2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a study according to OECD 422 the NOAEL for the reproductive performance in males and females and for the development of F1 offsprings was 1000 mg/kg bw (reference 7.8.1-1).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-08 to 2017-12-01

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd.Han

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest, Cserkesz u. 90
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
13 weeks (both sexes)
- Weight at study initiation: males: 359 - 434 g
females: 209 - 250g
- Fasting period before study: no
- Housing: before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female/cage; pregnant females: individually; males after mating: 2/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 27 days

DETAILS OF FOOD AND WATER QUALITY:
Food was changed at weekly intervals. Fresh drinking water was given daily. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Contents of the standard diet for rats and mice was guaranteed by the supplier. Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS: The substance was solved in distilled water in concentrations of 20, 60 and 200 mg/L.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle: 5 mL/kg body weight
- Batch no.: 1610-5527; 1701-5524; 1702-5502 (supplied by Parma Produkt Kft., Budapest, Hungary)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation or 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed by HPLC-UV in the Analytical Laboratory of the Test Facility twice during the study. Five aliquots of approx. 5 mL of each formulation (20, 60 and 200 mg/mL) and five aliquots of approx. 5 mL control substance (vehicle) were taken and analyzed. The samples were stored at room temperature before the analysis.
Concentration of the test item in the dosing formulations varied in the range of 97 - 108 % of the nominal concentrations.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. Recoveries of the test item from the vehicle were within the acceptance criteria (relative to nominal concentrations: 99 % at 1 mg/mL and 98 % at 200 mg/mL).
Stability and homogeneity in distilled water over the range of relevant concentration has been demostrated at room temperature for at least five days (recovery was 102 % of starting concentrations at 1 mg/mL and 103 % at 200 mg/mL), i.e. the maximum age of solutions administered.

Duration of treatment / exposure:

Males: dosed for 55 days (14 days pre-mating and 1-14 days mating plus 26 – 39 days of post-mating period until the necessary number of pregnant female animals was evident)
Females: dosed for 56 to 67 days (14 days pre-mating, through 1-18 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:

daily

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:

12 male and 12 female per dose and control

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 dose range finding study in rats. The test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day in this DRF study. There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food consumption, hematology, blood coagulation and clinical chemistry, necropsy findings, organ weights) after the 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.
The high dose was chosen as limit dose with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

- Fasting period before blood sampling for clinical biochemistry: Yes (approx. 16 hours)

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: general clinical observations (once per day), signs of morbidity and mortality (twice per day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (starting on Day 0) and at termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

FOOD EFFICIENCY:
- The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period.

OTHER: Further observations relevant for IUCLID section 7.5 were conducted. Please refer to the respective entry for details.

Oestrous cyclicity (parental animals):

The estrous cycle was monitored by examining vaginal smears each day before the treatment started for each animal being considered for the study for two weeks. The estrous cycle was evaluated and considered at randomization. However, some animals that failed to exhibit typical 4-5 days cycles were included in the study due to the large number of animals with irregular cycle.
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 smears were also prepared on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
- testis weight
- epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality,runts, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, T4 level (PND 13)

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were carefully examined for gross abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 55
- Maternal animals: All surviving animals 14 days post-partum

GROSS PATHOLOGY: Yes
- external appearance
- adrenal glands
- aorta
- bone with bone marrow and joint (femur)
- brain (representative regions: cerebrum, cerebellum, pons and medulla oblongata)
- eyes (lachrymal gland with Harderian glands)
- female mammary gland
- gonads (testis with epididymides, ovaries, uterus with fallopian tube and vagina)
- gross lesions
- heart
- kidneys
- large intestines (caecum, colon, rectum, incl. Peyer's patches)
- liver
- lungs (with main stem bronchi)
- Lymph nodes (submandibular, mesenteric)
- muscle (quadriceps)
- esophagus
- pancreas
- pituitary
- prostate
- salivary glands (submandibular)
- sciatic nerve
- seminal vesicle with coagulating gland
- skin
- small intestines (representative regions: duodenum, ileum, jejunum)
- spinal cord (at three levels: cervical, mid-thoracic and lumber
- spleen
- sternum
- stomach
- thymus
- thyroid + parathyroid
- trachea
- urinary bladder

