Trisiloxane, 3-ethyl-1,1,1,3,5,5,5-heptamethyl-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the results of a screening study performed according to OECD guideline 421 and under GLP principles, the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/day, based on decreased food consumption during lactation, respectively. The NOAEL for males was 600 mg/kg bw/day, as no adverse effects were observed up to and including the highest dose tested.

The NOAEL for reproductive toxicity was 50 mg/kg bw/day, based on increased gestational length, post-implantation loss, increased total litter loss post-partum. 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March to 02 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Wistar Han:RccHanTM:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: 12 weeks at start of treament
Weight: male 311-359 g, female 185-220 g at start of treatment
Acclimatisation: 6 days
Housing: initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet) ad libitum
Water: mains drinking water was supplied from polycarbonate bottles attached to the cage
Environmental enrichment: provided in the form of wooden chew blocks and cardboard fun
tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
Environmentmental conditions: Temperature: 22 ± 3 °C
Humidity: 50 ± 20%
Ventilation: 15 air changes/h
Lighting: 12 h light/dark cycles

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was administered once daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of corn oil.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Due to an error in the formulation instructions animals receiving 50 or 200 mg/kg bw/day received a higher than intended dosage (100 and 300 mg/kg bw/day) on Day 15 of the study only. The higher than intended dosage for these animals on this single occasion was considered to have not affected the overall purpose and integrity of the study.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to thirteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated analytical procedure was established for the determination of test substance in vehicle. The homogeneity and stability of the test formulations was determined at nominal concentrations of 12.5 and 150 mg/mL. The concentration of test substance in the formulations was determined at intervals throughout the study

Duration of treatment / exposure:

Males: at least Day 1 to Day 44 of the study
Females: Day 1 to Day 4 post partum

Frequency of treatment:

Daily

Details on study schedule:

i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 45.
vi. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized.
The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories

Parental animals: Observations and examinations:

Clinical Observations:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.
Body weights:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food consumption:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water consumption:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.

Pregnancy and parturition:
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays.
The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages
of the spermatogenic cycle. The examination was conducted in order to identify treatment-related e
ffects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic g
erm cells and sloughing of spermatogenic cells into the lumen.
Any cell or stage-specificity of testicular findings was noted.

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded.
Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Postmortem examinations (parental animals):

Necropsy:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ weights:
The epididymides and testes were removed from terminal kill adult males dissected free from fat

Histopathology:
Samples of the following tissues were preserved from all animals from each dose group, in buffered
10% formalin, except where stated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries, Testes, Mammary gland (females only), Uterus/Cervix, Macroscopic abnormalities, Vagina, Pituitary preserved in Modified Davidsons fluid. The tissues from control and 600 mg/kg bw/day dose group animals and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 600 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell or stage-specificity of testicular findings were noted.
Since there were no indications of treatment-related changes in the tissues scheduled to be retained for this study, examination was not extended to include animals in the low and intermediate groups.

Statistics:

Analysis of:
Body Weight, Body Weight Change, Food Consumption , Pre- Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights using the decision tree (ProvantisTM Tables and Statistics Module):
Data transformations performed using most suitable method.
Homogeneity of variance from means using Bartlett’s test.
Intergroup variances using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data analyzed to find the lowest treatment level that showed a significant effect using Williams Test or Shirley Test . If no dose response was found but the data shows nonhomogeneity of means, data analyzed by a stepwise Dunnett’s or Steel test to determine significant difference from control. Where data unsuitable for these analyses, pair-wise tests using Student t-test or MannWhitney U test.
Data not analyzed by Provantis assessed separately using the R Environment for Statistical Computing. Initially, distribution of the data by Shapiro-Wilk normality test, then assessment of homogeneity of data using Bartlett’s test. Where appropriate, parametric analysis of the data incorporating ANOVA, if significant, followed by pair-wise comparisons Dunnett’s test. Where parametric analysis of data unsuitable, non-parametric analysis of the data performed incorporating Kruskal-Wallis test which if significant followed by Mann-Whitney "U" test. Dose response relationships investigated by linear regression. Where data unsuitable for these analyses pair-wise tests performed using Student t-test or Mann-Whitney U test Reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting)
analyzed using non-parametric analyses.
Probability values (p):
p<0.01 **
p<0.05 *
p>0.05 (not significa

