Bis(triethoxysilylpropyl)amine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr - 30 Sep 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(triethoxysilylpropyl)amine

EC Number:

236-818-1

EC Name:

Bis(triethoxysilylpropyl)amine

Cas Number:

13497-18-2

Molecular formula:

C18H43NO6Si2

IUPAC Name:

bis(triethoxysilylpropyl)amine

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: CRL: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 11 weeks old males; 13 - 14 weeks old females
- Weight at study initiation: 271 - 306 g males; 200 - 238 g female
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to five animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm), as were males during the post-mating period. Recovery males and females were housed as such during the entire study period.
During the mating phase, Main males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating and lactation phases, Main females were individually housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: SM R/M-Z (pelleted) from SSNIFF® Spezialdiäten GmbH provided ad libitum
- Water: Municipal tap water provided ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
- Periodic analysis of the water was performed. For the diet, results of analysis for nutritional components and environmental contaminants were provided by the Supplier. There were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22
- Humidity (%): 46 to 79
- Air changes (per hr): At least 10
- Photoperiod: 12-hours light and 12-hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: dried and de-acidified corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The first day of dosing was designated as Day 1. The dose volume for each animal was based on the most recent body weight measurement. The dose formulations were stirred continuously during dose administration and doses were given using a plastic feeding tube.

VEHICLE
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Supplier: SigmaAldrich, Steinheim, Germany

Details on mating procedure:

- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Maximum of 14 consecutive days
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0 post-coitum
- In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible) will be used for re-mating.
- After successful mating each pregnant female was individually housed in Makrolon (MIII type) plastic cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations was performed at the Analytical Department of the Test Facility using a validated UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) method.
Accuracy and homogeneity were determined for all formulations prepared for use in Week 1 and for Group 2 formulation prepared for use in Week 5.
Accuracy
The concentrations analysed in the formulations of Group 2 (12.5 mg/mL), Group 3 (37.5 mg/mL) and Group 4 (125 mg/mL) were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90-110% of target concentration).
No test item was detected in the Group 1 formulation.
Homogeneity
The formulation of Group 4 was homogeneous (i.e. coefficient of variation ≤ 10%). The coefficient of variation for Group 2 analysed on 12 May 2021 was above the criterion (i.e. 12%). An Out of Specification investigation was performed to determine the cause of the relatively high variation, but no analytical reason could be identified. Therefore, it was decided to perform additional analysis of Group 2 on 11 Jun 2021 (Week 5). The additional analysis of the Group 2 revealed accuracy and homogeneity in agreement with targets (i.e. mean sample concentration results were within or equal to 90-110% of target concentration and coefficient of variation ≤ 10%).

Duration of treatment / exposure:

- Main and Recovery males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period (for Main males) up to and including the day before scheduled necropsy of Main males.
- Main females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete oestrous cycles), 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 were dosed during littering.
- Recovery females: during the same period as Main females, until at least the day before the first scheduled necropsy of Main females.

Frequency of treatment:

Once daily

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:

10 males and 10 females for the control, low-, mid-, and high-dose groups; 5 males and 5 females in the control and high-dose recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of the test substance in rats, and in an attempt to produce graded responses to the test item. In the Dose Range Finder study, mortality was observed at 1000 mg/kg bw/day in both sexes. At 250 and 500 mg/kg bw/day, lower body weight gain and food consumption were observed in females. Hunched posture, erected fur and abnormal breathing sounds were noted at 500 mg/kg bw/day. At 250 mg/kg bw/day, hunched posture was noted. Based on these results, dose levels for the Main study were set on 50, 150 and 500 mg/kg bw/day for the low, mid and high dose groups, respectively.
- Post-exposure recovery period in satellite groups: 14 days (males) and 20 days (females). For logistical reasons, the Recovery Period for females was 20 days. As the animals were still allowed at least 14 days of recovery, this was considered to have no impact on the study and interpretation of the results.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily, up to the day prior to necropsy. Mortality was observed at least twice daily beginning upon arrival through termination/release.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration of the test item and weekly during the Treatment and Recovery Period.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1 of treatment (prior to dosing) and weekly thereafter; Mated Main females: 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 for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No (food consumption was quantitatively measured per cage).