HISTOPATHOLOGY : Yes (control and high dose group, not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle groups)
- ovaries
- uterus
- vagina
- testes
- epididymides
- prostate
- seminal vesicle with coagulating gland
- thymus (one male in low dose group due to hemorrhage)
The tissues indicated above were prepared for microscopic examination and weighed, respectively.
In particular, stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure were examined.

organ weights:
For all male adult animals:
- brain
- testes
- epididymides
- prostate
- seminal vesicle with coagulating gland
For five males and females randomly selected from each group:
- adrenals
- brain
- heart,
- kidneys
- liver
- spleen
- thymus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed latest at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY: Pups euthanized at day 13 post-partum, or shortly thereafter, and dead pups were carefully examined for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS: none

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index

Offspring viability indices:

- post implantation mortality
- post-natal mortality
- survival index
- sex ratio

Clinical signs:

no effects observed

Description (incidence and severity):

Daily:
No treatment-related signs of systemic toxicity were detected at any dose level at the daily clinical observations (100, 300 and 1000 mg/kg bw/day, male or female).
Brownish fur around the right eye was detected in one male animal at 300 mg/kg bw/day and alopecia was observed on the chest of one female in the same group. These findings are considered to be chance findings occurring commonly in experimental rats and not related to the test item. There were no clinical signs in any other animal.

Weekly:
The behavior and physical condition of animals was not affected by the test item at any dose level (100, 300 or 1000 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Findings were limited to brownish fur around the right eye and alopecia on the chest in the same two animals at 300 mg/kg bw/day (1/12 male on days 27, 34 and 41; and 1/12 dam on gestation day 21 and on lactation days 0, 4 and 13) and they were considered as not related to treatment with the test item. All other animals were symptom-free during the study at the detailed weekly clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no mortality in any group during the course of study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related effects were noted on the mean body weight and body weight gain values at 100, 300 or 1000 mg/kg bw/day. There were no significant differences between the control and test item treated groups in the body weight or body weight gain of male animals during the pre-mating, mating or post mating periods and of female animals during the pre-mating, gestation or lactation periods.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

There were no test item related differences in the mean daily food consumption in any test item treated group when compared to the control during the study.
Statistical significance was noted for the slightly lower mean daily food consumption in female animals at the 1000 mg/kg bw/day group between lactation days 0 and 4. The difference with respect to their control was small (-13 %) and transient. Therefore, this single variation in the 1000 mg/kg bw/day group in the lactation days 0-4 period was considered to be a chance finding and not test item related.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no pathological changes in the examined hematological parameters in any of the groups (male and female, 100, 300 or 1000 mg/kg bw/day).
The examined hematological parameters were comparable in male animals of control and all test item treated groups.
In the female animals of all dose groups statistical significance was observed for some parameters with respect to their controls as follows: at the lower mean percentage of monocytes (MONO) and basophil granulocytes (BASO) and at the higher mean percentage of reticulocytes (RET) at 100 mg/kg bw/day; higher mean percentage of lymphocytes (LYM) and reticulocytes, lower mean percentage of monocytes and basophil granulocytes at 300 mg/kg bw/day; lower mean percentage of neutrophil granulocytes (NEU) and monocytes and higher mean percentage of lymphocytes at 1000 mg/kg bw/day.
These sporadic statistical differences are considered to be of no toxicological relevance because the differences between the control and test item treated groups were small or they were due to relative higher or lower values of control group compared to the historical control, the values were within the historical controls and/or they were not related to doses.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
In the animals of all dose groups statistical significance was observed for some parameters with respect to their controls as follows: higher mean sodium (Na+) concentration at 100 mg/kg bw/day (male); elevated concentrations of urea at 300 mg/kg bw/day (female) and glucose at 100, 300 and 1000 mg/kg bw/day (female).
These sporadic statistical differences are considered to be of no toxicological relevance because the differences between the control and test item treated groups were small or they were due to relative lower values of control group compared to the historical control, the values were within the historical controls and/or they were not related to doses.

There were no test item related adverse effects in the free T4 levels at 100, 300 or 1000 mg/kg bw/day (male).
Slight but statistically significant difference with respect to the control in parental male animals was considered toxicologically not relevant in the absence of a clear dose dependency.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (100, 300 and 1000 mg/kg bw/day, control).