Reproductive indices:

Mating performance and fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals paired/Number of animals mated x 100
Pregnancy Index (%) = Number of animals mated/Number of pregnant females x 100
Gestation and parturition data:
parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of pregnant females/Number of females delivering live offspring x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated
for each female/litter as follows:
Pre–implantation loss (%) = Number of corpora lutea - Number of implantation sites/ Number of corpora lutea x100
Post–implantation loss (%) = Number of implantation sites - Total number of offspring born/Number
of implantation sites x100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100
iii. Sex Ratio (% males) Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Total number of male offspring/ Number of offspring x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 600 mg/kg bw/day, increased post-dosing salivation was apparent for all animals with this sign first being apparent on Day 19 and 20 for males and females respectively. Similar increased post-dosing salivation was apparent at a lower frequency for four males and seven females at 200 mg/kg bw/day. Such increased post-dosing salivation is often observed when dosing via the oral gavage route and, at the frequency observed during this study, was considered to reflect slight distaste or irritancy of the test item formulations.
There were no other clinical signs observed for either sex at 200 or 600 mg/kg bw/day throughout the study.
There were no clinical signs apparent throughout the study for either sex at 50 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

One control female and two females at 600 mg/kg bw/day were killed before their scheduled termination. Control female number 15 was killed for animal welfare considerations after showing decreased respiration rate and noisy, laboured and gasping respiration on Day 4. Necropsy revealed oillike fluid (considered to be vehicle) in the stomach and gaseous distension of the stomach. This death was considered to be a result of an intubation error during dose administration.
Female 93, receiving 600 mg/kg bw/day, showed hunched posture, pilo-erection and appeared to be having difficulties with parturition, with one dead pup appearing to be stuck in the vagina, on Day 44 of the study. The animal was killed for animal welfare considerations and necropsy confirmed the earlier observation for the vagina, although no other macroscopic observations were apparent.
Female 95, receiving 600 mg/kg bw/day, showed hunched posture, pilo-erection, staining around the eyes, tiptoe gait and lethargy around the time of expected parturition (Day 39 of the study) and was killed for animal welfare considerations. Necropsy revealed 12 dead foetuses in the uterus, but no other macroscopic observations were apparent.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 600 mg/kg bw/day, food consumption during lactation (Days 1-4) was slightly lower than control for females rearing offspring to termination but assessment of food consumption has to be treated with caution due to the low group size. The difference in food intake was statistically significant when these data were supplemented with data from females failing to rear offspring to scheduled termination and probably reflects the lack of litter demand for these females.

Water consumption and compound intake (if drinking water study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animal 21 (control) showed signs of a false pregnancy with prominent corpora lutea, mucinification of the vagina and secretory activity in the mammary gland. A cyst was also present in the cervix. This female had successfully given birth to, and maintained six offspring to scheduled necropsy. Mammary Gland
At 600 mg/kg bw/day fewer females showed signs of lactation and this is considered to be due to the increased number of total litter losses observed.
Liver
Mild centrilobular hepatocyte hypertrophy was noted in 3/4 livers examined from Group 4 females. This finding is suggestive of an adaptive response due to enzyme induction in the liver. There were no treatment-related pathologic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating:
Mating performance, as assessed by pre-coital interval, was unaffected by treatment

