Reaction mass of 2,2'-(vinylenedi-p-phenylene)bis[5-methylbenzoxazole] and 2,2'-(vinylenedi-p- phenylene)bisbenzoxazole and 2-[4-[2-[4-(benzoxazol-2- yl)phenyl]vinyl]phenyl]-5-methylbenzoxazole

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL for reproductive performance of male/female rats: 900 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 May 2 to September 4, 2019

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:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Han: WIST

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 nulliparous and non-pregnant: yes
- Age at study initiation: 76 - 85 d (M and F)
- Weight at study initiation: 347 - 429 g (M); 211 - 262 g (F); weight variation did not exceed ± 20 % of the mean weight
- Housing: before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female / cage; mated females: individually; males after mating: 2 animals / cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

other: 1 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance was formulated in the vehicle in concentrations of 18, 60 and 180 mg/ml. Formulations were prepared beforehand not longer than for three days and stored at 5 ± 3 °C until use.

DIET PREPARATION
Animals received ssniff® SSNIFF Rat/Souris Elevage E, 10 mm autoclavable complete feed and ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum.
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.

VEHICLE
- Concentration in vehicle: 1 %
- Amount of vehicle: 5 ml/kg
- Lot/batch no.: CM2901147

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation
- Proof of pregnancy: vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female was caged: individually

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 10 ml (first occasion) or 5 ml (second occasion) of each formulation and five aliquots of 10 ml (first occasion) or 5 ml (second occasion) control substance (vehicle) were taken and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 93 and 102 % of the nominal values at both analytical occasions.
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 94 and 107 % at ca. 1 mg/ml and between 102 and 108 % at ca. 300 mg/ml).
Test item proved to be stable in 1 % methylcellulose at the intended concentrations at room temperature for one day and at 5±3°C for three days.

Duration of treatment / exposure:

36-64 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 (occurring after 20 days of acclimatisation without dosing).

Frequency of treatment:

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

Dose / conc.:

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

Remarks:

group 1 (control)

Dose / conc.:

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

Remarks:

group 2

Dose / conc.:

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

Remarks:

group 3

Dose / conc.:

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

Remarks:

group 4

No. of animals per sex per dose:

Number of animals/group: 12 animals/sex in the control and dose groups (at least 8 pregnant female animals per group were expected)
Number of groups: 4 (3 dose levels + 1 control group)

Control animals:

yes, concurrent vehicle

Details on study design:

Study design

a) Males
A PM M ↓
Acclimatization period Pre-mating period Mating Post mating period Blood sampling, necropsy, organ weighing
20 days 14 days 1-16 days 6-21 days day 36


b) Females
A PM M G ↓ PP/PN ↓
Acclimatization period Pre-mating period Mating Gestation period** Delivery Lactation period Blood sampling on lactation day 13; necropsy on lactation days 13-16
20 days* 14 days 1-16 days 22-days 13-16 days days 51-64

* pre-treatment examination of estrous cycle included
** for not delivered and non-pregnant female animals necropsy was conducted on day 40.

Dosing of both sexes begun after 20 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy. Rats of this strain reach full sexual maturity at the age of 10 weeks. The mating phase started after 14-days treatment (pre-mating) period.
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). Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Males were dosed for 36 days (14 days pre-mating and 1-16 days mating plus 6-21 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 1-16 days mating period and throughout pregnancy and at least up to and including day 12 post-partum or the day before sacrifice. The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.
Blood samples for assessment of serum thyroid hormones (FT4 and TSH) were collected from all parental male and female animals. All animals were subjected to a full detailed gross necropsy. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all males were weighed. F1 offspring were observed for clinical signs, litter weight, gender, body weight, anogenital distance and nipple retention. Blood samples for serum FT4 and TSH assessment were pooled from 2-7 pups per litter on post-natal day 4 (where it was feasible) and from 3-7 pups on post-natal day 13.

