Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step (if any): No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: for 14 days
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance administered neat. No analytical verification conducted as not needed.
Duration of treatment / exposure:
Main and Recovery Males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.
Frequency of treatment:
7 days per week
Details on study schedule:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: hree times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pre-test period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken from Main females to determine the stage of estrous. This was done for all Main females, except for females with total litter loss.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
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, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanised following completion of the mating period (a minimum of 28 days of administration)
- Maternal animals: All surviving animals that delivered were euthanised on PND 14-16.

GROSS NECROPSY
- Gross necropsy consisted of a full post mortem examination, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [# 1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13-15 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of sex determination both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index, precoital index, gestation index, fertility index, duration of gestations, post-implantation survival index, live birth index, percentage of live males at first litter check, percentage of live females at first litter check, viability index, lactation index.
Offspring viability indices:
viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg bw/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery 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):
At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg be/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, now achieving a level of statistical significance in 250 mg/kg be/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.

The group mean data for motor activity, comprising total movements and ambulations, showed a large variation between the groups in both males and females.

Whereas the group mean values for total movements and ambulations in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulations were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulations, when compared to controls.

The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulations in the latter animals, but not achieving a level of statistical significance in comparison with controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
All females had regular cycles of 4 days. During the mating period, extended di-estrus occurred in two control females (nos.58 and 59) until mating. Both females delivered normal litters. Extended di-estrus was also observed in one 250 mg/kg bw/day treated female no.88 during the mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testis and epididymis weight were unchanged following treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not to be affected by treatment. One 250 mg/kg bw/day treated female showed no evidence of mating, which was considered an incidental finding and not related to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced motor activity in main and recovery females at 250 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect on reproductive parameters
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
A pale and cold appearance and a wound on the right leg was observed for the pup of control litter no.51 before it went missing on Day 5.
From Day 9-10 of lactation onwards all pups in litter no.93 (at 250 mg/kg bw/day) showed alopecia.
The nature and incidence of these and other clinical signs remained within the range considered normal, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg bw/day) was found dead on Day 2. For control females, 6 pups (out of three litters, nos.51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At birth, body weights of male and female pups in litters at 250 mg/kg bw/day were higher (slightly over 10%) in comparison with that in the other groups, achieving levels of statistical significance when compared to controls. During lactation this difference remained, but had reduced to less than 10% on lactation Day 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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for all stillborn pups in litter 84.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, were related to the death or confirmed the in-life observation of the pup.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to 250 mg/kg bw/day had no effect on areola/nipple retention.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed at any dose level.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, no reproductive or developmental toxicity was observed up to the maximum dose of 250 mg/kg bw/day. A NOAEL of ≥250 mg/kg bw/day Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers was established for reproductive toxicity.