Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2018 - 30 Sept 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Serum thyroid hormone not measured in pups on day 4

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2018 - 30 Sept 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

recommended by guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12-13 weeks
- Weight at study initiation: males: 345 - 420 g; females: 217 - 277 g
- Housing: in groups of 5 animals/sex/cage, type IV polysulphone cages or double decker IVC cages during premating period for males and females and during post-mating period for males; males and females co-housed during mating period in ratio 1:1; females housed in type III H polysulphone cages after confirmation of mating; Altromin saw fibre used as bedding in each cage
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: 18 - 23 July 2018

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at the testing facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 7 Aug 2018 To: 29 Sept 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance. The appropriate amount of vehicle was added to obtain the final concentration of the test item. A solution with the vehicle was prepared by subjecting the test item-vehicle suspension for approximately 60 min at 40 °C to an ultrasonic bath and 45 min stirring using a heating plate at 40 °C. To keep formulations fluid during the daily administration, they were kept under magnetic stirring and warm (≤ 40 °C) using a heating plate. Based on the results of stability testing, the test item formulations were prepared at least once every 7 days. The prepared formulation was stored protected from light and at room temperature.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Consultation with the sponsor based on the test item’s characteristics and testing guideline
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): MKCF8882, MKCD1021
- Expiry dates: 12 November 2018 (MKCF8882), 24 November 2018 (MKCD1021)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As the test item was not shown to be homogenous, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples). The samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 180922) and until then stored under appropriate conditions based on available stability data.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating period, 14 days mating period)
Females: 63 days (14 days premating period, 14 days mating period, approx. 22 days through gestation period until post-natal day 12)

Frequency of treatment:

7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a dose range finding study (BSL Munich Study No. 181975) and in consultation with the sponsor. The animals in the dose range finding study showed no mortality and only slight test item-related clinicial signs. Test item-treated animals showed no gross difference in body weight development and no difference in food consumption compared to the control animals. The test item-treatment was found to have no effects on reproductive indices as well as pup-related parameters. Thus, the dose levels proposed to be used in the higher tier study are 100, 300, and 1000 mg/kg/day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first exposure, at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 h of parturition (day 0 post-partum), on post-natal day (PND) 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION: YES
- Time schedule: on the same days as body weight measurements, not measured during mating period in males and females and post-mating period in males

FOOD EFFICIENCY: NO

OPHTHALMOSCOPIC EXAMINATION: Yes
No further details reported.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals by using anesthesia (ketamine/xylazine).
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters summarised in table 1 were examined. Refer to 'Any other information on materials and methods incl. tables' below.
- Details on coagulation:
- Time schedule for examinations: at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters tested: prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters summarised in table 2 were examined. Refer to 'Any other information on materials and methods incl. tables' below.

URINALYSIS: Yes
- Time schedule for collection of urine: 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters summarised in table 3 were examined. Refer to 'Any other information on materials and methods incl. tables' below.

NEUROBEHAVIOURAL EXAMINATION / / FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: males: in the week before first treatment and during the last week of treatment in 5 randomly selected males; females: during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: The wet weight of organs (table 4, refer to 'Any other information on materials and methods incl. tables' below) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
- Further details: All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes
- Parameters summarised in table 5 were examined. Refer to 'Any other information on materials and methods incl. tables' below.
- Further details: Full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.
Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p < 0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Females during first premating week: absolute body weight increased to a level of approx. 2 % above controls; non adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

MORTALITY:
No mortality occurred in the during the treatment period of this study.

CLINICAL OBSERVATIONS:
Moving the bedding was seen in all female animals of the 300 mg/kg bw/day and 1000 mg/kg bw/day groups transiently during gestation and postnatal periods. A single 100 mg/kg bw/day female was observed moving the bedding on few gestation days. This clinical sign was observed immediately after the dose administration and therefore is considered to be a sign of a local reaction to the test item rather than a systemic adverse effect, and is not toxicologically relevant. Moving the bedding was not observed in male animals of the dose groups. Single or occasional findings that are assumed to be related to the gavage technique or that are considered incidental were: abnormal breathing, lacrymation, regurgitation of the test item, increased salivation, diarrhea, transient piloerection, scratches, crust or wounds. Hairless areas occurred in a few animals of all groups. This is usually observed in this strain and is not assumed to be toxicologically relevant.