Immunological findings:

not examined

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 examinations did not reveal any test item related alterations in the organs or tissues of male or female animals at 1000 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals; full histopathology in selected animals).
In one animal (control male; 1/12) decreased amount of secretum in the seminal vesicle (one side) was observed, which was considered as an individual disorder without toxicological significance.
In other male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (spermatogonia, spermatocytes, 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, seminal vesicles, and coagulating glands was normal in all animals as well.
In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. 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.
A few lesions were noted but they are considered to be common observations within the range of findings for animals of this strain and age and without pathological significance:
- dilatation of uterine horns at 1000 mg/kg bw/day (1/12);
- pulmonary alveolar emphysema (minimal degree): 2/5 males and 2/5 females in the control, 2/5 males in the high dose group);
- alveolar histiocytosis: in one control male (1/5) animal;
- hyperplasia of bronchus associated lymphoid tissue (BALT): 1/5 control female and 2/5 high dose female animals;
- pulmonary alveolar emphysema (minimal degree): 2/5 males and 2/5 females in the control, 2/5 males in the high dose group;
- acute hemorrhage in the thymus: 1/1 at 100 mg/kg bw/day, male, 1/5 high dose female;
Pulmonary emphysema and acute hemorrhage were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Alveolar histiocytosis is a common incidental finding in elder rats and consists of small groups of alveolar macrophages with abundant foamy (phospholipids-containing) cytoplasm. These cells are often sub-pleural or located in the more peripheral regions of the lungs.
Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance.
Dilatation of the uterine horns is indicative of a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose levels (100, 300 or 1000 mg/kg bw/day).
There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous during the pre-mating period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects on testis weight and epididymis weight were observed.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the test item at 100, 300 or 1000 mg/kg bw/day (male and female).
There was a statistically significant difference between the control and test item treated groups of male animals at 300 and 1000 mg/kg bw/day in copulatory indices. One pair of both of these groups failed to mate during the 14-day period resulting in lower copulatory indices, which, however was considered to be not related to the test item as values met well the historical controls.
There were no differences between the control and test item treated groups in female animals in the pre-coital interval and conceiving days. The gestation indices of the test item treated groups (100, 300 and 1000 mg/kg bw/day) exceeded that of the control as one control female (1/12) did not deliver. Thus, this statistically significant difference with respect to the control was considered to be indicative of biological variation and not related to the test item.

The percentage of post-implantation loss of 16 % (23/145) was slightly increased (reaching statistical significance) in the 1000 mg/kg bw group compared to control (4 % = 5/136). However, the percentage of post-implantation loss for the historical control is 10 % (179/1737; no range provided in report) and thus the difference between the historical control and the highest treatment group is considerably smaller than to the control group. This indicates that an actual test substance related effect is questionable. The mean number of post-implantation loss (1.9/dam; SD=2.6, N=12) exceeded the control (0.5/dam; SD=0.9, N=11) without statistical significance. Also for this value the historical control mean (1.3/dam; SD=1.5; N = 143 dams) is considerably more similar to the highest dose group. Furthermore, no dose response relationship was observed for both, percentage of post-implantation loss (100 mg/kg bw/d: 8 % = 12/144, 300 mg/kg bw/d: 5 % = 6/131) and for mean number of post-implantation loss (100 mg/kg bw/d: 1.0/dam; SD=1.3; N = 12 dams, 300 mg/kg bw/d: 0.5/dam; SD=0.8; N = 12 dams). The mean number of total birth and live pups/litter correlated with these findings with respect to their control. However, the mean number of liveborn or viable pups were comparable to the historical control mean. As the observed differences were of a slight degree, without a clear dose-response relationship and the values remained comparable with the historical control data with a slightly higher difference for the percentage of post-implantation loss, a test item related effect is not considered.
The number of pregnant females and dams delivered, the mean number of implantations, duration of pregnancy, and pups births (total, live, still birth, viable) and live birth index were comparable in all groups (100, 300 and 1000 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related effects were observed.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of dead offspring per litter was similar in all groups (0 or 1). There were no significant differences between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day) 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 pup weights as well as the mean pup weight gain were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.
Slight changes - without statistical significance - were detected in the mean litter weights and litter weight gain; however, these differences were in good correlation with the number of pups per litter within the doses.

