3-(2,4-bis(4-((5-(4,6-bis(2-aminopropylamino)-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-disulfonaphthalen-3-yl)azo)phenylamino)-1,3,5-triazin-6-ylamino)propyldiethylammonium lactate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26 to April 29, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29th July, 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 80 – 85 days; females 80 – 85 days
- Weight at study initiation: 372 – 423 g for male animals; 216 – 245 g for female animals. The weight variation did not exceed +- 20 per cent of the mean weight
- Housing: before mating 2 animals of the same sex/cage; during mating 1 male and 1 female / cage; sperm positive females individually; males after mating 2 animals / cage
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
- Water (e.g. ad libitum): tap water for human consumption ad libitum
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air-exchanges/ hour by a central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.
IN-LIFE DATES: From: January 26, 2022 To: March 25, 2022

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (Distilled water) in concentrations of 25, 50 and 100 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for four days and stored at room temperature (15 – 25 °C) until use.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL of each formulation and five aliquots 5 mL control substance (vehicle) were taken and analyzed.
Date of sampling: January 26 and March 10, 2022;
Date of analysis: January 26-27 and March 10-11, 2022;
Concentration of the test item in the dosing formulations varied in the range of 92 and 110 % of the nominal values at both analytical occasions. Results and details of analysis are attached to this Report (Appendix 17).
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: between 102 and 106 % at 0.5 mg/mL and at 125 mg/mL, respectively).
Blue PE 3811/Cartasol Blue K5R proved to be stable in distilled water at the intended concentrations at room temperature for four days.

Duration of treatment / exposure:

The experimental period involved 20 days of acclimatization (including 14 days for examination of estrous cycle) and 36-58 days treatment/observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment was considered as day 0 of examination.
Males were dosed for 44 days (14 days pre-mating and 1-14 days mating period plus 16-29 days of post-mating period until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.

Frequency of treatment:

The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours).

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary repeated dose toxicity study with Blue PE 3811/Cartasol Blue K5R in rats.
The high dose is chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals or representing the limit dose according to the respective OECD guideline.
The low dose is chosen to induce no toxic effect. The mid dose is interpolated geometrically.
A control and three dose groups were involved in the study
- Fasting period before blood sampling for clinical biochemistry:

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the administration at approximately the same time, considering the peak period of anticipated effects after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of parental animals was determined with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum.
Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
- The food consumption was determined weekly by reweighing the given and non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 0, 7, 13, and post-mating days 27, 34, 41 and 43 for male animals; pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment started from each animal was being considered for study for two weeks.
Animals exhibited typical 4-5 days cycles were included in the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smear also was prepared on the day of the necropsy.
Each morning a vaginal smear were prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope.

Sperm parameters (parental animals):

Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups.

Litter observations:

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition was complete, day 0), and on post-natal day 13 with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
The anogenital distance of each pup was determined on post-natal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on post-natal day 4 and the litter weight was calculated for evaluation on post-natal day 4.
The number of nipples/areolae in male pups was counted on post-natal day 13.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were dosed for 44 days (14 days pre-mating and 1-14 days mating period plus 16-29 days of post-mating period until the necessary number of pregnant female animals was evident); then they were sacrificed.
- Maternal animals: Females were dosed for 14 days pre-mating, through 1-14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice. The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.

GROSS NECROPSY
Gross necropsy was performed on each animal one day after the last treatment. Animals were euthanized by exsanguination after verification of deep narcosis by Release 300 mg/mL solution for injection were opened and the appearance of the all tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with pharynx.

Dead pup and pups selected for thyroid gland preservation (one male and one female pup per litter, where it was feasible) were subjected to gross macroscopic observations.
Pups euthanized on post-natal day 13 or shortly thereafter, were carefully examined externally for gross abnormalities.
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.


HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Absolute organ weight was recorded. Relative (to body and brain weight) organ weights were calculated and reported. The thyroid weight was not determined as it was not relevant. Paired organs were weighed together.

Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
These organs were also processed and examined histologically in non-pregnant female and male this female cohabited with in the mid dose group.
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

The following parameters were examined and evaluated:

Parental males

 Clinical observations
 Body weight †
 Body weight gain †
 Food consumption †
 Percentage of pairings
 Percentage of fertile males †
 Percentage of infertile males †
 * Male copulatory index †
 * Male fertility index †
 Thyroid hormone (FT3, FT4 and TSH) level†
 Necropsy findings
 Organ weights (absolute and relative to the body and brain weights) †
 Histopathology findings

Parental females

 Clinical observations
 Body weight †
 Body weight gain †
 Food consumption †
 Percentage of pairings /sperm positive females
 Percentage of pregnant females †
 Percentage of sperm positive, but non - pregnant females
 Percentage of non-mated females
 * Female copulatory index †
 * Female fertility index †
 * Gestation index †
 Duration of pregnancy (days) †
 Number of implantations / dams †
 * Post-implantation mortality †
 Number of dams with live pups on lactation days 0, 4 and 13
 Necropsy findings
 Histopathology findings