FUNCTIONAL OBSERVATIONS:
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

OPHTHALMOLOGY:
No opthalmoscopic alterations were seen at the end of the treatment period in any of the animals.

BODY WEIGHT DEVELOPMENT:
A statistically significant higher body weight gain was observed in females in the 1000 mg/kg bw/day group during the first premating week. This is not considered to be toxicologically relevant as the absolute body weight of these animals only increased to a level of approx. 2% above controls. Body weights of male animals were in the normal range of variation throughout the treatment period of the study and there were no statistically significant differences between dose groups and controls.

FOOD CONSUMPTION:
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study. Slightly and statistically significant higher food intake was seen in female animals of the 1000 mg/kg bw/day group during the first 2 weeks of gestation and the last week of the postnatal period (Day 4 to 13). This is not considered to be an adverse effect.

HAEMATOLOGY AND COAGULATION:
A statistically significant increased rate of large unstained cells (78% above controls) was found in the blood of females of the 1000 mg/kg bw/day group. Large unstained cells are assumed to be progenitor cells of the bone marrow released into circulation at a low rate. In absence of changes in white and red blood cells and histopathological findings in bone marrow, this is assumed not to be of biological relevance. Moreover, all values were in the range of historical control data. In addition, there were no significant differences in haematological parameters between dose groups and control group and all were within the normal range of variation. There was no statistically significant effect of the test item on coagulation parameters (PT, aPTT) determined at the end of the treatment period.

CLINICAL BIOCHEMISTRY:
There was no biologically relevant effect of the test item on clinical biochemistry parameters determined at the end of the treatment period of this study. A slight, but statistically significant higher serum urea level (26% above controls) found in female animals of the 300 mg/kg bw/day group was found. Due to the slightness and the lack of a dose-dependency this is not considered to be of toxicological relevance. Moreover, this did not coincide with any abnormalities observed in the renal system.

URINALYSIS:
The urinalysis performed in male and female animals sacrificed at the end of treatment period showed no test item treatment-related effect in all treatment groups. All urinary parameters were in the normal range of variation.

PATHOLOGY:
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental.

ORGAN WEIGHTS:
There were no statistically significant differences in organ weights between dosed and control animals. A tendency towards slightly higher absolute and relative (to body weight) adrenal weight in male (15% above controls, respectively) and female animals (23 and 27% above controls, respectively) of the 1000 mg/kg bw/day group is possibly related to a secondary stress response caused by gavaging of the test item formulation. A high mean thymus weight found in females of the 100 mg/kg bw/day group is caused by a markedly higher thymus weight of animal no. 54. There were no relevant findings from the clinical pathology or histopathology. A tendency towards higher ovary weight in all dose groups (between 26 and 29% above controls) is not considered to be toxicologically relevant as there were no corresponding histopathological findings.

HISTOPATHOLOGY:
No test item related changes were noted. All recorded findings were deemed to be incidental or fell within the range of background alterations that may be recorded in Wistar rats. There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology were observed. No treatment-related effect on interstitial cell structure was noticed.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to and including the highest dose tested

Key result

Critical effects observed:

no

Tables summarising individual animal data are attached as pdf documents to the technical dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

EC Number:

947-918-6

Molecular formula:

not applicable, UVCB substance.

