clinical signs (ataxia and splayed gait) and test item-related body weight loss in the first days of treatment, with an overall body weight gain of less than 50% of the controls, were observed in male and female animals. There were no clearly adverse effects in clinical pathology, organ weights or at necropsy. No clear effects were observed at 250 mg/kg bw/day. Based on these results, it was considered that the high dose may be above the maximum tolerated dose (MTD).
- Rationale for animal assignment: All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the pre-exposure period.
- Fasting period before blood sampling for clinical biochemistry: All animals were fasted overnight.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were made once a day (except the days when detailed clinical observations were made).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were performed once before the start, before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment.
- The animals were monitored outside the home cage in a standard arena for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parental females were weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the parental female animals measured on GD3, 10 and 17 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly.

OTHER: Heamatology, clinical chemistry, urinalysis and neurobehavioural examination (for details please refer to endpoint repeated dose toxicity: oral)

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments started. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles with coagulating glands weight, stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), postnatal mortality, presence of gross anomalies, body weights (PND0, 4 and 13), behavioural abnormalities, anogenital distance (AGD, PND0), presence of nipples/areolae in male pups (PND13).

For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
- from up to two pups per litter on PND4
- from up to two pups per litter on PND13

GROSS EXAMINATION OF DEAD PUPS:
yes, the pups found dead and intact (not cannibalized or autolyzed) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized under pentobarbital anaesthesia, followed by exsanguination after 28 days.
- Maternal animals: Females showing no-evidence of copulation were sacrificed as practical, 25-26 days after the day of presumed mating. Dams were euthanized under pentobarbital anaesthesia, followed by exsanguination on PND14.

GROSS NECROPSY
- After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
At the time of termination, the weight of the following organs from all adult animals were recorded: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, and thyroids with parathyroids. Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights.

For the adult animals, a detailed histological examination was performed as follows: on the selected list of retained organs in the control and high-dose groups (selected 5 animals/sex/group); the trachea and lungs of one low-dose female because macroscopic findings (abnormalities) were seen at necropsy; all kidney samples of mid- and low-dose males, plus the remaining control and high-dose males; and retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high-dose groups, and of all males and all females that failed to sire / conceive or unable to delivering healthy pups.
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow). Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4. All surviving pups were terminated on PND13.
- Pups sacrificed on PND4 and/or PND13 were carefully examined externally for gross abnormalities.

Statistics:

SPSS PC+4.0 program: The heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If a significant result was obtained, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

SAS v9.2 software package: The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests. Where both tests showed no significant heterogeneity, an ANOVA / ANCOVA (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests showed significance, a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.

For pathology data, first a Chi-squared test was used; the system then checked the significance of the result against a user-defined value (0.05) and where suitable, also performed pairwise tests. The Fisher’s Exact Test was performed replacing the Chi-squared test as the final evaluation method if the group size was <5.

Reproductive indices:

Male mating index = (number of males with confirmed mating)/(total number of males cohabited) X 100
Female mating index = (number of sperm-positive females)/(total number of females cohabited) X 100
Male fertility index = (number of males impregnating a female)/(total number of males cohabited) X 100
Female fertility index = (number of pregnant females)/(number of sperm-positive females) X 100
Gestation index = (number of females with live born pups)/(number of pregnant females) X 100

Offspring viability indices:

Survival index = (number of live pups at designated time)/(number of pups born) X 100
Pre-implantation mortality = (number of corpora lutea - number or implantations)/(number of corpora lutea) X 100
Intrauterine mortality = (number of mplantations - number or live borns)/(number of implantations) X 100
Total mortality = (number of implantation - number of viable pups [PND 0/4/13])/(number of implantations) X 100
Sex ratio - female = (number of female pups [PND 0/4/13])/(number of pups examined [PND 0/4/13]) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were observed during the study in all high-dose (500 mg/kg bw/day) males and females. Treatment-related effects consisted of splayed gait, which was observed in all high dose males from Day 2 to Day 27. High-dose females displayed splayed gait from Day 2 until the end of the lactation period. Slightly or moderately decreased activity was recorded for 4 high-dose males on Day 14 and/or Day 15 and for 1 high-dose female on Day 14.
No clinical signs were observed for mid- (150 mg/kg bw/day) and low-dose (50 mg/kg bw/day) animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related effect (reduction) on body weight gain was detected during the study in the high-dose males (500 mg/kg bw/day), while no such effect was observed in high-dose females or in other groups.
There were no statistically significant differences in the absolute body weight values of the control and test item treated groups.