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:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects in the free T4 levels at 100, 300 or 1000 mg/kg bw/day (offspring sampled on postnatal day 13).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances (AGD; absolute and normalized) were not affected by the test item at 100, 300 and 1000 mg/kg bw/day.
Statistical significances were observed at the longer anogenital distances (absolute and normalized as well) in male pups of the 1000 mg/kg bw/day treated dams, at the shorter absolute anogenital distance in female pups at 1000 mg/kg bw/day as well as at the shorter normalized anogenital distance in female pups at 100, 300 and 1000 mg/kg bw/day. These minor changes were considered to be indicative of biological variations 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 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.

Organ weight findings including organ / body weight ratios:

not examined

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 changes in the organs or tissues of stillborn offsprings (3/3 control, 4/4 at 100 mg/kg bw/day, 2/2 at 300 mg/kg bw/day, 2/2 at 1000 mg/kg bw/day) subjected to necropsy on postnatal day 0 or in dead offsprings (1/1 control; 1/1 at 300 mg/kg bw/day) subjected to necropsy on postnatal day 0 or 1, respectively.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution
There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related effects were observed.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Tabulary summary of reproductive and developmental results:

Observations	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Pairs started (n)	12	12	12	12
Estrous cycle - mean length in days	4.1	4.2	4.0	4.4
- irregular cycle	1/12	2/12	1/12	1/12
Females showing copulation evidence	12	12	12	12
Females achieving pregnancy	12	12	12	12
Conceiving days 0 -5 (n)	12	12	12	11
Conceiving days 6 -11 (n)	0	0	0	1
Pregnancy <= 21 days (n)	0	0	0	0
Pregnancy = 22 days (n)	11	12	11	11
Pregnancy >= 21 days (n)	0	0	1	1
Dams with live young born (n)	11	12	12	12
Dams with live young at day 4 (n)	11	12	12	12
Implants/dam (mean)	12.4	12.0	10.9	12.1
Live pups/dam at birth (mean)	11.9	11.0	10.4	10.2
Live pups/dam at day 4 (mean)	11.8	11.0	10.3	10.2
Sex ratio (m/f) at birth	5.6/6.2	4.6/6.4	4.7/5.8	4.8/5.4
Sex ratio (m/f) at day 4	5.6/6.2	4.6/6.4	4.7/5.7	4.8/5.4
Litter weight at birth (mean; g)	73.7	68.9	66.5	63.3
Litter weight at day 4 (mean; g)	131.4	122.3	116.4	112.7
Pup weight at birth (mean; g)	6.3	6.3	6.5	6.3
Pup weight at day 4 (mean; g)	11.2	11.3	11.5	11.4
Normalised anogenital distance at day 4 (mean in mm; m/f)	2.8/1.9	2.8/1.9	2.9/1.8	3.0/1.8
Pup weight at day 13 (mean; g)	32.6	31.2	32.2	32.4
Number of nipples -male pups at day 13	0	0	0	0
ABNORMAL PUPS
Dams with 0/1/>=2	0/0/0	0/0/0	0/0/0	0/0/0
LOSS of OFFSPRINGS
Pre-natal/post implantation (implantations minus live birth)
Females with 0	8	7	8	5
Females with 1	2	0	2	2
Females with 2	1	3	2	2
Females with >= 3	1	2	0	3
Post-natal (live birth minus alive at post-natal day 13)
Females with 0/1/>=2	10/1/0	12/0/0	11/1/0	12/0/0

Conclusions:

In a study according to OECD 422 the NOAEL for the reproductive performance in males and females and for the development of F1 offsprings was 1000 mg/kg bw.

Executive summary:

The possible effects on reproduction and development of the test substance was investigated in an experimental study according to OECD test guideline 422 under GLP-conditions.

Four groups of Wistar rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day.

The suitability of distilled water as vehicle for the test item was analytically verified up front as the test item was stable in distilled water at room temperature for at least five days.

The concentration of the test item in the dosing formulations administered to the animals was confirmed twice during the study.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 55 days). Females were additionally exposed through the gestation period and up to lactation days 14 - 19, i.e. up to the day before necropsy (altogether for 56 to 67 days). 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.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (T4) from 2-6 pups per litter on post-natal day 4, from all dams and 2-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. T4 levels were measured in parental male animals and in offspring sampled on postnatal day 13.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, blood coagulation, clinical chemistry, gross necropsy, and organ weighing as well as full histopathology in the control and high dose groups.

There was no mortality in any group during the course of study.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations. At the same intervals, the behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period.