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: 5/sex/group for selected animals
- Parameters checked in Tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
On the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/sex/group for selected animals
- Parameters checked in Table 3 were examined.

SERUM HORMONES: Yes (Thyroxine (T4))
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: Males from all dose groups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected 5 Main males and all Recovery males were tested once during Week 4 of treatment and the selected 5 Main females were tested once during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested.
- Dose groups that were examined: All
- Battery of functions tested included hearing ability, pupillary reflex, static righting reflex and grip strength

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and for Main females during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

If a treatment-related effect on oestrous cycle was suspected based on the data obtained during the first 14 days of treatment, daily vaginal lavage was performed for Recovery females during the treatment-free recovery period to investigate reversibility of this effect.

End of Treatment - on the day of necropsy, a vaginal lavage was also be taken from Main females to determine the stage of oestrus. This was done for all Main females, except for females that have to be euthanized in extremis or die spontaneously.

Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed for all selected control and Group 4 (500 mg/kg bw/day) males, and all Main males that failed to sire. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: yes
- If yes, maximum of 8 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Mortality (the number of live and dead pups was determined on PND 1 and daily thereafter), clinical observations (at least once daily, up to the day prior to necropsy), individual body weights (on PND 1, 4, 7 and 13), sex (on PND 1 and 4), anogenital distance (AGD, on PND 1) and areola/nipple retention (all male pups in each litter on PND 13)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined

Postmortem examinations (parental animals):

SACRIFICE
- Main males (which sire or fail to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Recovery males: After the recovery period of at least 14 days, which is at least 14 days after the scheduled necropsy of Main males.
- Main females which delivered: PND 14 - 16.
- Main females which failed to deliver: With evidence of mating, post-coitum Days 25 - 27. Without evidence of mating, approximately 24 - 26 days after the last day of the mating period.
- Recovery females: After the recovery period of at least 14 days, which is at least 14 days after the first scheduled necropsy of Main females.

GROSS PATHOLOGY and ORGAN WEIGHTS: Yes (see Attachment C, attached background material)

HISTOPATHOLOGY: Yes (see Attachment C and Table 4)

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded.
- All remaining pups were euthanized on PND 14-16. Sex was determined 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. Any external abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection), and was be preserved in 10% buffered formalin.

GROSS NECROPSY
- Gross necropsy consisted of an examination of the external surface of the body.

HISTOPATHOLOGY / ORGAN WEIGTHS
A histopathological evaluation and organ weights were not evaluated for offspring.

SERUM HORMONES: Yes (Thyroxine (T4))
- Time of blood sample collection: PND 14-16 pups. Blood was also collected from two surplus pups per litter at PND 4 for possible thyroid hormone analysis.

Statistics:

All statistical tests was conducted at the 5% significance level. All pairwise comparisons was conducted using two-sided tests and was reported at the 1% and 5% levels.

For mortality, clinical signs, body weights, food consumption, functional tests, organ weights, reproduction parameters, and observations regarding pups, parametric variables were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. For non-parametric variables, data were compared using a Steel-test (many-to-one rank test).

For clinical pathology (haematology, coagulation and clinical chemistry), Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene's test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett's or Dunn's test, respectively. For incidence, an overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Reproductive indices:

Mating index (%): (Number of females mated/Number of females paired) X 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/Number of females mated) X 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) X 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) X 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeds the number of implantation sites recorded.