Fertility:
Fertility, as assessed by pregnancy rate, was unaffected by treatment at 50, 200 or 600 mg/kg bw/day.
At 200 mg/kg bw/day, mating resulted in 4/12 females failing to achieve pregnancy, however, as 11/12 females at 600 mg/kg bw/day achieved pregnancy, this lower than expected pregnancy rate was considered to be incidental and unrelated to treatment.
Gestation length:
For females at 600 mg/kg bw/day, there was a shift towards a longer gestation period compared to control and there appeared to be an association with this longer gestation length and subsequent total litter loss post partum. Gestation length appeared to be unaffected by treatment at 50 or 200 mg/kg bw/day, although one female at 200 mg/kg bw/day showed a long gestation period of 24½ days and subsequently showed total litter loss post partum.
Litter responses:
At 600 mg/kg bw/day, two females were killed around the time of expected parturition and five females showed total litter loss post partum before scheduled termination resulting in only four females successfully maintaining a litter to scheduled termination on Day 5 of lactation/age. Interpretation of results at this dosage has to be treated with caution in view of the small group size.
At 200 mg/kg bw/day, only eight females achieved pregnancy and one female subsequently showed total litter loss post partum, resulting in seven females maintaining a litter to scheduled termination.
At 50 mg/kg bw/day, eleven females achieved pregnancy but one female was not observed to give birth, resulting in ten females maintaining a litter to scheduled termination. A total of eleven control females maintained a litter to scheduled termination.
Litter size and viability:
The following assessment is generally based on litters maintained to scheduled termination but, consideration of data from litters showing total litter loss post partum, was also included. At 600 mg/kg bw/day, mean corpora lutea count was slightly lower than control for females maintaining their offspring to termination (Day 5 of age), but when all littering females were taken into account, mean corpora lutea was similar to control. This finding was therefore considered to be incidental and unrelated to treatment. There was no obvious effect of treatment on pre-implantation loss. Although no post-implantation loss was apparent for females that maintained their offspring to termination, postimplantation loss was higher than control when all littering females were taken into account. Live birth
index and viability index for females maintaining their offspring to termination was only slightly lower than control, however, when all littering females were taken into account offspring survival at these time points was clearly lower than control. Lower mean litter size compared to control was apparent on Day 1, and attained statistical significance on Day 4; litter size was even lower when all littering females were taken into account. There was no obvious effect on sex ratio throughout, indicating that there was no selective effect on survival for either sex.
Overall a total of five females showed total litter loss post partum and additionally two dams were terminated around the time of parturition. At 200 mg/kg bw/day, mean corpora lutea count, preimplantation loss and mean number of implantations appeared unaffected by maternal treatment. Post-implantation loss for all littering females was slightly higher than control, but this was principally
due to the one female that showed total litter loss post partum, as post implantation loss was lower
than control for females maintaining their offspring to termination (Day 5 of age).
Subsequent litter size at birth, live birth index and litter size on Day 1 and viability index and litter size on Day 4 of age was unaffected by maternal treatment for females that maintained offspring to termination (Day 5 of age). Viability index on Day 4 was lower than control when the female that showed total litter loss post partum was included. While values for this female appear atypical when compared with the rest of the litters at this dosage, there were a number of similar females at the higher dosage of 600 mg/kg bw/day, and therefore an association with treatment could not be discounted.
At 50 mg/kg bw/day, there was no effect of maternal treatment on mean numbers of corpora lutea or implantations, pre- or post- implantation loss, litter size at birth, Day 1 or Day 4 or on offspring survival indices at Day 1 or Day 4 of age.
Offspring growth and development:
At 600 mg/kg bw/day, there no obvious effect of treatment on mean offspring body weight on Day 1; mean values were marginally higher than control probably reflecting the longer gestation period and therefore relative older post-coital age of the offspring at this dosage. Mean litter weight was statistically significantly lower than control reflecting the lower litter size compared to control. Subsequent body weight gain was slightly lower than control resulting in a marginally lower mean body weight for surviving offspring by Day 4. Differences from control in mean litter weight were more pronounced by Day 4 reflecting both the lower offspring body weight gain and lower litter size at this dosage. Additionally, at 600 mg/kg bw/day, there was a higher incidence, compared to control, of offspring showing
clinical signs indicating that the offspring were failing to thrive; small, cold, weak and no milk visible in stomach and an increase in the number of dead or missing
offspring. The mean success rate at assessment of surface right for the offspring at Day 1 of age was slightly lower than control at this dosage.
At 200 mg/kg bw/day, there was no obvious effect of maternal treatment on mean offspring body weight on Day 1. Subsequent body weight gain was slightly lower than control, but this resulted in only a marginally lower body weight at Day 4 and all differences from control failed to attain statistical significance. Additionally, there was a higher incidence of offspring showing clinical signs of small and/or no milk visible in stomach. There was no obvious effect on the mean success rate at assessment of surface righting for the offspring at Day 1 of age.
At 50 mg/kg bw/day, there was no obvious effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4. There was no obvious effect of treatment on the incidence of clinical signs for offspring on the study or on the mean success rate at assessment of surface right for the offspring at Day 1 of age.