A greater than 40% reduction in the body weight gain of high-dose males was detected on the first week of treatment compared to the control data (the difference was statistically significant at p<0.01). Although the animals showed recovery, the reduction (by 17%) in the total body gain during the entire treatment period (Days 0-27) was still statistically significant (p<0.05) compared to control. No similar effect was seen in high-dose females or in mid- or low-dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related reduced food consumption was observed only in the first week of treatment in high-dose males and females (500 mg/kg bw/day). There were no test item related differences in the mean daily food consumption of mid- or low-dose groups.
A 16% and 8% reduction in food consumption was noted in the high-dose males and females, respectively in the first week of treatment. For males, the difference compared to the control value was statistically significant at p<0.01; for females the difference compared to the control value was statistically significant at p<0.05 level). However, it was only a transient effect, the animals recovered quickly and food consumption returned to the control level, and no statistically significant difference was recorded in the high-dose males at the end of the treatment period, or in the high-dose females in the gestation or 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):

There were no differences in males and females of the test item treated groups compared to control that were statistically significant or could be considered toxicologically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly increased urea concentration was observed in high-dose males (500 mg/kg bw/day), this finding might be test item related and connected to the kidney findings recorded during histopathology evaluation. There were no other changes in the evaluated serum chemistry parameters that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in urea concentration was seen in high-dose males (500 mg/kg bw/day) when compared to the control. However, the observed values were within the historical control range. No significant change was seen in mid- or low- dose males, or in any female groups (although it should be noted that values for all groups including control showed higher mean value than the upper limit of the historical control range). There were no statistically significant changes in other parameters (creatinine, calcium, potassium, phosphate and sodium; although creatinine in high-dose males decreased by 12% compared to control), but the relationship to test item treatment is plausible based on the observed histopathology findings in the kidney samples of high-dose males. No statistically significant changes were recorded for any other parameters in the test item treated males and females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly (p<0.01) lower urinary pH was detected in high-dose males when compared to control, the mean pH was close to the lower end of the historical control range. The specific gravity of the urine of high-dose males was significantly higher than control (p<0.05). Although the absolute difference was small in this case, but slight or moderate presence of protein in the urine was noted in all high-dose male samples while 3/5 of the control samples were negative and two control samples showed slight protein content. These findings may be related to the test item treatment based on the observed histopathology findings in the kidney samples of high-dose males. The pH of the mid-dose male urine samples was also significantly (p<0.05) lower than control in case of mid dose males. No similar changes in pH or specific gravity were seen in the low dose males, or high-, mid- or low-dose females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Although splayed gait was observed for all high-dose animals after dosing, the signs recovered each day by the afternoon. There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.
No test item related effect was noted in the Irwin test.
No statistically significant or biologically relevant differences were noted during the quantitative assessment of landing foot splay test or grip strength of any dose groups when compared to control during the last week of treatment.
In case of motor activity measurements (SMART), all dose groups of males and females had a normal locomotor activity; in all cases the initial activity was high, with generally reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. The high-dose females showed slightly lower data than control females (due to one control female showing higher than usual activity), but there was no statistical significance between dose group when evaluating the total travelled distance (period of 0-60 minutes). Despite the observation of splayed gait (shortly after treatment each day) the quantitative assessment of activity did not show any clear effect of treatment on movement, although the high-dose males and females had slightly low activity in the first 5-minute period. Taking into account the single female control animal with a high activity, and the lack of a clear dose effect, the single statistical difference was considered as not being a test item related effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed in the kidney of the high-dose males (500 mg/kg bw/day). These findings correlated with increase of organ weights. No test item-related changes were seen in high-dose females.

Minimal to moderate eosinophilic droplets within the cortical tubules (cytoplasm and lumen) were present in 5/5 examined high-dose males. Droplets were mainly multifocal and bilaterally distributed in the cortex. Droplets tended to be rounded form or polyangular of varying size. In 3/5 of these males, minimal to slight focal/multifocal unilateral/bilateral tubule degeneration (with some regenerated
tubules) was developed. The presence of degeneration suggests the effect in high-dose males was adverse.