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) X 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) X 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) X 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) X 100
Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: During the treatment period, reflux was noted on a single occasion in three individual males at 500 mg/kg bw/day (of which one was decedent Male No. 37) and in one male at 150 mg/kg bw/day. In addition, rales, gasping with flat posture or piloerection were incidentally noted in a few males treated at 500 mg/kg bw/day and in one male at 150 mg/kg bw/day.
No clinical signs were noted during the recovery period.
Main females: At 500 mg/kg bw/day, slight lethargy, flat posture and uncoordinated movements were noted in 4/10 females on the first day of dosing. In addition, quick breathing, laboured respiration, rales and/or gasping were noted in individual females on one or two consecutive days after a few days and after four to five weeks of dosing. Hunched posture and/or piloerection were noted occasionally in a several females across all dose groups during the mating and lactation periods, with a slightly higher prevalence in the 500 mg/kg bw/day group.
Reflux was noted in one female treated at 150 mg/kg bw/day on one occasion.
Recovery females: During the treatment period, reflux was noted in one control female and in one female treated at 500 mg/kg bw/day on one occasion.
At 500 mg/kg bw/day, lethargy, flat posture and uncoordinated movements were noted for one female on the first day of dosing. Hunched posture and/or piloerection were occasionally noted in all Recovery females at 500 mg/kg bw/day throughout the treatment period. Hunched posture was also noted in two control females at the end of the mating period.
During the last week of the recovery period, hunched posture, rales and piloerection were noted in a single female. In the absence of other corroborative findings in this animal, and considering this occurred well after the end of the treatment period and/or was also noted in one control animal, this was considered unrelated to treatment.
All subsets: Salivation seen directly after dosing among all animal subsets in all test item-treated groups was observed in a dose-related manner and 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 rather than a sign of systemic toxicity.
No additional findings were noted for any of the subsets during the weekly arena observations in this study.
Incidental findings that were noted included scabs, bent tail apex and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of five animals of the 500 mg/kg bw/day group (one male and four females) did not survive until the end of the study period. From these five animals, three deaths were considered unrelated to treatment with the test item and for two animals, a test item-relationship is considered unlikely but could not be fully excluded.
Female No. 91 had severe clonic muscle spasms directly after dosing and subsequently died on Day 24 of study (post-coitum Day 7). On the days prior to its death, a hunched posture and/or piloerection was noted. At necropsy, reddish foci were present on the thymus (microscopic correlate was congestion) and glandular stomach (microscopic correlate was minimal haemorrhages). This animal was not pregnant. Based on the microscopic evaluation, the cause of death could not be determined. Therefore, a test item-relationship could not be excluded.
Female No. 95 was found dead on Day 11 of lactation (Day 50 of study). No relevant clinical signs were noted before its death. At necropsy, reddish foci were present in the thymus (microscopic correlate was congestion) and the liver was enlarged (without microscopic correlate). Based on the microscopic evaluation, the cause of death could not be determined. Therefore, a test item-relationship could not be excluded.
Male No. 37 was sacrificed in extremis on Day 10 of study. Directly after dosing, this animal had a reflux of the given dose and was lethargic, moderately salivating, breathing with difficulty with rales and gasping, and had red staining on the snout. At necropsy, the gastro-intestinal tract was distended with gas from stomach to caecum, and reddish, isolated foci were noted on the thymus, correlating with the haemorrhages found during microscopic evaluation. Together with an area of moderate fibroplasia in the thymus, the cause of moribundancy was most likely a procedural treatment error and unrelated to treatment with the test item.
Female No. 89 was euthanized on Day 1 of lactation (Day 38 of study), as it had a total litter loss. At the single incidence, and in the absence of changes in the reproductive organs of this animal and other reproductive effects, this was considered to be unrelated to treatment with the test item.
Female No. 99 was sacrificed in extremis on Day 4 of the Recovery Period (Day 55 of study) due to severe exophthalmos of the right eye, with microscopically marked hemorrhage and acute inflammation. This was considered related to the blood collection procedure (i.e., from the retro-orbital sinus) and therefore, this death is unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: At 500 mg/kg bw/day, body weight gain was slightly lower during the mating period compared to controls (not statistically significant), resulting in a lower body weight at end of treatment compared to controls (4% lower). Body weight gain was still slightly lower during the recovery period (not statistically significant), resulting in a lower body weight at end of recovery compared to controls (6% lower).