* = Formulas for calculations are given below
† = Statistical analysis was performed

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy was performed for offspring selected for thyroid gland preservation.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following parameters were examined and evaluated:
Offspring

 Litter weight on post-natal days 0, 4 and 13 †
 Mean body weight gain per litter between post-natal days 0-4, 4-13 and for overall post-natal days †
 Number of live births per litter, and number of viable pups per litter on post-natal days 0, 4 and 13 †
 * Survival Index of pups on post-natal day 13 †
 * Sex ratio % (on post-natal days 0 and 13). †
 Normalized anogenital distance †
 Number of nipples/areolae in male pups †
 Thyroid hormone (FT3, FT4 and TSH) level of pups on post-natal day 13 †

* = Formulas for calculations are given below
† = Statistical analysis was performed

Reproductive indices:

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males: ("Number"  "of"  "males"  "impregnating"  a "female" )/"Total Number of males with confirmed mating"  x 100
Females ("Number"  "of"  "pregnant"  "females" )/"Number of sperm positive females"  x 100

Gestation index: Measure of pregnancy that provides at least one live pup
("Number"  "of"  "females with live born pups" )/"Number of pregnant females"  x "100"

Offspring viability indices:

Post-implantation/ pre-natal mortality (intrauterine mortality)
("Number"  "of"  "implantations - Number of liveborns" )/"Number of implantations"  x 100

Post-natal mortality /Extra-uterine mortality
("Number"  "of"  "liveborns -Number of live pups on PN13" )/"Number of liveborns"  x 100

Survival Index
("Number"  "of"  live pups on post-natal day 13)/"Number of pups born"  x 100

Sex ratio
("Number"  "of"  "pups"  "examined"  - "Number"  "of"  "males " ("females" ))/"Number of pups examined"  x 100

Normalized anogenital distance
"Absolute anogenital distance" /"(Body weight)1/3"

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations.
The behavior and physical condition of animals were considered to be normal in all male and female animals in the control, at 250, 500 and 1000 mg/kg bw/day during the clinical observations.
There were no clinical signs in male animals in the control, 250, 500 or 1000 mg/kg bw/day groups during the entire observation period (pre-mating, mating and post-mating periods).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality in the control, 250, 500 or 1000 mg/kg bw/day groups during the course of study (pre-mating, mating and post-mating period in male animals and in non-pregnant females, pre-mating, mating, gestation and lactation periods in female animals).
One female animal (No. 127) at control dose was accidentally over-anesthetized at the blood sampling for thyroid hormone determination on Day 51. This animal was processed in the same way as the animals at terminal necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not affected by the test item in male or in female animals at 250, 500 or 1000 mg/kg bw/day during the entire observation period.
The mean body weight was similar in the male animals in the control and test item administered groups from Day 0 up to and including Day 43. Statistical significance with respect to the control was observed at the slightly lower mean body weight gain of male animals at 1000 mg/kg bw/day between Days 27 and 34. This minor change however had no influence on the mean body weight or on the summarized mean body weight gain.
Therefore, this finding in male animals was considered to have no toxicological relevance.
In the female animals, the mean body weight was comparable to their control at 250, 500 and 1000 mg/kg bw/day during the pre-mating, gestation and lactation periods. There was no statistical significance differences during the entire observation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related changes in the food consumption of male or female animals at any dose level (250, 500 and 1000 mg/kg bw/day).
The mean daily food consumption was similar in the control and test item treated (250, 500 and 1000 mg/kg bw/day) male animals during the entire observation period (pre-mating and post-mating).
The mean daily food consumption was comparable in female animals in the control, 500 and 1000 mg/kg bw/day groups during the entire observation period (pre-mating and gestation periods).
The mean daily food consumption was slightly – but statistically significantly – higher than in the control group in female animals at 250 mg/kg bw/day between lactation days 0 and 4.

Food efficiency:

not specified

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

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences with respect to the control in the thyroid hormone (FT3, FT4 and TSH) levels in parental male animals or in PN13 offspring at 250 or 1000 mg/kg bw/day.
Statistically significantly lower mean TSH thyroid hormone level was observed in PN13 offspring at 500 mg/kg bw/day and statistically not but lower mean TSH thyroid hormone level (-11%) was observed at 1000 mg/kg bw/day without toxicological meaning. In case of at 500 mg/kg bw/day the mean values were within to the historical control range. Thus, these differences were judged to be toxicologically not relevant. Furthermore,dose dependent were not found between the dose groups of parent male and offspring animals.
One pup (Animal no: 222P) at dose level 250 mg/kg bw/day the TSH level was extremely higher therefore the value of TSH resulting had highly elevated (39%) in this group.
This difference was considered to have no toxicological meaning.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological investigation did not reveal toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive organs of experimental male and female animals at control and 1000 mg/kg bw/day.
In the male animals the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.
The histological picture of epididymides, (storage of mature spermatozoa) was normal in all cases as well.
In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Test item influence on the estrous cycle was not detected at any dose level (250, 500 and 1000 mg/kg bw/day).
All examined parameters of estrous cycle – percentage of animals with regular/ irregular cycle, mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous – were comparable in animals appointed for the control and test item dose groups during the pre-treatment period.
There were no significant differences between the control and test item treated animals in the investigated parameters of estrous cycle during the two weeks pre-mating period.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 250, 500 or 1000 mg/kg bw/day.
Statistically significant difference with respect to the control were detected at the lower copulatory index (92%) in male and female animals administered with 1000 mg/kg bw/day as one animal (no. 408) failed to mate their partner during the two-weeks mating period. This minor change was considered to be not related to the test item because similar incidences also occur in non-treated experimental rats of this strain.

There were no significant differences between the control and test item treated groups in the fertility index of male animals.
The fertility index of female animals (percentage of pregnant) was slightly lower (92%) than in the control at 500 mg/kg bw/day as one sperm positive female (1/12) was not pregnant.
Statistically significant difference with respect to the control were detected at the lower percentage of pregnant females (92%) at 500 mg/kg bw/day, sperm positive female (1/12) was not pregnant.
These minor changes in reproductive parameters were considered to be indicative of biological variation and not related to the test item as values met well the historical control moreover lower fertility was detected in the mid dose groups but not in the high dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between post-natal days 0 and 13.
At 500 and 1000 mg/kg bw/day, the percentage of offspring with signs (cold) was slightly higher than in the control group. Furthermore the percentage of offspring with signs (not suckled) was noted only in the control group.
These signs and differences with respect to the control were considered to be toxicologically not relevant as were not associated with depression of the development of the offspring.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
There were no statistically significant differences between the control and test item treated groups (250, 500 or 1000 mg/kg bw/day) in the mean number of live, viable or dead offspring per litter on post-natal days 0, 4 or 13.
There were no significant differences between the control and test item treated (250, 500 or 1000 mg/kg bw/day) groups in the survival indices.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and mean pup weights as well as the mean litter weight gain and pup weight gain were similar in the control and in all test item treated groups (250, 500 and 1000 mg/kg bw/day) on post-natal days 0, 4 and 13.
Considering the offspring’s body weight in females separately, statistically significant difference with respect to the control was detected at the lower mean weight of female pups at 250, 500 and 1000 mg/kg bw/day on post-natal day 4.

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on post-natal days 0, 4 or 13.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not affected by the test item treatment at 250, 500 and 1000 mg/kg bw/day.
Statistical significances were observed at the shorter absolute anogenital distances in female offspring at 250 and 500 mg/kg bw/day when compared to the control. These minor changes were considered to be indicative of biological variations in the lack of dose relationship, and not related to the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control or 250, 500 or 1000 mg/kg bw/day groups on post-natal day 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
There were no macroscopic findings in male and female pups at 250, 500 or 1000 mg/kg bw/day subjected to necropsy on post-natal day 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: offspring development

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 offspring development: 1000 mg/kg bw/day

Executive summary:

The objective of this study was to obtain initial information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 250, 500 and 1000 mg/kg bw/day compared to control animals according to OECD 421.

The dose setting was agreed with the Sponsor. Doses are selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 250, 500 and 1000 mg/kg bw/day doses in concentrations of 25, 50 and 100 mg/mL corresponding to a 10 mL/kg bw dosing volume. A group of vehicle (Distilled water) treated animals (n= 12/sex) served as a control.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating as well as on the day of the necropsy. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 3-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and 4-8 pups per litter on post-partum/ post-natal day 13 and from all parent male animals and from non-pregnant and not mated females at termination on Day 36/41.
Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Han:Wistar rats at 250, 500 or 1000 mg/kg bw/day doses administered by oral gavage and the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female rats at the doses of 250, 500 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 offspring development: 1000 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effects on developmental toxicity

Description of key information

NOAEL for F1 offspring development: 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The objective of this study was to obtain initial information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 250, 500 and 1000 mg/kg bw/day compared to control animals according to OECD 421.

The dose setting was agreed with the Sponsor. Doses are selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 250, 500 and 1000 mg/kg bw/day doses in concentrations of 25, 50 and 100 mg/mL corresponding to a 10 mL/kg bw dosing volume. A group of vehicle (Distilled water) treated animals (n= 12/sex) served as a control.

Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Han:Wistar rats at 250, 500 or 1000 mg/kg bw/day doses administered by oral gavage and the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female rats at the doses of 250, 500 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 offspring development: 1000 mg/kg bw/day

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), "Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.

Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects."

 

The available experimental data are adequate for classification and labelling and the substance is not classified for reproductive and developmental toxicity according to the CLP Regulation (EC 1272/2008).