IUPAC Name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

recommended by guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12-13 weeks
- Weight at study initiation: males: 345 - 420 g; females: 217 - 277 g
- Housing: in groups of 5 animals/sex/cage, type IV polysulphone cages or double decker IVC cages during premating period for males and females and during post-mating period for males; males and females co-housed during mating period in ratio 1:1; females housed in type III H polysulphone cages after confirmation of mating; Altromin saw fibre used as bedding in each cage
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: 18 - 23 July 2018

DETAILS OF FOOD AND WATER QUALITY:
Certificates of food, water and bedding are filed for two years at the testing facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 7 Aug 2018 To: 29 Sept 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance. The appropriate amount of vehicle was added to obtain the final concentration of the test item. A solution with the vehicle was prepared by subjecting the test item-vehicle suspension for approximately 60 min at 40 °C to an ultrasonic bath and 45 min stirring using a heating plate at 40 °C. To keep formulations fluid during the daily administration, they were kept under magnetic stirring and warm (≤ 40 °C) using a heating plate. Based on the results of stability testing, the test item formulations were prepared at least once every 7 days. The prepared formulation was stored protected from light and at room temperature.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Consultation with the sponsor based on the test item’s characteristics and testing guideline
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): MKCF8882, MKCD1021
- Expiry dates: 12 November 2018 (MKCF8882), 24 November 2018 (MKCD1021)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As the test item was not shown to be homogenous, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples). The samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 180922) and until then stored under appropriate conditions based on available stability data.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating period, 14 days mating period)
Females: 63 days (14 days premating period, 14 days mating period, approx. 22 days through gestation period until post-natal day 12)

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a dose range finding study (BSL Munich Study No. 181975) and in consultation with the sponsor. The animals in the dose range finding study showed no mortality and only slight test item-related clinicial signs. Test item-treated animals showed no gross difference in body weight development and no difference in food consumption compared to the control animals. The test item-treatment was found to have no effects on reproductive indices as well as pup-related parameters. Thus, the dose levels proposed to be used in the higher tier study are 100, 300, and 1000 mg/kg/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
once before first exposure, at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
All animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 h of parturition (day 0 post-partum), on post-natal day (PND) 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
YES
- Time schedule: on the same days as body weight measurements, not measured during mating period in males and females and post-mating period in males

FOOD EFFICIENCY: NO

OPHTHALMOSCOPIC EXAMINATION: Yes
No further details reported.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
at the end of the treatment prior to or as part of the sacrifice of the animals by using anesthesia (ketamine/xylazine).
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters summarised in table 1 were examined.
Refer to 'Any other information on materials and methods incl. tables' below.
- Details on coagulation:
- Time schedule for examinations: at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters tested: prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals:
5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters summarised in table 2 were examined.
Refer to 'Any other information on materials and methods incl. tables' below.

THYROID HORMONE (T4) ANALYSIS:
Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis.

URINALYSIS: Yes
- Time schedule for collection of urine:
5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters summarised in table 3 were examined.
Refer to 'Any other information on materials and methods incl. tables' below.

NEUROBEHAVIOURAL EXAMINATION / / FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations:
males: in the week before first treatment and during the last week of treatment in 5 randomly selected males; females: during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating was observed. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented.

Sperm parameters (parental animals):

Parameters examined in P male animals:
Testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating gland), for the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

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:
possible cause of death was not determined for pups born or found dead.

THYROID HORMONE (T4) ANALYSIS:
Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All F0 male animals were sacrificed following completion of the mating period and 28 days of dose administration.
- Maternal animals: All F0 female animals were sacrificed on post-natal day 13

GROSS PATHOLOGY: Yes
- Organ weights: The wet weight of organs (table 4, refer to 'Any other information on materials and methods incl. tables' below) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
- Further details: All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes
- Parameters summarised in table 5 were examined. Refer to 'Any other information on materials and methods incl. tables' below.
- Further details: Full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on post-natal day 13.

GROSS NECROPSY
- Gross necropsy consisted of: careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.
Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p < 0.05 was considered as statistically significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Females during first premating week: absolute body weight increased to a level of approx. 2 % above controls; non adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

ESTROUS CYCLES:
The test item had no biologically significant effect on the estrous cycle analysed during the two weeks premating period after the first administration in treatment groups when compared to the controls. There were no significant differences in the length or sequence of cycle stages between the dose groups and the control group.