No effect on body weight (gain) was noted in males treated at 50 and 150 mg/kg bw/day.

Main females: At 500 mg/kg bw/day on Day 13 of lactation, mean body weight was 8% lower compared to controls.

No test item-related changes in body weights and body weight gain were observed up to 150 mg/kg bw/day. Any other changes in body weight gain were considered to be incidental and unrelated to treatment in the absence of a dose-related trend.

Recovery females: Body weights and body weight gains were considered to be unaffected by treatment with the test item throughout the study.

The apparent lower body weight gain at 500 mg/kg bw/day between Days 8 and 19 of the recovery period was considered unrelated to treatment and was likely an effect of a slightly higher starting body weight for recovery females compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: Food consumption was considered to be unaffected by treatment with the test item throughout the study up to 500 mg/kg bw/day.

Main females: At 500 mg/kg bw/day, food consumption was 24% lower (relative: 20% lower) over post-coitum Days 17-20 compared to controls and 19% lower (relative: 12% lower, not statistically significant) over Days 7-13 of lactation compared to controls.

Food consumption was considered to be unaffected by treatment with the test item up to 150 mg/kg bw/day.

Recovery females: A trend towards a lower food consumption (absolute and relative) was noted in females treated at 500 mg/kg bw/day throughout the treatment and recovery period, without achieving statistical significance. Given the small magnitude of change, this was considered not toxicologically relevant.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males: At 500 mg/kg bw/day, neutrophil (NEUT), monocyte (MONO) and large unstained cells (LUC) counts were increased compared to control values (1.90, 2.35 and 1.91x to control, respectively). In addition, a trend towards increased white blood cell (WBC), eosinophil (EOS) and basophil (BASO) counts was noted at 500 mg/kg bw/day, as well as a trend towards increased neutrophil and monocyte counts at 150 mg/kg bw/day.

Red blood cell (RBC) count was increased at 500 mg/kg bw/day (1.06x to control).

After a 14-day recovery period, neutrophil, monocyte and basophil counts were still increased (1.25, 1.48 and 2.00x to control, respectively), without achieving statistical significance. In addition, reticulocyte (RETIC) count was decreased at the end of the recovery period (0.88x to control).

No toxicologically relevant changes were noted in haematological parameters at 50 mg/kg bw/day.

Main females: Reticulocyte counts were decreased from 50 mg/kg bw/day onwards (0.55, 0.68 and 0.43x to control at 50, 150 and 500 mg/kg bw/day, respectively, not statistically significant at 150 mg/kg bw/day). Mean corpuscular volume (MCV) was decreased at 500 mg/kg bw/day (0.94x to control) and red blood cell counts were increased (1.07x to control, not statistically significant). In addition, a trend towards increased neutrophil, monocyte and basophil counts could be observed from 150 mg/kg bw/day onwards, however without a dose-related response.

Any other changes in haematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Recovery females: At end of treatment, increases in white blood cell counts (1.58x to control), mainly attributed to increased numbers of neutrophils, lymphocytes and monocytes (2.11, 1.47 and 2.31x to control, respectively), were noted in females treated at 500 mg/kg bw/day. In addition, a trend towards higher eosinophil, basophil and large unstained cells was noted. A trend towards a decrease in reticulocyte counts (0.95x to control) and an increase in red blood cell counts (1.05x to control) was noted.

After a 20-day recovery period, counts of white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and LUC were still increased, but without achieving statistical significance, except for eosinophil counts (1.64x to control). A trend towards a decrease in reticulocyte counts (0.86x to control) and an increase in red blood cell counts (1.06x to control) was still noted.