All other changes are regarded as incidental or a common background and included: minimal to slight extramedullary haematopoiesis in the spleen of five control and seven high-dose animals; minimal inflammatory cell infiltrate in the prostate of two control, one low-dose and one high-dose male; minimal inflammatory cell infiltrate in the heart of one high-dose male; minimal hepatocellular macrovesicular vacuolation in the liver of a control male; severe tubular atrophy and degeneration in the left testis and moderate ductal atrophy in the left epididymis of a high-dose male; and moderate oedema in the lungs of a low-dose female.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis, thyroid gland weights and available histopathology data.
Compared to the relevant control values, statistically significant decrease (by 20%, p<0.01) in the thyroxine hormone (T4) concentration was noted for parental males. However, the mean value of parental males was within the historical control range. The difference was most probably caused by one low value (28.7 ng/mL, outside the historical control range of 34.3-60.7 ng/mL) in the high-dose group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect of test item on oestrus cycles was noted. Each female selected for the treatment showed acceptable cycles (mean cycle length was in the range of 3.97-4.00 days for each group) before starting the treatment period. There was no effect on test item on the oestrus cycle of females (mean cycle length was in the range of 3.92-4.15 days for each group after the treatment started).

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating, fertility or gestation (summary data are shown in Tables 2-3).

The mating index (males and females) was 100% in all groups. The fertility index was in the normal range (83%-100%) for all males and females. A total of five females were non-pregnant: two females in the control group, two females in the low-dose group and one female in the high-dose group, but this incidence was considered as acceptable The gestation index was 100% in all groups.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation) for all females in the control or test item treated
groups. The mean duration of mating was 2.30, 2.30, 2.25 and 2.82 days in control, low-, mid- and high-dose groups, respectively.

No effect of treatment was noted during the gestation, parturition and post-partum period (Table 4).
The mean length of gestation was 22.40, 22.40, 22.92 and 23.18 days in control, low-, mid- and high-dose groups, respectively. The individual data (in the range of 22-24 days) were in line with the normal physiological range. These data indicated no effect of test item (despite of the statistical significance in the mid-dose group (p<0.05) or high-dose group (p<0.01)).
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant differences were noted (although a slightly lower value was detected in the high-dose group).
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose groups. The observed post-natal and total mortality values were higher in the high-dose group than in the control (due to two litters with full mortality, while only one such a litter was recognized in the control group). However, this difference was considered not clearly showing a test item related effect.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

urinalysis

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Based on the external evaluation, almost all the pups were normal. Clinical signs were observed in two cases. Hyperextension of the left hind limb was noted for a control pup found dead on PND 0. One pup in the high-dose group was cold to touch on PND5, however it was normal by the next day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no clear test item effect on mortality or survival of the pups.
There were no significant differences or effects that could be clearly ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups as detailed in Tables 5 and 6.
In three cases, there were no surviving pups at the end of the observation period: all pups of a control female died by PND5 and all pups of two high-dose females died by PND3 or PND1. The number of viable pups on PND 0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group (Tables 5 and 6), thus there was no treatment-related effect on the viability of pups at those time points (higher percentage of pups born was viable in the test item treated group than in the control group).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls (Table 7).
When evaluated per litter basis, the mean litter body weights on PND 0, PND 4 and PND 13 and body weight gain in the relevant periods showed no toxicologically significant differences compared to controls in the F1 generation. Occasionally, slightly higher or slightly lower values were observed in the low-, mid- and high- dose groups,but without statistical significance and without dose response, thus those values were considered as not being test item related effect. In summary, there were no effects of treatment on pup weights or weight gains.

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when compared to control (as shown in Table 8).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.
There were no effects on the nipple retention: no nipples/areolae were counted for any male pups on PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences on absolute and relative (to body) thyroid gland weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were seen in unscheduled deaths of F1 pups. No test item-related macroscopic findings were recorded in F1 offspring generation euthanized and examined externally at scheduled termination on PND 13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.