Test item related changes of the body weight or body weight gain were not detected. The body weight development was not disturbed and was comparable in the control and test item treated groups.

The mean daily food consumption was not affected by the test item in male or female animals at all test item groups during the entire study (pre-mating, post-mating, gestation and lactation periods).

There were no test item related differences between the control and test item treated groups in the gonadal function, mating behavior, conception, pregnancy, and parturition of male or female animals (100, 300 and 1000 mg/kg bw/day).

The post-implantation loss was statistically slightly increased in dams at 1000 mg/kg bw/day compared to the control group.

Clinical pathology examinations (hematology, blood coagulation, clinical chemistry and thyroid hormone levels) did not reveal toxicological relevant alterations related to the test item.

Specific macroscopic alterations related to the test item were not found at necropsy.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at the highest dose of 1000 mg/kg bw/day.

During full histopathology, there were no changes related to the test item in organs or tissues of selected animals (male or female) at 1000 mg/kg bw/day.

No adverse findings on the offspring’s development were detected (mortality, clinical signs and body weight, anogenital distance and nipple retention in male pups, necropsy).

Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Hsd.Han: Wistar rats at 100, 300 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 rats at the doses of 100, 300 or 1000 mg/kg bw/day and in parental females at the doses of 100 or 300 mg/kg bw/day.

The pre-natal loss (post-implantation loss; percentage and mean) was slightly higher in dams at 1000 mg/kg bw/day than in the control group. As the observed differences were of a slight degree, without a clear dose-response relationship and the values remained comparable with the historical control data, a test item related effect is not considered.

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 NOAEL for the reproductive performance in males and females and for the development of F1 offsprings was 1000 mg/kg bw.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted according to guideline and GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The possible effects on reproduction and development of the test substance was investigated in an experimental study according to OECD test guideline 422 under GLP-conditions.

Four groups of Wistar rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day.

The suitability of distilled water as vehicle for the test item was analytically verified up front as the test item was stable in distilled water at room temperature for at least five days.

The concentration of the test item in the dosing formulations administered to the animals was confirmed twice during the study.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 55 days). Females were additionally exposed through the gestation period and up to lactation days 14 - 19, i.e. up to the day before necropsy (altogether for 56 to 67 days). 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.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (T4) from 2-6 pups per litter on post-natal day 4, from all dams and 2-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. T4 levels were measured in parental male animals and in offspring sampled on postnatal day 13.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, blood coagulation, clinical chemistry, gross necropsy, and organ weighing as well as full histopathology in the control and high dose groups.

There was no mortality in any group during the course of study.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations. At the same intervals, the behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period.

Test item related changes of the body weight or body weight gain were not detected. The body weight development was not disturbed and was comparable in the control and test item treated groups.

The mean daily food consumption was not affected by the test item in male or female animals at all test item groups during the entire study (pre-mating, post-mating, gestation and lactation periods).

There were no test item related differences between the control and test item treated groups in the gonadal function, mating behavior, conception, pregnancy, and parturition of male or female animals (100, 300 and 1000 mg/kg bw/day).

The post-implantation loss was statistically slightly increased in dams at 1000 mg/kg bw/day compared to the control group.

Clinical pathology examinations (hematology, blood coagulation, clinical chemistry and thyroid hormone levels) did not reveal toxicological relevant alterations related to the test item.

Specific macroscopic alterations related to the test item were not found at necropsy.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at the highest dose of 1000 mg/kg bw/day.

During full histopathology, there were no changes related to the test item in organs or tissues of selected animals (male or female) at 1000 mg/kg bw/day.

No adverse findings on the offspring’s development were detected (mortality, clinical signs and body weight, anogenital distance and nipple retention in male pups, necropsy).

Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Hsd.Han: Wistar rats at 100, 300 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 rats at the doses of 100, 300 or 1000 mg/kg bw/day and in parental females at the doses of 100 or 300 mg/kg bw/day.

The pre-natal loss (post-implantation loss; percentage and mean) was slightly higher in dams at 1000 mg/kg bw/day than in the control group. As the observed differences were of a slight degree, without a clear dose-response relationship and the values remained comparable with the historical control data, a test item related effect is not considered.

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 NOAEL for the reproductive performance in males and female and for the development of F1 offsprings was 1000 mg/kg bw.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on toxicity to reproduction, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.