All animals: Coagulation parameters were considered not to have been affected by treatment up to 500 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: At end of treatment, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity was increased in males treated at 500 mg/kg bw/day (2.19 and 2.43x to control, respectively). Inorganic phosphate (PHOS) levels were decreased from 50 mg/kg bw/day onwards (0.84, 0.75 and 0.85x to control for the 50, 150 and 500 mg/kg bw/day groups, respectively), without a dose-response relationship.

After a 14-day recovery period, ALT and AST activities were increased even more (3.37 and 4.29x to control), although not statistically significant, due to high variance caused by extreme ALT and AST levels in Male No. 47 (481 and 721 U/L, respectively). Inorganic phosphate levels had not recovered after a treatment-free period (0.83x to control, not statistically significant).

Any other changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Main females: At 500 mg/kg bw/day, ALT and AST activity was (markedly) increased in 3 out of 5 females, resulting in a mean increase of 1.56 and 8.53x to control, respectively (not statistically significant due to high variance).

Other changes in treated females when compared to controls, were considered to have arisen as a result of slightly high control values and were considered to be of no toxicological significance in the absence of a treatment-related distribution.

Recovery females: At end of treatment, ALT and AST activity was increased in females treated at 500 mg/kg bw/day (2.33 and 5.32x to control, respectively). In addition, potassium levels were increased (1.14x to control).

After a 20-day recovery period, mean ALT and AST activity was increased even further (6.85 and 8.44x to control, respectively), with extreme values in ALT and AST levels in Female No. 96 (1124 and 1405 U/L, respectively). Mean bile acid levels were also increased (2.43x of control and 1.81x to end of treatment value). Potassium levels were similar to end of treatment value (1.02x) but the magnitude of change to control was lower (1.08x to control, not statistically significant).

Other changes in treated females when compared to controls, were considered to have arisen as a result of slightly low control values and were considered to be of no toxicological significance.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

At end of treatment, a decrease in total T4 in males at 500 mg/kg bw/day was noted (0.79x of control, not statistically significant); mean values within the range of historical control data*. At the individual level, 3/10 values of males at 500 mg/kg bw/day were below the range of the concurrent control values. Given the magnitude of change, a relationship with the test item could not be excluded. However, no adverse effects on thyroid histopathology and no test item- related changes in thyroid weight were recorded.

*Historical control data (2017 - 2021): Total T4 in males: Mean: 4.63 µg/dL, P5-P95: 3.06-6.48 (N=916)

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment up to 500 mg/kg bw/day.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. In Recovery females treated at 500 mg/kg bw/day, hind grip strength was slightly lower at the end of treatment compared to control values. As values remained well within historical control data for nulliparous females of this age and strain, this change was considered to be not toxicologically relevant.
Motor activity was considered not to be affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

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 microscopic findings were noted in the lung, liver, mesenteric lymph node of males and females and in the thymus and skeletal muscle of females. See Tables 6 and 7.

Lung: Diffuse alveolar macrophages were observed in males up to slight degree and in females up to moderate degree, starting at a dose of 150 mg/kg bw/day. After a recovery period of 14/20 days, minimal diffuse alveolar macrophages were present in both sexes, suggesting partial recovery.

Liver: Microgranulomas were observed in males at a dose of 500 mg/kg bw/day and in females starting at 150 mg/kg bw/day, both sexes up to slight degree. These microgranulomas consisted of small clusters of macrophages and/or Kupffer cells, scattered throughout the liver. After a recovery period of 14/20 days, the incidence and severity of microgranuloma in males was similar whereas in females the severity was increased, suggesting that no recovery occurred.
Mesenteric lymph nodes: An increased incidence and/or severity of macrophage foci was observed in Main males starting at 150 mg/kg bw/day and in Main females at 500 mg/kg bw/day, both sexes up to marked degree. The minimal severity noted in 2/5 Main females at 150 mg/kg bw/day was considered to be within background. After a recovery period of 14/20 days, incidences and severity of macrophage foci in males and females were similar, suggesting that no recovery occurred.