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

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

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

Table 2: Summary of reproductive parameters (males)

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of males used for mating	12	12	12	12
Number of successful mating	12	12	12	12
Number of fertile males	10	10	12	11
Male mating index (%)	100	100	100	100
Male fertility index (%)	83	83	100	92

 

Table 3: Summary of reproductive parameters (females)

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of females used for mating	12	12	12	12
Number of sperm-positive females	12	12	12	12
Number of females with no implantation sites	2	2	0	1
Number of pregnant females	10	10	12	11
Pregnant females with live born(s)	10	10	12	11
Pregnant females not delivered living pups	0	0	0	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	83	83	100	92
Female gestation index (%)	100	100	100	100

Table 4: Summary of reproduction parameters

	Dose groups
Parameters	Control	Low	Mid	High	Statistics
Number of evaluated females	10	10	12	11
Number of implantations, mean	14.60	14.90	15.17	12.73	NS
Pre-natal mortality, mean	1.60	0.40	0.33	0.64	NS
Pre-natal mortality (%), mean	10.00	2.41	2.09	10.10	NS
Number of pups born, mean	14.40	14.60	14.92	12.27	NS
Number of live born pups, mean	13.00	14.50	14.83	12.09	NS
Number of living pups on PND13, mean	10.60	12.30	12.58	9.36	NS
Post-natal mortality, mean	0.60	0.20	0.25	1.09	NS
Post-natal mortality (%), mean	12.01	1.21	1.52	21.99	NS
Total mortality, mean	2.20	0.60	0.58	1.73	NS
Total mortality (%), mean	13.88	3.59	3.62	25.27	NS
Duration of pregnancy (days)	22.40	22.40	22.92*	23.18**	DC

 Notes: Data (group mean values) were rounded to two decimal places. Pre-natal mortality was also called “intrauterine mortality” in the Study Plan. Post-natal mortality PND0-13 and total mortality on PND13 are shown in the table. Five females had no implantation sites, they were excluded from calculation. For post-natal mortality and total mortality number calculation the pups killed for blood sampling on PND4 were excluded.
Statistical significance compared to control: * = p<0.05, ** = p<0.01
DC: Duncan’s Multiple Range Test, NS: Statistically not significant compared to control

Table 5:Summary of survival (offspring)

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose	Statistics
Number of evaluated litters	10	10	12	11
Number of implantations, mean	14.60	14.90	15.17	12.73	NS
Number of pups born, mean	14.40	14.60	14.92	12.27	NS
Number of live born pups, mean	13.00	14.50	14.83	12.09	NS
Post-natal mortality on PND0-4, mean	0.40	0.10	0.25	0.91	NS
Post-natal mortality on PND0-4 (%), mean	7.96	0.63	1.52	21.10	NS
Total mortality on PND4, mean	2.00	0.50	0.58	1.55	NS
Total mortality on PND4 (%), mean	12.55	3.00	3.62	24.39	NS
Post-natal mortality on PND0-13, mean	0.60	0.20	0.25	1.09	NS
Post-natal mortality on PND0-13 (%), mean	12.01	1.21	1.52	21.99	NS
Total mortality on PND13, mean	2.20	0.60	0.58	1.73	NS
Total mortality on PND13 (%), mean	13.88	3.59	3.62	25.27	NS
Survival index on PND0	91.16	99.41	99.54	93.18	NS
Sex ratio (%) on PND0	54.75	47.56	50.45	48.03	NS
Survival index on PND4	92.04	99.38	98.48	78.90	NS
Sex ratio (%) on PND4	54.53	47.98	50.67	51.62	NS
Survival index on PND13	89.17	99.33	100.00	98.59	NS
Sex ratio (%) on PND13	47.57	46.83	48.82	46.86	NS

Notes: Data (group mean values) were rounded to two decimal places. Sex ratio means shows the ratio of females. Survival index was calculated in comparison with the end of previous period (on PND0 was compared to the number of pups born, on PND4 it was compared to the number of live born pups, on PND13 it was compared to the number of pups after culling on PND4).
NS: Statistically not significant compared to control

 

Table 6:Summary of mortality (offspring)