Positive control:

no

Parental animals: Observations and examinations:

Mortality
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

Clinical observations
General clinical observations were made on parental animals once a day, after the administration at approximately the same time, considering the peak period of anticipated effects after dosing.
Clinical 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
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 measurement
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 20, 27 and 34 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).

Examination of placental sign
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If negative on day 13, the examination was repeated on day 14 of gestation.

Observation of the delivery process
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations and any evidence of abnormal deliveries were recorded. The duration of gestation was recorded and is calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Clinical biochemistry
Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) as follows:
- from 2-7 pups per litter on post-natal day 4 (if it was feasible) - pup blood was pooled by litter;
- from all dams and 3-7 pups per litter on post-natal day 13;
- from all parent male animals at termination on day 36;
- from all not delivered and non-pregnant female animals at termination on day 40.

Blood samples collected for thyroid hormones (FT4 and TSH) measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A (no anticoagulant). Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. All samples for FT4 and TSH determination were stored between minus 15 and minus 30 °C until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones (FT4 and TSH). Further assessment of FT4 and TSH in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in post-natal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (FT4 in ng/dl and TSHin µIU/ml) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.

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):

Parameters examined in P male parental generation:
- detailed histological examinations were performed on testes, epididymides, prostate, and seminal vesicles with coagulating glands – 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.

Postmortem examinations (parental animals):

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 Isofluran CP®.
After examination of the external appearance, the cranial, thoracic and abdominal cavities 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.
All organs showing macroscopic lesions were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above).

Organ weight
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.

Histopathology
Detailed histological examinations were performed on the ovaries, uterus, testes, epididymides, prostate, and seminal vesicles with coagulating glands – 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 dam with full litter death, in not mated male animal, in non-pregnant female animals and males these females cohabited with in the low and mid dose groups:
- males: 206, 210, 305, 309;
- females: 226, 230, 329, 331;
In addition, the following organs were processed and examined due to the macroscopic findings:
- kidneys: 305, 311, 408, 229, 425, 428;
- liver: 224;
- skin: 121, 230;
- spleen: 331;
- kidneys in offspring: 322/M;
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.

Postmortem examinations (offspring):

Stillborn pups 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.

Statistics:

The following parameters were examined and evaluated statistically

Parental males
Body weight, body weight gain, food consumption, percentage of fertile males, percentage of infertile males, copulatory index, fertility index, thyroid hormone (FT4 and TSH) level, organ weights (absolute and relative to the body and brain weights)

Parental females
Body weight, body weight gain, food consumption, percentage of pregnant rats, percentage of non-mated females, copulatory index, fertility index, gestation index, duration of pregnancy (days), number of implantations / dams, post-implantation mortality

Offspring
Litter weight on PN days 0, 4 and 13, mean body weight gain per litter between PN days 0-4, 4-13 and for overall PN days, no. of live births per litter, and no. of viable pups per litter on PN days 0 and 13, survival index of pups on PN day 13, sex ratio % (on PN days 0 and 13), normalized anogenital distance, no. of nipples/areolae in male pups, thyroid hormone (FT4 and TSH) level of pups on PN day 13.

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
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) is 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:

Copulatory index: measure of animals’ ability to mate
Males = 100 × no. of males with confrimed mating / total no. of males cohabited
Females = 100 × no. of sperm positive females / total no. of females cohabited

Fertility index: measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males = 100 × no. of males impregnating a female / total no. of males with confirmed mating
Females = 100 × no. of pregnant females / no. of sperm positive females

Gestation index: measure of pregnancy that provides at least one live pup
100 × no. of females with live born pups / no. of pregnant females

Offspring viability indices:

Post-implantation / pre-natal mortality (intrauterine mortality) = 100 × (no. of implanattions - no. of liveborns) / no. of implantations

Post-natal mortality / extra-uterine mortality = 100 × (no. of liveborns - no. of livepups on PN 13) / no. of liverborns

Survival Index = 100 × no. of live pups on PN 13 / no. of liveborns

Sex ratio = 100 × (no. of pups examined - no. of males (females)) / no. of pups examined