Thymus: An increased severity of lymphoid depletion up to slight degree (minimal degree is considered to be background) was observed in Main females at 500 mg/kg bw/day. After a recovery period of 20 days, 2/5 females at 500 mg/kg bw/day showed minimal lymphoid depletion which is regarded to be within background range.

Skeletal muscle: An increased incidence and severity of mononuclear inflammatory infiltrates (minimal degree is considered normal background) and degeneration of myofibers was observed in females at 500 mg/kg bw/day up to moderate degree. After a recovery period of 20 days, the severity of inflammatory cell infiltrates was at minimal degree and no degeneration of myofibers was present.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item. All females had regular cycles of 4 to 5 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not affected by treatment with the test item. All females showed evidence of mating, so mating indices were 100% for all groups.

Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days, except for one control female that showed evidence of mating after 11 days. As this occurred in the control group, this was unrelated to treatment with the test item.

Number of implantation sites was considered not to be affected by treatment with the test item. The mean numbers of implantation sites were 13.3, 11.8, 13.5 and 13.4 for the control, 50, 150 and 500 mg/kg bw/day groups, respectively.

Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 100% for the control, 50 and 150 mg/kg bw/day groups and 90% for the 500 mg/kg bw/day group, respectively.
One female at 500 mg/kg bw/day (No. 91), which was found dead on post-coitum Day 7, had no implantations. In the absence of any reproductive/developmental toxicity and at the single incidence, this was considered not to be related to treatment with the test item.

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
Except for one female at 500 mg/kg bw/day (No. 89), all pregnant females had live offspring. The gestation indices were 100% for the 50, 150 groups and 89% for the 500 mg/kg bw/day group, respectively.

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

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 92, 93 and 87% for the control, 50, 150 and 500 mg/kg bw/day groups, respectively. The slightly lower post-implantation survival index at 500 mg/kg bw/day was still within historical control range and therefore considered toxicologically irrelevant.

Litter size was considered not affected by treatment with the test item.
Mean live litter sizes were 11.9, 10.9, 12.6 and 10.6 living pups/litter for the control, 50, 150 and 500 mg/kg bw/day groups, respectively.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

clinical biochemistry

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

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 at this dose level

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: hepatobiliary system and musculoskeletal system

Organ:

liver

myofibres

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item. The nature and incidence of clinical signs noted (abnormal posture of the leg, wounds and scabs, broken/bent tail) were within the range considered normal for pups of this age and/or occurred in the control group, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 98, 100, 100 and 90% for the control, 50, 150 and 500 mg/kg bw/day groups, respectively.
Female No. 89 (500 mg/kg bw/day) had a total litter loss on Day 1 of lactation, as all ten pups were found dead at first litter check. Due to severe cannibalism of all pups, the number of pups should be interpreted with caution.
In addition to the pups mentioned above, two pups of the control group (one each in Litter Nos. 55 and 57) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since both dead pups were part of the control group.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment with the test item.
Viability indices were 99% for the control and 100% for the 50, 150 and 500 mg/kg bw/day groups, respectively.
One pup of the control group (Litter No. 60) was killed in extremis on PND 1, as this pup had a blue appearance and was lethargic. No toxicological relevance was attributed to this finding since this concerned a single control pup.

The lactation indices were 100% for the control, 50 and 150 mg/kg bw/day groups and 88% for the 500 mg/kg bw/day group, respectively.
The lower lactation index at 500 mg/kg bw/day was caused by Litter No. 95: as the dam was found dead on Day 11 of Lactation, all remaining eight pups (after culling) of this female were necropsied on PND 11 for animal welfare reasons. As the cause of death was not related to the condition of the pups, the lower lactation index for this treatment group was considered to be unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, male, female and combined pup body weights on PND 13 were lower compared to the concurrent control value (11, 10 and 10% lower, respectively), without reaching statistical significance for females alone. Values were below the historical control range for both sexes.
Historical control data for pup body weights (2017 - 2020):
Mean pup body weight on PND 13 for males: 30.7 grams, P5-P95: 26.0-35.3 (N=4611)
Mean pup body weight on PND 13 for females: 29.8 grams, P5-P95: 25.1-34.7 (N=4628))

Body weights of pups were considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. The statistically significantly higher pup body weights at 50 mg/kg bw/day on PND 1 and 4 were considered a chance finding, as the opposite effect would be expected in case of a toxicological effect and as it occurred without a dose-response relationship.