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose	Statistics
Number of evaluated litters	10	10	12	11	NA
Number of pups born	144	146	179	135	NS
Number of cannibalized pups	0 / 0	0 / 0	0 / 0	0 / 0	NS
Number of autolyzed pups	1 / 1	1 / 1	1 / 1	2 / 2	NS
Number of stillborn pups	9 / 1	0 / 0	0 / 0	0 / 0	NS
Number of live born pups	134	145**	178**	133**	CH
Number of found dead pups (born alive)	4 / 1	0 / 0	0 / 0	0 / 0	NS
Number of living pups on PND0	130	145**	178**	133**	CH
Number of cannibalized pups (PND0-13)	6 / 4	2 / 2	2 / 2	7 / 3	NA
Number of autolyzed pups (PND0-13)	--	--	1 / 1	5 / 3	NA
Number of found dead, intact pups (PND0-13)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Total number of pups died (born alive)	10 / 4	2 / 2	3 / 3	12 / 5	NS
Culled for blood sampling on PND4	18 / 9	20 / 10	24 / 12	18 / 9	NS
Number of litters with living pups on PND13	9	10	12	9	NA
Number of viable pups on PND13	106	123	151	103	NS

Notes: Mortality numbers mean number of pups / number of affected litters.
PND0-13 means the lactation period, counted from the end of working day on PND0.
There were no living pups in litter #1510 by PND5, in litter #4501 by PND3 and in litter #4504 by PND1.
Statistical significance compared to control: * = p<0.05, ** = p<0.01
NA: Not applicable, CH: Chi square test, NS: Statistically not significant compared to control

Table 7: Body weight data (offspring)

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose	Statistics
Number of evaluated litters	10	10	12	11
Number of viable pups (PND0)	130	145	178	133	NA
Mean litter body weight (PND0), g	6.650	6.534	6.784	6.812	NS
Number of viable pups (PND4)	126	144	175	123	NS
Mean litter body weight (PND4), g	10.245	10.580	11.078	10.298	NS
Mean litter body weight gain (PND0-4), g	3.507	4.048	4.296	3.517	NS
Number of viable pups (PND13)	106	123	151	103	NS
Mean litter body weight (PND13), g	29.662	30.209	30.085	28.485	NS
Mean litter body weight gain (PND4-13), g	18.888	19.577	18.975	18.153	NS
Mean litter body weight gain (PND0-13), g	23.015	23.672	23.282	21.702	NS

Notes: Body weight / body weight gain data (litter mean values) were rounded to three decimal places.
All living pups of two high-dose females (#4501 and #4504) died before PND4, therefore PND4 and PND13 results for high-dose group were calculated from 9 litters. Similarly, all pups of a control female (#1510) died by PND5, therefore PND13 results for control group were calculated from 9 litters.
NS: Statistically not significant, NA: Not applicable

Table 8: Anogenital distance

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose	Statistics
Male pups
Number of evaluated male pups	54	65	78	54	NS
Anogenital distance, litter mean of males (mm)	3.67	3.67	3.69	3.79	NS
Minimum / Maximum value, litter mean (mm)	3.4 / 3.8	3.6 / 3.8	3.4 / 3.9	3.3 / 4.1
Female pups
Number of evaluated female pups	52	58	73	49	NS
Anogenital distance, litter mean of females (mm)	1.69	1.65	1.67	1.57	NS
Minimum / Maximum value, litter mean (mm)	1.4 / 1.9	1.5 / 2.0	1.5 / 1.9	1.1 / 2.2

Notes: Data (group mean or litter mean values) were rounded to two decimal places.
NS: Statistically not significant compared to control

 

Applicant's summary and conclusion

Conclusions:

In an oral gavage study conducted to OECD TG 422 and to GLP (reliability score 1) the NOAEL relating to repeated dose (parental systemic) effects was 150 mg/kg bw/day based on clinical signs, urinary analysis, kidney organ weight and histopathology. The NOAEL relating to reproductive toxicity was at least 500 mg/kg bw/day, as no significant adverse effects were observed in rats. The NOAEL for the F1 generation was 500 mg/kg bw/day because there were no adverse effects on the F1 offspring body weight, viability, clinical signs, physical or sexual development. No test item related macroscopic findings were recorded for F1 pups at necropsy.