Reaction mass of isomers disodium 8-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-5-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, disodium 5-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-8-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL (male rats)= 1000 mg/kg body weight/day
NOAEL (female rats)= 300 mg/kg body weight/day
NOAEL (fetal and developmental toxicity)=1000 mg/kg body weight/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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

Qualifier:

according to guideline

Guideline:

other: Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 27 July 1995

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10-11 wks (male and females); (F1) x wks
- Weight at study initiation: (P) Males: 277-315 g; Females: 186-216 g; (F1) Males: x-x g; Females: x-x g
- Housing: individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: free access to diet for rats and mice
- Water: free access to tap water, sulphur acidified to a pH of ca. 2.8
- Acclimation period: at least 5 days under laboratory conditions

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

The vehicle used in this study was aqua ad injectionem (Diprom).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the definite volume of sterile water was added. The preparation was sonicated for 10-15 min and finally the concentration of solution was adjusted by dilution with sterile water.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION ANALYSIS:
Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).
All formulation samples were analysed on the day of sample collection and were stored at -20° C. These samples were analysed after the completion of the toxicity study.

Duration of treatment / exposure:

54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

once daily

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high dose (HD)

Dose / conc.:

300 mg/kg bw/day

Remarks:

medium dose (MD)

Dose / conc.:

100 mg/kg bw/day

Remarks:

low dose (LD)

Dose / conc.:

0 mg/kg bw/day

Remarks:

control group (C)

No. of animals per sex per dose:

10 male and 10 female animals per group, 80 animals (40 males and 40 females)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: According to the results of the 28-Day Repeated dose oral toxicity study in Wistar rats (BSL project no. 131051) no mortality occurred and no severe clinical signs were observed during the course of the study. Increased in liver weight was observed at the end of the treatment period at 300 and 1000 mg/kg bw/day. However, after completion of the treatment, liver weights recovered. Preliminary histopathologic data do not indicate effects on sexual organs. Considering the experimental design and the aim of this study and in consultation with the sponsor the doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C)
- Rationale for animal assignment (if not random): Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Females showing signs of abortion or premature delivery prior to the scheduled day of necropsy were sacrificed and subjected to a thorough macroscopic examination.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

OTHER: Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by toe tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

SACRIFICE
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 4 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24432, expiry date: 01/2015 and Serumwerk, lot no: 00512, expiry date: 07/2014).
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (LD 53, MD 67 and HD 76) were sacrificed on presumed gestation day 26 considering the last day of mating as last possible sperm positive day or using the sperm-positive vaginal smear as an evidence of mating and gestation day 0.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were preserved in modified Davidson’s Solution.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 24 to 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery.

HISTOPATHOLOGY / ORGAN WEIGHTS
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanized due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 μm and stained with hematoxylin and eosin and were examined in Control and HD animals. Female reproductive organs were also evaluated in all females of LD and MD groups. Any gross lesion macroscopically identified was examined microscopically in all animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.
The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.
The gestation length, pre coital interval, the number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, litter size and litter weights were summarized in tabular form.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights and food consumption were also presented as figures. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and are presented as percentage.

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software
(p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Male: There were no clinical signs of toxicological relevance noted in male animals during the treatment period. Some males treated at the highest dose (1000 mg/kg/day) showed only slight and transient signs of local reaction (salivation and action of pushing the head through the bedding) after treatment.
Female: There were no clinical signs of toxicological relevance noted in the females during the treatment period which reached terminal sacrifice. Some female animals treated at the highest dose (1000 mg/kg/day) and two MD female animals showed slight and transient signs of local reaction (salivation and action of pushing the head through the bedding) after treatment. In three female HD animals (No. 71, 74 and 79) that were sacrificed earlier on lactation day 0 or 1, adverse signs of toxicity with abnormal breathing due to swollen tongue or cheek were observed and one animal in moribund status showed clonic convulsions and tremors. In one further female found dead on Gestation day 22 shortly after littering (No. 80), a swollen tongue was observed at necropsy. In these 4 animals, littering seemed to be a trigger of the deteriorated health condition, as no preceding signs of toxicity were observed before littering.