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):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item up to 500 mg/kg bw/day was considered to have no effect on areola/nipple retention. For all of the examined male pups, no nipples were observed on PND 13.

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 with the test item.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Sex ratio was considered not to be affected by treatment with the test item.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

2

Key result

Dose descriptor:

LOAEL

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:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 5. Mean Percent Male Spleen Weight Differences from Control Groups

	Main Males					Recovery Males
Dose level (mg/kg bw/day):	50		150		500	500
SPLEEN
Absolute	-5		9		22*	-1
Relative to body weight	0		15		31**	3

 *: P≤0.05, **: P≤0.01

Table 6. Summary Test Item-Related Microscopic Findings – Males

	Main Males				Recovery Males
Dose level (mg/kg bw/day):	0	50	150	500	0	500
LUNG^#	5	5	5	6*	5	5
Diffuse alveolar macrophages
Minimal	-	-	1	2	-	3
Slight	-	-	-	2	-	-
LIVER^#	5	5	5	6	5	5
Microgranuloma
Minimal	-	-	-	1	-	1
Slight	-	-	-	3	-	4
MESENTERIC LYMPH NODE^#	5	5	5	6	5	5
Increased macrophage foci
Minimal	-	-	-	-	-	1
Slight	-	-	-	-	-	1
Moderate	-	-	1	3	-	1
Marked	-	-	-	1	-	1

^# = Number of tissues examined from each group.

* = Male 37 included.

Table 7. Summary Test Item-Related Microscopic Findings – Females

	Main Females				Recovery Females
Dose level (mg/kg bw/day):	0	50	150	500	0	500
LUNG^#	5	5	5	6*	5	5^$
Diffuse alveolar macrophages
Minimal	-	-	1	1	-	2
Slight	-	-	3	2	-	-
Moderate	-	-	-	3	-	-
LIVER^#	5	5	5	6	5	5^$
Microgranuloma
Minimal	-	-	-	1	-	1
Slight	-	-	1	5	-	-
Moderate	-	-	-	-	-	3
Marked	-	-	-	-	-	1
MESENTERIC LYMPH NODE^#	5	5	5	6	5	5^$
Increased macrophage foci
Minimal	-	-	2	-	3	-
Slight	-	-	-	2	-	1
Moderate	-	-	-	2	-	3
Marked	-	-	-	2	-	1
THYMUS^#	5	5	4	6	5	5^$
Depletion lymphoid
Minimal	-	1	-	1	-	2
Slight	-	-	-	2	-	-
SKELETAL MUSCLE^#	5	5	5	6	5	5^$
Infiltrate inflammatory cell
Minimal	-	1	-	2	2	-
Slight	-	-	-	2	-	-
Moderate	-	-	-	1	-	-
Degeneration myofiber
Minimal	-	-	-	3	-	-
Slight	-	-	-	1	-	-
Moderate	-	-	-	1	-	-

^# = Number of tissues examined from each group. .

* = Female 95 included.

^$= Female 99 was sacrificed after 4 days of recovery.

Applicant's summary and conclusion

Conclusions:

A well reported oral combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (reliability score 1), conducted according to the current guideline (OECD 422) and in accordance with GLP, identified a NOAEL (systemic) for parental animals of 150 mg/kg bw/day in rats based on mortality, markedly increased levels of liver enzymes (ALT and AST) and skeletal muscle degeneration in females. The reproductive NOAEL in parental animals was at least 500 mg/kg bw/day based on a lack of reproductive effects observed in the study. The developmental NOAEL was 150 mg/kg bw/day, based on lower pup body weights at 500 mg/kg bw/day.