Normalized anogenital distance = absolute anogenital distance / (body weight)^(1/2)

Clinical signs:

effects observed, non-treatment-related

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 90, 300 and 900 mg/kg bw/day during the clinical observations.
There were no clinical signs in male animals in the control, 90, 300 or 900 mg/kg bw/day groups during the entire observation period (pre-mating, mating and post-mating periods).
Clinical signs were noted for some female animals as follows:
- brownish discharge in the vagina in not delivered female animal (1/12) in control group on gestation day 23;
- alopecia on the skin of fore limbs and chest in one dam (1/11) in control group between lactation days 0-12 and between lactation days 6-12, respectively;
- scar on the neck in one of the non-pregnant females at 90 mg/kg bw/day (1/2) during the post-mating period – between days 30 and 39;
Brownish discharge in vagina was presumably due to failed delivery of one control female animal.
Dermal alterations (scar and alopecia) are common in experimental rats of this strain occurring in not treated animals.
The mentioned signs were only detected in the control and low dose treated animals and there were no related findings. Therefore, these clinical signs were judged to be toxicologically not relevant.

Mortality:

no mortality observed

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 90, 300 or 900 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 34. Statistical significance with respect to the control was observed at the slightly lower mean body weight gain of male animals at 300 and 900 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 90, 300 and 900 mg/kg bw/day during the pre-mating, gestation and lactation periods. Statistical significance with respect to the control was only noted for the higher mean summarized mean body weight gain of female animals administered with 90 mg/kg bw/day during the pre-mating period. This slight change in the mean body weight gain was considered to be toxicologically not relevant as it had no influence on the mean body weight and similar finding was not detected in animals at the higher doses.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum thyroid hormones: there were no statistically significant differences with respect to the control in the thyroid hormone (FT4 and TSH) levels in parental male animals.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the organs of animals subjected to histopathological examination, no lesions appearing only in the treated animals in function of applied doses, thus unanimously attributable to the test item were detected.
Hyperplasia of hematopoietic cells and lymphoid cells in the spleen associated with focal enlargement was observed in one female animal at 300 mg/kg bw/day (1/1). Extramedullary hematopoiesis in the red pulp and development of lymphoid cells in the white pulp is normally present in the spleen of rats. Hyperplasia of hematopoietic cells or lymphoid cells are commonly observed in the spleen associated with different proliferative stimuli and is considered as slight reversible lesion. Hyperplasia was observed in one mid dose treated dam and was considered as an incidental finding, without toxicological significance.
Focal fibrosis in the Glisson’s capsule of liver (1/1 female at 90 mg/kg bw/day) is due to the diaphragmatic hernia, in association with the mechanical irritation of serous capsule of the liver.
One or both sided pyelectasia was detected with low incidence at 90 mg/kg bw/day (1/1 female), at 300 mg/kg bw/day (2/2 male) and at 900 mg/kg bw/day (1/1 male and 2/2 female) groups. This renal finding – without other pathological lesions, degeneration, inflammation, fibrosis etc.) – is common in laboratory rats without toxicological significance.
Histological examinations revealed atrophy of hair follicles in one control dam (1/1) and subacute dermatitis in other one at 90 mg/kg bw/day (1/1). These dermal changes were without inflammation, parasites or fungal infection and were considered as individual diseases.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Test item influence on the estrous cycle was not detected at any dose level (90, 300 and 900 mg/kg bw/day).
Statistical significance was detected at the lower percentage of regular cycles in animals appointed for the 90 and 300 mg/kg bw/day when compared to the animals appointed for the control group. All other examined parameters of estrous cycle were comparable in animals appointed for the control and test item dose groups during the pre-treatment period.
The percentage of animals with regular cycles was slightly lower than in the control group at 90, 300 and 900 mg/kg bw/day during the pre-mating period. Statistical significance was noted for the less mean number of days in pre-estrous in animals at 90 mg/kg bw/day group during the pre-mating period.
These findings were considered to be indicative of biological variation and independent from the treatment. There was no dose dependency during the pre-mating period and the changes in the estrous cycle had no influence on the mating or reproductive performance of these animals.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the male animals belonging to the 900 mg/kg bw/day group (12/12) and to the control group (12/12) and at 90 and 300 mg/kg bw/day (2/2, both doses), the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism.
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 was normal in all cases as well.