Mortality:

mortality observed, treatment-related

Description (incidence):

Male: No mortality occurred in the control or any of the dose groups during the treatment period of this study in the male animals.
Female: In the control and lower dose groups (LD, MD) no mortality was recorded in female animals. At the highest dose, two animals were found dead (one in the pre-mating phase and one shortly after littering), and 4 animals were sacrificed earlier due to deteriorated health (three on postnatal day 0 or 1, and one in the premating phase). One death was attributed to misgavage (No. 78) and for the 5 other decedents histopathological evaluation confirmed a test item related cause of death. For 2/5 decedents (No. 75 and No.80) the cause of death was considered probably test item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male: No adverse test-item effects were observed in mean body weight and gains and food consumption during the treatment period in male animals.
Female: In HD female animals a tendency towards lower weight gain in the first week of pre-mating and the first two weeks of gestation was observed without reaching statistical significance. At the end of gestation body weight was approximately 9% below controls in the female HD group. In female animals, during the pre-mating and gestation period a dose dependent effect was observed on food consumption. In the second week of pre-mating, the food consumption was between 15 and 19% lower in the test-item treated groups as compared to control, these effects reaching statistical significance. In the first week of treatment, in female HD animals the food consumption was already 15% lower as compared to control. During gestation the same tendency was observed and during the second week of gestation, in MD and HD animals the food consumption was respectively 13 to 23% lower respectively as compared to control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in the control and test item treated groups.
In male animals no effect on food consumption was observed in the two pre-mating weeks in all test-item treated groups as compared to control group.
In female animals, during the pre-mating and gestation period a dose dependent effect was observed on food consumption. In the second week of pre-mating, the food consumption was between 15 and 19% lower in the test-item treated groups as compared to control, these effects reaching statistical significance. In the first week of treatment, in female HD animals the food consumption was already 15% lower as compared to control. During gestation the same tendency was observed and during the second week of gestation, in MD and HD animals the food consumption was respectively 13 to 23% lower as compared to control animals. During lactation no clear effect was seen, but it is worth noting that in
HD group, only 3 animals were remaining for evaluation.
In light of the slightly reduced weight gain and the test-item related intercurrent deaths observed in the HD female group, the dose dependent observed effect on food consumption in female animals is considered as test item related and of toxicological relevance.

Haematological findings:

no effects observed

Description (incidence and severity):