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects up to and including the highest dose level (600 mg/kg bw/day)

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased food consumption during lactation

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg/kg bw/day, there was a higher incidence, compared to control, of offspring showing clinical signs indicating that the offspring were failing to thrive; small, cold, weak and no milk visible in stomach and an increase in the number of dead or missing offspring. The mean success rate at assessment of surface right for the offspring at Day 1 of age was slightly lower than control at this dosage.
At 200 mg/kg bw/day there was a higher incidence of offspring showing clinical signs of small and/or no milk visible in stomach. There was no obvious effect on the mean success rate at assessment of surface righting for the offspring at Day 1 of age.
At 50 mg/kg bw/day there was no obvious effect of treatment on the incidence of clinical signs for offspring on the study or on the mean success rate at assessment of surface right for the offspring at Day 1 of age.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

For females maintaining their offspring to termination at 600 mg/kg bw/day, mean corpora lutea count was slightly lower than control but, as the mean number of corpora lutea for all littering females at this dosage was similar to control, this finding was considered incidental and unrelated to treatment. There was no obvious effect of treatment on pre-implantation loss. No post-implantation
losses were apparent for females that maintained their offspring to termination and live birth index and viability index for these females was only slightly lower than control; however, higher mean postimplantation loss and lower live birth and viability indices were apparent when all littering females were included. Mean litter size was lower than control on Day 1 and Day 4 but sex ratio appeared unaffected throughout, indicating that there was no selective effect on survival for either sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 600 mg/kg bw/day, there no obvious effect of treatment on mean offspring body weight on Day 1;
mean values were marginally higher than control probably reflecting the longer gestation period and therefore relative older post-coital age of the offspring at this dosage. Mean litter weight was statistically significantly lower than control reflecting the lower litter size compared to control. Subsequent body weight gain was slightly lower than control resulting in a marginally lower mean body weight for surviving offspring by Day 4. Differences from control in mean litter weight were more pronounced by Day 4 reflecting both the lower offspring body weight gain and lower litter size at this dosage. At 200 mg/kg bw/day, there was no obvious effect of maternal treatment on mean offspring body weight on Day 1. Subsequent body weight gain was slightly lower than control, but this resulted in only a marginally lower body weight at Day 4 and all differences from control failed to attain statistical significance.
At 50 mg/kg bw/day, there was no obvious effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4.

Histopathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings apparent for offspring dying during the study and offspring at termination did not indicate any underlying effect of maternal treatment on offspring development and were consistent with the in-life clinical signs apparent for the offspring.

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of a screening study performed according to OECD guideline 421 and under GLP principles, the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/day, based on decreased food consumption during lactation, respectively. The NOAEL for males was 600 mg/kg bw/day, as no adverse effects were observed up to and including the highest dose tested.

The NOAEL for reproductive toxicity was 50 mg/kg bw/day, based on increased gestational length, post-implantation loss, increased total litter loss post-partum. The NOAEL for offspring toxicity was found to be 50 mg/kg bw/day, based on reduced body weight and body weight gain of the pups, and clinical signs evident of indicating failure to thrive.