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 90, 300 or 900 mg/kg bw/day.
Statistically significant difference with respect to the control were detected at the lower copulatory index in male animal administered with 300 mg/kg bw/day as one animal (no. 305) failed to mate their partner during the two-weeks mating period.
The fertility index (percentage of fertile males) was slightly lower than in the control group in male animals at 90 and 300 mg/kg bw/day because of infertile mating of two or three pairs, respectively. The percentage of male animals estimated to be infertile was thus slightly higher than in the control group at 90 and 300 mg/kg bw/day.
The fertility index of female animals (percentage of pregnants) was slightly lower than in the control at 90 and 300 mg/kg bw/day, while the percentage of dams delivered (gestation index) exceeded the control at 90, 300 and 900 mg/kg bw/day.
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 low and mid dose groups but not in the high dose group.

Delivery data of dams
There were no differences in the evaluated delivery parameters of dams between the control and test item treated groups (90, 300 or 900 mg/kg bw/day).
The mean number of implantation sites per dams were comparable in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, mean number of implantations, mean number of births (total, live and stillborn), in the live birth indices, in pre- and post-natal loss or in total loss.

Histopathology of reproductive female organs
In the female animals belonging to the 900 mg/kg bw/day (12/12) and control (12/12) groups, as well as to the 90 and 300 mg/kg bw/day (2/2, both doses) groups, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. 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 were normal in all cases as well.
In one female animal in the control group (1/12 not giving birth), the dilatation of uterine horn and acute hemorrhage into horn were individual disorders.
Dilatation of uterine horn in one dam (1/2 at 300 mg/kg bw/day) – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrous phase) of uterus without pathological significance.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

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.
The percentage of offspring with signs (cold, not suckled) was low and similar in the control, 90 and 900 mg/kg bw/day groups. At 300 mg/kg bw/day, the percentage of cold and not suckled pups was slightly higher than in the control group.
Hemorrhage was noted for one pup at 300 mg/kg bw/day on the day of birth and this pup was cannibalized (missing pup) up to the next day.
These signs and differences with respect to the control were considered to be toxicologically not relevant as the signs were transient and were not associated with depression of the development of the offspring.

Mortality / viability:

mortality observed, non-treatment-related

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 (90, 300 or 900 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 (90, 300 or 900 mg/kg bw/day) groups in the survival indices.

Body weight and weight changes:

effects observed, non-treatment-related

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 (90, 300 and 900 mg/kg bw/day) on post-natal days 0, 4 and 13.
If evaluated male and female pups separately, statistical significance with respect to the control was detected at the slightly lower mean body weight of male and female pups at 300 mg/kg bw/day and at the slightly higher mean body weight of female pups at 90 mg/kg bw/day on post-natal day 4. These minor changes were considered to be toxicologically not relevant and not related to the treatment as these were detected at the low and mid dose groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum thyroid hormones: there were no statistically significant differences with respect to the control in the thyroid hormone (FT4 and TSH) levels in PN13 offspring at any dose levels.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances (absolute and normalized in male or female offspring) were not affected by the test item treatment at 90, 300 and 900 mg/kg bw/day.
Statistical significances were observed at the higher absolute and normalized anogenital distances in male and female offspring at 90 and 900 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):

Nipple retention (male) was not affected by the test item treatment at 90, 300 and 900 mg/kg bw/day.
Nipples/areoles were not visible in any of the examined male offspring in the control or 90, 300 or 900 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 stillborn male pup at 90 mg/kg bw/day subjected to necropsy on post-natal day 0.
One side pyelectasia was noted for one offspring at 300 mg/kg bw/day (1/20) groups at the terminal necropsy. Pyelectasia is a common finding in laboratory rats without toxicological significance. This renal change in offspring was not accompanied by other pathological lesions, degeneration, inflammation, fibrosis etc. – as supported by histological examinations.