On lactation day 4, in the test-item treated female animals for which blood sampling has been performed no toxicologically relevant differences as compared to control were observed. The statistically significant higher % of basophiles in HD female animals as compared to control (0.37 % versus 0.15 %) observed is not assumed to be biologically relevant, as the values remained within the normal range of variation for female animals of this strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On lactation day 4, in the test-item treated female animals for which blood sampling has been performed all parameters with exception of total bile acids did not reveal any differences as compared to control group values. In two of the 3 HD females measured total bile acid level was higher as compared to control values. The mean HD group value was statistically significantly higher as compared to the mean control group value (2 x increases). In the MD group, some single animals had also higher values as compared to control. The values in the HD and MD groups remained within the normal range of variation for female animals of this strain. In view of the centrilobular hepatocellular hypertrophy observed in all HD decedents at histopathology, this higher level observed in HD females is considered to be a non-adverse adaptive finding.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Male: No test item-related histological findings were observed in male reproductive organs in this study.
Female: In two HD female decedents (No. 75, No. 78) submitted to necropsy during the early treatment phase, both animals were found not to be pregnant and had a dark discoloured thymus. In addition, No. 78 had also dark discoloured ileum, Peyer's patch, lung, trachea and uterus, and its thoracic cavity was blood-filled, for this animal histological evaluation confirmed an incidental cause of death.
Rats Nos. 71, 74 and 79 were sacrificed moribund towards the end of the treatment phase. The predominant macroscopic findings in one or several of these animals were fluid distension of salivary glands, mandibular lymph node and periocular tissue, swollen tongue and swollen skeletal muscle of the cheek, as well as fluid-filled thoracic cavity with small thoracic organs. Female No. 80 was found dead towards the end of the treatment phase (gestation day 22, found dead shortly after littering) and presented also an enlarged tongue, as well as dark discoloured lung and thymus.
Six females treated at 1000 mg/kg/day died or were sacrificed moribund before the end of the treatment phase. One spontaneous death (No. 78) was attributed to misgavage, and the cause of death of the other five decedents was considered to be clearly (3/5; No. 71, 74, and 79) or probably (2/5; No. 75 and No. 80) test item-related. Among the numerous histopathological findings noted in the decedents, mixed cell inflammation with edema of skeletal muscle and other tissues at the head region, corroborating macroscopic swollen tongue and cheek muscles, were the most prominent findings in the three decedents (No. 71, 79 and 80). Other findings comprised multifocal tubular necrosis of the renal medulla, multifocal mixed cell (peri)vasculitis in the lung and uterus, acute thymic degeneration and single cell death. All six decedents presented a centrilobular hepatocellular hypertrophy of the liver and sinus histiocytosis of one or several lymph nodes, and 4/6 decedents showed a diffuse cortical hypertrophy of the adrenal gland.
At terminal sacrifice, no clearly test item-related pathological finding was detected in female reproductive organs at 1000 mg/kg/day, and 3/4 high dose females killed at terminal sacrifice showed typical post-partum histomorphology of their reproductive organs. One high dose female was found not to be pregnant at terminal sacrifice and did not show typical indication of sexual cycling. It presented a minimal multifocal mixed cell (peri)vasculitis in the uterus as well as uterine distension. A test item relationship of these findings was considered possible.
There was no indication of any test item-related effect on female reproductive organs at 100 and 300 mg/kg/day, and the isolated non-pregnant females treated at 100 and 300 mg/kg/day showed physiological sexual cycling.

Reproductive performance:

no effects observed

Description (incidence and severity):

Females: No treatment-related effect was observed for the precoital interval or for the duration of gestation in the test-item treated groups as compared to control. Successful mating resulted in 10/10 pregnancies in the C group, 9/10 pregnancies in the LD group, 9/10 pregnancies in the MD group and 7/8 pregnancies in HD group. No effects on fertility index and delivery index were observed in the test item treated groups as compared to control group.
The groups mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group.
On lactation day 4, no toxicologically relevant differences were observed in haematological parameters measured in the test-item treated female animals as compared to control animals.
On lactation day 4 differences were observed in clinical biochemistry. In two of the three HD females total bile acid level was higher as compared to control values. The mean HD group value was statistically significantly higher as compared to the mean control group value (2 x increases). The values in the HD group remained within the normal range of variation for female animals of this strain. In view of the centrilobular hepatocellular hypertrophy observed in all HD decedents at histopathology, this higher level observed in HD females is considered to be a non-adverse adaptive finding.


There were no effect of toxicological relevance noted for copulation index, fertility index and delivery index. A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the HD (88%) as compared to 100% in control, as one of the 8 HD female mated was not pregnant. This rate remains within the fertility index commonly observed for animals of this strain.
The viability index was reduced in the HD group (-50%) when compared with the control group, as three female HD animals were euthanized with their pups on lactation day 0 or 1 and their pups counted as dead.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