PRECOITAL INTERVAL AND DURATION OF GESTATION:
There was a minimal but statistically significant shorter duration of gestation in all dose groups. Due to the slightness of this effect, the lack of a dose-dependency and the absence of developmental abnormalities this is not considered to be toxicologically relevant. Moreover, values were within the historical control data range. A tendency of minimally longer precoital interval in the 1000 mg/kg bw/day group is not considered to be toxicologically relevant as it fell within the historical control data range.

PRE- AND POST-NATAL DATA:
A statistically significant higher number of corpora lutea and implantation sites (16% above controls, respectively) were found in the 1000 mg/kg bw/day group, when compared to controls. This is consistent with the higher number of total pups and live pups on PND 0. No considerable difference in pre-implantation loss between dose groups and control group and a statistically significant lower post-implantation loss in the 1000 mg/kg bw/day group compared to control group were not considered toxicologically relevant.

REPRODUCTIVE INDICES:
There were no test item-related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group. All values were within the range of historical control data.

MORTALITY:
No mortality occurred in the during the treatment period of this study.

CLINICAL OBSERVATIONS:
Moving the bedding was seen in all female animals of the 300 mg/kg bw/day and 1000 mg/kg bw/day groups transiently during gestation and postnatal periods. A single 100 mg/kg bw/day female was observed moving the bedding on few gestation days. This clinical sign was observed immediately after the dose administration and therefore is considered to be a sign of a local reaction to the test item rather than a systemic adverse effect, and is not toxicologically relevant. Moving the bedding was not observed in male animals of the dose groups. Single or occasional findings that are assumed to be related to the gavage technique or that are considered incidental were: abnormal breathing, lacrymation, regurgitation of the test item, increased salivation, diarrhea, transient piloerection, scratches, crust or wounds. Hairless areas occurred in a few animals of all groups. This is usually observed in this strain and is not assumed to be toxicologically relevant.

FUNCTIONAL OBSERVATIONS:
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

OPHTHALMOLOGY:
No opthalmoscopic alterations were seen at the end of the treatment period in any of the animals.

BODY WEIGHT DEVELOPMENT:
A statistically significant higher body weight gain was observed in females in the 1000 mg/kg bw/day group during the first premating week. This is not considered to be toxicologically relevant as the absolute body weight of these animals only increased to a level of approx. 2% above controls. Body weights of male animals were in the normal range of variation throughout the treatment period of the study and there were no statistically significant differences between dose groups and controls.

FOOD CONSUMPTION:
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study. Slightly and statistically significant higher food intake was seen in female animals of the 1000 mg/kg bw/day group during the first 2 weeks of gestation and the last week of the postnatal period (Day 4 to 13). This is not considered to be an adverse effect.

HAEMATOLOGY AND COAGULATION:
A statistically significant increased rate of large unstained cells (78% above controls) was found in the blood of females of the 1000 mg/kg bw/day group. Large unstained cells are assumed to be progenitor cells of the bone marrow released into circulation at a low rate. In absence of changes in white and red blood cells and histopathological findings in bone marrow, this is assumed not to be of biological relevance. Moreover, all values were in the range of historical control data. In addition, there were no significant differences in haematological parameters between dose groups and control group and all were within the normal range of variation. There was no statistically significant effect of the test item on coagulation parameters (PT, aPTT) determined at the end of the treatment period.

CLINICAL BIOCHEMISTRY:
There was no biologically relevant effect of the test item on clinical biochemistry parameters determined at the end of the treatment period of this study. A slight, but statistically significant higher serum urea level (26% above controls) found in female animals of the 300 mg/kg bw/day group was found. Due to the slightness and the lack of a dose-dependency this is not considered to be of toxicological relevance. Moreover, this did not coincide with any abnormalities observed in the renal system.

URINALYSIS:
The urinalysis performed in male and female animals sacrificed at the end of treatment period showed no test item treatment-related effect in all treatment groups. All urinary parameters were in the normal range of variation.

PATHOLOGY:
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental.