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 post-natal days 0, 4 or 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under test conditions, test item did not cause signs of systemic toxicity in parental male and female Han: Wistar rats at 90, 300 or 900 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 90, 300 or 900 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, NOAELs were determined as follows:
NOAEL for systemic toxicity of male/female rats: 900 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 900 mg/kg bw/day
NOAEL for F1 offspring development: 900 mg/kg bw/day

Executive summary:

Method

The study was run according to OECD guideline 421, as a screening test on reproductive toxicity of test item. Male and female rats were repeatedly dosed orally (by gavage) at doses of 90, 300 and 900 mg/kg bw/day.

Possible effects on male and female reproductive performance were assessed in terms of gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to post-natal day 13 associated with administration of repeated maternal doses.

Four groups of Han:WIST rats consisting of 12 animals per group and sex were dosed orally (by gavage) once daily at 0 (vehicle only), 90, 300 and 900 mg/kg bw/day doses with a 5 ml/kg bw dosing volume. A group of vehicle (1 % methylcellulose) treated animals (n= 12/sex) served as a control.

The concentration of the test item in the dosing formulations administered to animals was checked two times during the study and found to vary in the acceptable range between 93 % and 102 % of the nominal values.

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 36 days). Females were additionally exposed through the gestation period and up to lactation days 12-15, i.e. up to the day before necropsy (altogether for 51-64 days). Non-pregnant and not delivered female animals were administered for 40 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 as well as on the day of the necropsy.

Blood samples were collected for determination of serum levels of thyroid hormones (FT4 and TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and 3-7 pups per litter on post-partum/ post-natal day 13 and from all parent male animals and from non-pregnant and not delivered females at termination on day 40.

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.

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

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the possible subsequent histopathological examination. Gross necropsy was performed for offspring selected for thyroid gland preservation.

Histopathology examination was performed on ovaries, uterus, testes, epididymides, prostate, seminal vesicles with coagulating gland in the control and high dose groups. These organs were also processed and examined histologically in not mated male animals and in not delivered and non-pregnant females and males these females cohabited with in the low and mid dose groups.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the control, low, mid and high dose groups including offspring.

Results

Mortality

There was no test item related mortality at any dose level (90, 300 and 900 mg/kg bw/day).

 

Clinical observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, either at the daily or at the detailed weekly clinical observations.

 

Body weight and body weight gain

The body weight development was not affected by the test item in male or in female animals at any dose level during the entire treatment period.

 

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at any doses during the entire study.

 

Estrous cycle

A test item influence on the estrous cycle was not detected at any dose level.

 

Delivery data of dams

There were no toxicologically relevant differences between the control and test item treated groups in the evaluated delivery parameters of dams.

 

Reproductive performance

There were no test item related differences between the control and test item treated groups in the examined parameters of reproductive performance of male or female animals.

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in parental male animals or in PN13 offspring.

 

Necropsy

Specific macroscopic alterations were not detected in male or female animals in any group.

 

Organ weight

The weight of testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole was similar in the control group and at any dose level.

 

Histopathology

Histopathological examinations did not reveal pathological alterations in the investigated organs or tissues in male or female animals at 900 mg/kg bw/day (uterus, testes, epididymides, prostate, seminal vesicles with coagulating gland).

 

Offspring

The offspring’s development was not affected at any dose level. No adverse effect on mortality, clinical signs, body weight or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distances (male and female) or nipple retention (male) were not influenced.

 

Conclusion

Based on these observations, NOAELs were determined as follows:

NOAEL for systemic toxicity of male/female rats: 900 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 900 mg/kg bw/day

NOAEL for F1 offspring development: 900 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

900 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality and reliability

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 study was run according to OECD guideline 421. Groups of 12 rats/sex were repeatedly dosed orally (by gavage) at doses of 0 (vehicle only), 90, 300 and 900 mg/kg bw/day.