other: mortality

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect of the litter data was observed such as the total number of pups born, number of male and females, sex ratio, live pups on PND 0. On PND 4 only three litters from HD females were left. The total number of live pups was not different as compared to the other groups. It is nevertheless to be noted that the pup viability index in the HD group showed a 50% reduction as compared to control group due to the death of three female HD animals on lactation day 0 or lactation day 1. These females were euthanized for animal welfare reasons, and their pups were therefore sacrificed as well and counted as dead. The number of still births and runts were low and similar in all groups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant effect on survival of the pups from PND 0 to PND 4 was observed in the LD and MD treated groups when compared with controls. In one LD female (Nr.59), approximately 50% of the pups did not survive up to PND 4. This finding is considered incidental.
In the HD group, as 3 females were euthanized either on PND 0 or 1, their pups were counted as dead as well, and the overall survival rate of the pups in the HD group was therefore around 50% in this group. This low survival rate on PND 4 accounts for a maternal toxicity and does not reflect any developmental toxicity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The statistical analysis of the litter weight data did not reveal any significant differences between the treatment and control groups.
On PND 4 in the three remaining litters of the HD group, total, male and female litter weights were similar as compared to the other test-item treated groups and to the control groups.

No treatment-related effect was observed on the litter data and litter weight data such as the total number of pups born, number of males and females, sex ratio, live pups on PND 0, total litter weight, male and female litter weights. On PND 4 the total number of live pups was 50 % statistically lower in the HD groups as compared to the control group, but this was due to maternal toxicity and not related to developmental toxicity. The pups of the females that were euthanised due to maternal toxicity were immediately sacrificed. The number of still births and runts were low and similar in all groups. There were no relevant gross abnormalities in the pups. The viability index (number of live offspring on day 4 / number of live offspring at birth x100) was lower in the HD group when compared to the control (50% versus 99%). This lower viability index was due to maternal toxicity and is not due to developmental toxicity (3 females were sacrificed on lactation day 0 or 1).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Table 1. 

 

Group	gender: female	Day and cause of death	Clinical findings preceding or at time of death
HD	6/10	Euthanasia: No. 71 (LD1), No. 74 (LD1), No. 75 (PD5), No. 79 (LD0) Found dead: No. 78 (PD8), No. 80 (GD22)	No. 71 (LD1): severe piloerection, severe abnormal breathing, moving the bedding, swollen tongue and cheek No. 74 (LD1): severe piloerection, moderate salivation, half eyelid closure, moderate abnormal breathing, clonic convulsion, tremor, chromodacryorrhea, bloody vagina No. 79 (LD0): severe abnormal breathing, severe piloerection, swollen cheek, lacrimation, severe salivation, swollen tongue No. 80 (GD22): found dead with swollen tongue No. 75 (PD5): on PD4 biting of gavage cannula, the day after vocalization, moderate piloerection No. 78 (PD8): no signs of toxicity

Conclusions:

No effects of test item were found at dose levels of up to 1000 mg/kg body weight/day in male animals. The NOAEL of test item in the conditions of this study is considered to be 1000 mg/kg body weight/day in Wistar male rats for general and reproduction toxicity.
At a dose level of 1000 mg/kg in female animals, food consumption and at a lower level body weight were influenced by the treatment. Four female animals of the HD group were found dead or sacrificed moribund shortly after delivery (directly after littering, or postnatal day 0 or 1) among the 7 that were pregnant. All these animals showed signs of acute reaction (dyspnea, swollen tongue and face muscles, one animal with tremor and convulsions). These signs are typical of anaphylactic shock reactions observed in rodents. The stress under which the females are submitted at time of delivery seemed to be a trigger of these symptoms. Therefore the toxicity observed in female HD animals is not considered to be directly test item related. The physio-pathological mechanisms of the acute reactions observed is not known but could be influenced by the test item at the highest dose, as well as by the parturition, and any further possible causes cannot be excluded.
Based on the test item related deaths observed and the associated histopathological findings observed (myositis or mixed cell infiltrate(s) of the tongue, cheek skeletal muscle or salivary glands, multifocal mixed cell (peri)vasculitis in the lungs, acute thymic degeneration, dilated sinusoids of lymph nodes, multifocal tubular necrosis of the renal medulla), the NOAEL in this study is considered for parent female animals to be 300 mg/kg body weight/day with absence of impairment of the reproduction performances.
Concerning fetal and developmental toxicity the NOAEL in the conditions of this study is considered to be 1000 mg/kg body weight/day.