ORGAN WEIGHTS:
There were no statistically significant differences in organ weights between dosed and control animals. A tendency towards slightly higher absolute and relative (to body weight) adrenal weight in male (15% above controls, respectively) and female animals (23 and 27% above controls, respectively) of the 1000 mg/kg bw/day group is possibly related to a secondary stress response caused by gavaging of the test item formulation. A high mean thymus weight found in females of the 100 mg/kg bw/day group is caused by a markedly higher thymus weight of animal no. 54. There were no relevant findings from the clinical pathology or histopathology. A tendency towards higher ovary weight in all dose groups (between 26 and 29% above controls) is not considered to be toxicologically relevant as there were no corresponding histopathological findings.

HISTOPATHOLOGY:
No test item related changes were noted. All recorded findings were deemed to be incidental or fell within the range of background alterations that may be recorded in Wistar rats. There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology were observed. No treatment-related effect on interstitial cell structure was noticed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

LITTER DATA:
On post-natal day (PND) 0, the total number of pups (alive and dead) was statistically significantly higher in the 1000 mg/kg bw/day group, when compared to controls (35% above controls). On PND 0, 4 and 13 the number of live pups in this group was statistically significantly higher, when compared to controls (35%, 36% and 39% above controls). With respect to the genders, the numbers of male pups were statistically significantly higher in 1000 mg/kg bw/day group compared to controls on PND 0 and 4, whereas only a tendency towards a higher number of female pups was seen in this group. There were no test item treatment related effects on litter data including number of male pups on PND 13 and number of female pups, sex ratio, number of still births and runts. There were no statistically significant differences noted for these litter parameters and the values were comparable between dose groups and control group. The increased number of pups observed is not considered to be an adverse effect.

LITTER WEIGHT DATA:
There was a statistically significant higher litter weight in the 1000 mg/kg bw/day group on PND 0 which is related to the higher number of pups. The higher numbers also explain the tendency towards higher total litter weight on PND 4 and 13 and male litter weight on PND 0, 4 and 13.

PUP SURVIVAL DATA:
No test item-related effect was observed on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in all dose groups.

ANOGENITAL DISTANCE AND NIPPLE RETENTION:
Male pup weight and absolute anogenital distance were both significantly smaller in the presence of a higher number of pups per litter. However, there was no biologically relevant effect on relative anogenital distance of male pups of the 300 mg/kg bw/day and 1000 mg/kg bw/day groups, when compared to controls. This also appliesto male pups of the 100 mg/kg bw/day group where weight was slightly but statistically significantly lower compared to controls. Nipple retention was unaffected by treatment with the test item. A moderately but statistically significant shorter relative anogenital distance was observed in female pups of the 1000 mg/kg bw/day group (15% below controls). As was in males, a slightly higher number of female pups and slightly and statistically significant lower pup weight was observed. However, this is not considered sufficient to explain the significant difference. The values, however, were in the range of historical control data. No effect on relative anogenital distance was observed in the 100 mg/kg bw/day and 300 mg/kg bw/day groups.

THYROID HORMONE (T4) ANALYSIS:
No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight and thyroxine hormone (T4) level in males and PND 13 pups of the dose groups when compared to the controls.

PUP EXTERNAL FINDINGS:
A few pups were not found, found dead or still born on PND 0, i.e. 2 pups from control dam no. 41, 1 pup of control dam no. 42, 1 pup of 100 mg/kg bw/day group dam no. 51, 5 pups of 100 mg/kg bw/day group dam no. 54, 4 pups of 100 mg/kg bw/day group dam no. 55, 1 pup of 300 mg/kg bw/day group dam no. 63, 3 pups of 300 mg/kg bw/day group dam no. 64, 5 pups of 300 mg/kg bw/day group dam no. 69). No test item-related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few isolated findings like crust on left flank (pup no. 1 from control dam no. 49), crust on right flank (pup no. 10 from control dam no. 49), dark, no indication of suckling (pup no. 8 of dam no. 55), dark between eye and shoulder (pup no. 7 of dam no. 62), small (pup no. 1 of dam no. 69), dark snout (pup no. 7 of dam no. 74), small, dark snout (pup no. 2 of dam no. 75), small (pup no. 11 of dam no. 77) were observed. These external findings were considered to be spontaneous and not related to test item treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Tables summarising individual animal data are attached as pdf documents to the technical dossier.