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 36 days). Females were additionally exposed through the gestation period and up to lactation days 12-15, i.e. up to the day before necropsy (altogether for 51-64 days). Non-pregnant and not delivered female animals were administered for 40 days.

Observations included: mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. In addition, estrous cycle was monitored during pre-mating and mating period until evidence of mating as well as on the day of the necropsy; serum levels of thyroid hormones FT4 and TSH were measured in all damns on post-partum day 13 and from all parent male animals and from non-pregnant and not delivered females at termination on day 40.

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

Thyroid gland was preserved from all adult males and females for the possible subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, testes, epididymides, prostate, seminal vesicles with coagulating gland in the control and high dose groups. These organs were also processed and examined histologically in not mated male animals and in not delivered and non-pregnant females and males these females cohabited with in the low and mid dose groups.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of all groups.

There was no test item related mortality at any dose level (90, 300 and 900 mg/kg bw/day).

No test item related effects at any dose level were seen in terms of: mortality, clinical signs, changes in body weight and body weight gain, food consumption, estrous cycle, delivery of dams, reproductive performance, organ weight, serum thyroid hormones, relevant changes recorded by necropsy and hystopathology.

Based on these observations, NOAELs were determined as follows:

NOAEL for systemic toxicity of male/female rats: 900 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 900 mg/kg bw/day

Effects on developmental toxicity

Description of key information

NOAEL for F1 offspring development: 900 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

900 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality and reliability

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The study was run according to OECD guideline 421. Groups of 12 rats/sex were repeatedly dosed orally (by gavage) at doses of 0 (vehicle only), 90, 300 and 900 mg/kg bw/day.

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 36 days). Females were additionally exposed through the gestation period and up to lactation days 12-15, i.e. up to the day before necropsy (altogether for 51-64 days). Non-pregnant and not delivered female animals were administered for 40 days.

Observations were carried out on offspring. Blood samples were collected for determination of serum levels of thyroid hormones (FT4 and TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4 and 3-7 pups per litter on post-partum/ post-natal day 13.

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.

Thyroid gland was preserved from one male and one female pup per litter for the possible subsequent histopathological examination. Gross necropsy was performed for offspring selected for thyroid gland preservation.

Organs showing macroscopic findings were processed and examined histologically in animals of all groups including offspring.

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in parental male animals or in PN13 offspring.

The offspring’s development was not affected at any dose level. No adverse effect on mortality, clinical signs, body weight or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distances (male and female) or nipple retention (male) were not influenced.

NOAEL for F1 offspring development: 900 mg/kg bw/day

 

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

For the purpose of classification for reproductive toxicity, substances are allocated to one of two categories. Within each category, effects on sexual function and fertility, and on development, are considered separately.

CATEGORY 1

Known or presumed human reproductive toxicant Substances are classified in Category 1 for reproductive toxicity when they are known to have produced an adverse effect on sexual function and fertility, or on development in humans or when there is evidence from animal studies, possibly supplemented with other information, to provide a strong presumption that the substance has the capacity to interfere with reproduction in humans. The classification of a substance is further distinguished on the basis of whether the evidence for classification is primarily from human data (Category 1A) or from animal data (Category 1B).

Category 1A

Known human reproductive toxicant: the classification of a substance in Category 1A is largely based on evidence from humans.

Category 1B

Presumed human reproductive toxicant: the classification of a substance in Category 1B is largely based on data from animal studies. Such data shall provide clear evidence of an adverse effect on sexual function and fertility or on development 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 other toxic effects. However, when there is mechanistic information that raises doubt about the relevance of the effect for humans, classification in Category 2 may be more appropriate.

CATEGORY 2

Suspected human reproductive toxicant: 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.

Based on available experimental evidences, Fluorescent Brightner 219 is not classified for reproductive toxicity within the CLP Regulation (EC 1272/2008).