Executive summary:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 200 and 600 mg/kg bw/day.
A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil) over the same treatment period. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.
Pairing of animals within each dose group was undertaken on a one male: one female basis within each
treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their
offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on
all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 45 with termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues and macroscopic abnormalities was performed.
There were 3 mortalities during the study, one control female was killed for animal welfare reasons following mis-dosing and two females from the top dose group were killed for animal welfare reasons at or around parturition, one of these females was shown to have a dead pup stuck in the vagina with no other macroscopic observations and the other was found to have 12 dead fetuses in the uterus with no other macroscopic observations apparent.
Clinical observations were limited to increased post dose salivation at intervals in the high and mid dose
groups. There were no effects on bodyweight or food consumption except in the high dose females during lactation Days 1 -4 which was slightly lower than control. There were no effects on mating performance or fertility.
Treatment related effects were observed on gestation length, litter size and viability. In the high dose group there was a tendency towards longer gestation period which was apparently associated with
subsequent total litter loss post partum. One female in the mid dose group also showed an exended gestation period and subsequent total litter loss post partum. One female in the low dose group was not observed to give birth.
In the high dose group 4 (of 11), in the mid dose group 7 (of 8) and the low dose 10 (of 11) successfully maintained a litter to scheduled termination, with the low dose group observation being due to the female not observed to give birth. When all littering females were taken into account in the high dose group, post implantation losses were higher than control and live birth index, mean litter size and viability index were all lower than control.
In the mid dose group, when all litering females were taken into account, post implantation loss was slightly higher than control and viability index on Day 4 was slightly lower than control.
At the low dose there was no effect of maternal treatment on mean numbers of corpora lutea or implantations, pre- or post- implantation loss, litter size at birth, Day 1 or Day 4 or on offspring survival indices at Day 1 or Day 4 of age. However, one dam in this group failed to give birth.
At the high dose, mean litter weight was statistically significantly lower than control reflecting lower litter size compared to control. Subsequent body weight gain was slightly lower than control resulting in a marginally lower mean body weight for surviving offspring by Day 4. Lower mean litter weight on Day 4 reflected both lower offspring body weight gain and lower litter size at this dosage compared to control. Additionally, there was a higher incidence of offspring showing clinical signs indicating that the offspring were failing to thrive; small, cold, weak and no milk visible in stomach and an increase in the number of dead or missing offspring compared to control. The mean success rate for surface righting at Day 1 of age was slightly lower than control. At the mid dose, offspring body weight gain from Day 1 was slightly lower than control, but resulted in only a marginally lower body weight at Day 4. However, there was a higher incidence of offspring showing clinical signs of small and/or no milk visible in stomach. At the low dose, there was no obvious effect of treatment on offspring body weight or surfacing righting ability on Day 1 and the incidence of clinical signs and body weight gain to Day 4 of age. At the high dose, necropsy revealed enlarged livers for four females killed at scheduled termination, there were no necrosy findings for dying offspring.
At the mid and high dose levels absolute and body weight-relative testes weights were statistically significantly higher than control, no statistically significant differences from control were apparent for epididymal weights. At the low dose no significant differences from control were observed.
Mild centrilobular hepatocyte hypertrophy was noted in 3/4 livers examined from high dose females. This finding is suggestive of an adaptive response due to enzyme induction in the liver.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the results of a screening study performed according to OECD guideline 421 and under GLP principles, the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/day, based on decreased food consumption during lactation, respectively. The NOAEL for males was 600 mg/kg bw/day, as no adverse effects were observed up to and including the highest dose tested.

The NOAEL for offspring toxicity was found to be 50 mg/kg bw/day, based on reduced body weight and body weight gain of the pups, and clinical signs evident of indicating failure to thrive.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

An OECD 421 study was conducted in which the test item was administered by gavage to three groups, each of twelve male and twelve female

Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 200 and 600 mg/kg bw/day.

At the high dose, mean litter weight was statistically significantly lower than control reflecting lower litter marginally lower mean body weight for surviving offspring by Day 4. Lower mean litter weight on Day 4 reflected both lower offspring body weight gain and lower litter size at this dosage compared to control. Additionally, there was a higher incidence of offspring showing clinical signs indicating that the offspring were failing to thrive; small, cold, weak and no milk visible in stomach and an increase in the number of dead or missing offspring compared to control. The mean success rate for surface righting at Day 1 of age was slightly lower than control.

At the mid dose, offspring body weight gain from Day 1 was slightly lower than control, but resulted in only a marginally lower body weight at Day 4. However, there was a higher incidence of offspring showing clinical signs of small and/or no milk visible in stomach.