Executive summary:

The aim of this study was to assess the possible effects of on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.
During the period of administration, the animals were observed each day for signs of toxicity. Animals that died or were euthanized were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.
Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.
After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.
The males were sacrificed after completion of the mating period on treatment days 29 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.
During the course of the study it was decided to include in addition the determination of clinical pathology parameters (hematology and biochemistry) in the remaining female animals submitted to necropsy on lactation day 4 therefore blood was collected at necropsy for analysis. At necropsy, in male and female animals which reached the planned sacrifice, organ weights were measured for the reproductive organs. In addition the number of implantation sites and corpora lutea was recorded for each parental female at necropsy.  Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities.
A full histopathological evaluation of the tissues was performed on high dose and control animals and in non pregnant female animals of the low and medium dose animals. These examinations were extended to animals of all other dosage groups for the female reproductive organs. Any gross lesion macroscopically identified was examined microscopically in all animals.

The following doses were evaluated: Control (0 mg/kg bw), low dose (100 mg/kg bw), medium dose (300 mg/kg bw), high dose (1000 mg/kg bw).

Results

Parental end points
Male
No mortality occurred in the control or any of the dose groups during the treatment period of this study in the male animals. There were no clinical signs of toxicological relevance noted in male animals during the treatment period. Some males treated at the highest dose (1000 mg/kg/day) showed only slight and transient signs of local reaction (salivation and action of pushing the head through the bedding) after treatment.
No adverse test-item effects were observed in mean body weight and gains and food consumption during the treatment period in male animals. Gross pathological observation of male animals revealed no treatment-related findings. In male animals, there was no statistically significant difference in the absolute and relative reproductive organ weights of the treatment groups when compared with the control group. No test item-related histological findings were observed in male reproductive organs in this study.
Female
In the control and lower dose groups (LD, MD) no mortality was recorded in female animals. At the highest dose, two animals were found dead (one in the pre-mating phase and one shortly after littering), and 4 animals were sacrificed earlier due to deteriorated health (three on postnatal day 0 or 1, and one in the premating phase). One death was attributed to misgavage (No. 78) and for the 5 other decedents histopathological evaluation confirmed a test item related cause of death. For 2/5 decedents (No. 75 and No.80) the cause of death was considered probably test item related. There were no clinical signs of toxicological relevance noted in the females during the treatment period which reached terminal sacrifice. Some female animals treated at the highest dose (1000 mg/kg/day) and two MD female animals showed slight and transient signs of local reaction (salivation and action of pushing the head through the bedding) after treatment. In three female HD animals (No. 71, 74 and 79) that were sacrificed earlier on lactation day 0 or 1, adverse signs of toxicity with abnormal breathing due to swollen tongue or cheek were observed and one animal in moribund status showed clonic convulsions and tremors. In one further female found dead on Gestation day 22 shortly after littering (No. 80), a swollen tongue was observed at necropsy. In these 4 animals, littering seemed to be a trigger of the deteriorated health condition, as no preceding signs of toxicity were observed before littering. In HD female animals a tendency towards lower weight gain in the first week of pre-mating and the first two weeks of gestation was observed without reaching statistical significance.
At the end of gestation body weight was approximately 9% below controls in the female HD group. In female animals, during the pre-mating and gestation period a dose dependent effect was observed on food consumption. In the second week of pre-mating, the food consumption was between 15 and 19% lower in the test-item treated groups as compared to control, these effects reaching statistical significance. In the first week of treatment, in
female HD animals the food consumption was already 15% lower as compared to control. During gestation the same tendency was observed and during the second week of
gestation, in MD and HD animals the food consumption was respectively 13 to 23% lower respectively as compared to control animals.
No treatment-related effect was observed for the precoital interval or for the duration of gestation in the test-item treated groups as compared to control. Successful mating resulted in 10/10 pregnancies in the C group, 9/10 pregnancies in the LD group, 9/10 pregnancies in the MD group and 7/8 pregnancies in HD group. No effects on fertility index and delivery index were observed in the test item treated groups as compared to control group.