At the low dose, there was no obvious effect of treatment on offspring body weight or surfacing righting ability on Day 1 and the incidence of clinical signs and body weight gain to Day 4 of age.

Justification for classification or non-classification

In the OECD 421 study which was conducted in Wistar Han™:RccHan™:WIST strain rats, there was a dose-related incidence of increased gestation length, litter loss, and the failure of the surviving offspring to thrive at the 200 and 600 mg/kg bw/day dose levels, with no effects observed at the lowest dose of 50 mg/kg bw/day.

Specific positive findings included:

• Increased gestation length: There was a shift towards a longer gestation period compared to control (and subsequent total litter loss post-partum) at 600 mg/kg bw/day, with a similar outcome seen in one female at 200 mg/kg bw/day. 
• Litter loss: The need to sacrifice  in extremis two high dose females close to the time of expected parturition, with one female showing twelve dead fetuses in the uterus, provides further evidence of an adverse effect on pregnancy from treatment with the test item. There was also an observed association between increased gestation length and total litter loss post-partum (five dams surviving to parturition lost their entire litters post-partum), suggesting that delaying the onset of parturition could have been a factor in the poor survival of pups at or around the time of birth.
• Pup failure to thrive: In the surviving offspring, an increased incidence of failing to thrive (small, cold, weak and no milk visible in the stomach and an increase in the number of dead or missing offspring compared to control) was seen at 600 mg/kg bw/day, with a similar finding noted in offspring at 200 mg/kg bw/day.   

In the OECD 417 pharmacokinetic study conducted using the same batch of test material in the same strain of rat, the kinetics of [14C]Y-14877 were determined following 13-daily oral doses of non-radiolabelled Y-14877 followed by a single oral dose of [14C]Y-14877 on Day 14 at 50, 200 and 600 mg/kg bw/day (same dose levels used in OECD TG 421 study). 

 

No clinical signs attributable to the administration of [14C]Y-14877 were observed during the course of the study. The rate of absorption of radioactivity was not linear with increasing dose level. Circulating Y-14877 was metabolized to significantly more polar components at all dose levels, though metabolic profiles indicated that the biotransformation pathway of Y-14877 was not affected by dose level. The rate of biotransformation based on half-lives of radioactivity was not linear in whole blood or plasma with increasing dose. Excretion in urine decreased and in feces increased with increasing dose level in a nonlinear fashion. Excretion in expired air did not show a clear (linear) pattern with increasing dose. In summary, the toxicokinetics of Y-14877 following repeated oral dosing provide adequate information on its absorption, distribution, biotransformation (i.e. metabolism) and excretion and suggests these processes are not linear with increasing dose. The kinetically derived maximum dose appears to be below 200 mg/kg bw/day.

 

The results from the pharmacokinetic study demonstrate that the adverse reproductive effects (dose-related incidence of increased gestation length, litter loss, and the failure of the surviving offspring to thrive) observed at 200 and 600 mg/kg bw/day in the OECD TG 421 study only occurred in the presence of non-linear / saturation kinetics. Further, the report specifically concludes that the kinetically derived maximum dose (KMD) appears to be below 200 mg/kg bw/day. No effects were observed at the lowest dose of 50 mg/kg bw/day.

 

ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a (p435) states that “When conducting repeated dose toxicity studies, it is necessary to ensure that the observed treatment-related toxicity is not associated with the administration of excessive high doses causing saturation of absorption and detoxification mechanisms. The results obtained from studies using excessive doses causing saturation of metabolism are often of limited value in defining the risk posed at more relevant and realistic exposure levels where a substance can be readily metabolised and cleared from the body. It is suggested that a key element in designing better repeated dose toxicity studies is to select appropriate dose levels based on results from useful metabolic and toxicokinetic investigations.”

On this basis, the results obtained in the OECD421 study with doses above the kinetically derived maximum dose (<200mg/kg bw/day) cannot be considered reliable for purposes of hazard identification or risk assessment. Consequently, there is insufficient reliable information on adverse effects to fertility or developmental toxicity to justify a hazard classification in accordance with Regulation (EC) No 1272/2008 (as amended).