The groups mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group.
On lactation day 4, no toxicologically relevant differences were observed in haematological parameters measured in the test-item treated female animals as compared to control animals.
On lactation day 4 differences were observed in clinical biochemistry. In two of the three HD females total bile acid level was higher as compared to control values. The mean HD group value was statistically significantly higher as compared to the mean control group value (2 x increases). The values in the HD group remained within the normal range of variation for female animals of this strain. In view of the centrilobular hepatocellular hypertrophy observed in all HD decedents at histopathology, this higher level observed in HD females is considered to be a non-adverse adaptive finding.
In females, there was no statistically significant difference in the absolute and relative reproductive organ weights of the treatment groups when compared with the control group. Gross pathological observation of females at scheduled necropsy revealed no treatmentrelated findings.
In two HD female decedents (No. 75, No. 78) submitted to necropsy during the early treatment phase, both animals were found not to be pregnant and had a dark discoloured thymus. In addition, No. 78 had also dark discoloured ileum, Peyer's patch, lung, trachea and uterus, and its thoracic cavity was blood-filled, for this animal histological evaluation confirmed an incidental cause of death. Rats Nos. 71, 74 and 79 were sacrificed moribund towards the end of the treatment phase. The predominant macroscopic findings in one or several of these animals were fluid distension of salivary glands, mandibular lymph node and periocular tissue, swollen tongue and swollen skeletal muscle of the cheek, as well as fluid-filled thoracic cavity with small thoracic organs. Female No. 80 was found dead towards the end of the treatment phase (gestation day 22, found dead shortly after littering) and presented also an enlarged tongue, as well as dark discoloured lung and thymus.
Six females treated at 1000 mg/kg/day died or were sacrificed moribund before the end of the treatment phase. One spontaneous death (No. 78) was attributed to misgavage, and the cause of death of the other five decedents was considered to be clearly (3/5; No. 71, 74, and 79) or probably (2/5; No. 75 and No. 80) test item-related. Among the numerous histopathological findings noted in the decedents, mixed cell inflammation with edema of
skeletal muscle and other tissues at the head region, corroborating macroscopic swollen tongue and cheek muscles, were the most prominent findings in the three decedents (No. 71, 79 and 80). Other findings comprised multifocal tubular necrosis of the renal medulla, multifocal mixed cell (peri)vasculitis in the lung and uterus, acute thymic degeneration and single cell death. All six decedents presented a centrilobular hepatocellular hypertrophy of
the liver and sinus histiocytosis of one or several lymph nodes, and 4/6 decedents showed a diffuse cortical hypertrophy of the adrenal gland. At terminal sacrifice, no clearly test item-related pathological finding was detected in female reproductive organs at 1000 mg/kg/day, and 3/4 high dose females killed at terminal sacrifice showed typical post-partum histomorphology of their reproductive organs. One high dose female was found not to be pregnant at terminal sacrifice and did not show typical indication of sexual cycling. It presented a minimal multifocal mixed cell (peri)vasculitis in the uterus as well as uterine distension. A test item relationship of these findings was considered possible. There was no indication of any test item-related effect on female reproductive organs at 100 and 300 mg/kg/day, and the isolated non-pregnant females treated at 100 and 300 mg/kg/day showed physiological sexual cycling.
Developmental toxicity
No treatment-related effect was observed on the litter data and litter weight data such as the total number of pups born, number of males and females, sex ratio, live pups on PND 0, total litter weight, male and female litter weights. On PND 4 the total number of live pups was 50 % statistically lower in the HD groups as compared to the control group, but this was due to maternal toxicity and not related to developmental toxicity. The pups of the females that were euthanised due to maternal toxicity were immediately sacrificed. The number of still births and runts were low and similar in all groups. There were no relevant gross abnormalities in the pups. The viability index (number of live offspring on day 4 / number of live offspring at birth x100) was lower in the HD group when compared to the control (50% versus 99%). This lower viability index was due to maternal toxicity and is not due to developmental toxicity (3 females were sacrificed on lactation day 0 or 1).

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

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

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

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

 

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