Hexasodium 4,4'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis[5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-27 and 2015-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test) 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 10-11 weeks (males/females)
- Acclimatization period: 14 days
- Housing: a) individually in Makrolon type M III cages during the study period; b) male and female mating partners were housed together during in polycarbonate cages type III during overnights mating; c) pregnant animals and their litters were housed together until PND 4 (end of lactation); d) for motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: (P) From: 2015-05-27 To: 2015-05-12 (males), From: 2015-05-27 To: 2015-06-05 (females); (F1) From: 2015-05-18 To: 2015-05-22

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced daily.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study.

The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. Homogeneity was verified in 3 samples in the highest and lowest concentration (was used as a concentration control at the same time) at the beginning of the study; additional concentration control analyses were done in the mid concentration. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in drinking water. The concentrations of the test substance in drinking water were found to be in the range of 103-120 % of the nominal concentration.

Duration of treatment / exposure:

males: 29 days
females: 53 days

Frequency of treatment:

once daily

Details on study schedule:

see information on material and methods (table 1)

Remarks:

Doses / Concentrations:
12, 60 and 300 mg/kg bw/d
Basis:
other: test material

Remarks:

Doses / Concentrations:
10.6, 52.8 and 264.0 mg/kg bw/d
Basis:
other: content of test substance (88 g/100 g)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 14-day range finding study in which 0, 300 and 1000 mg/kg bw/day were tested.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: Any signs of morbidity, pertinent behavioral changes and signs of overt toxicity were examined. Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays), the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- The following parameter were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period; on study day 0 (start of administration period) and thereafter once a week at the same time of the day (in the morning)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week for male and female parental animals
- Exceptions: Food consumption was not determined during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20. Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

OTHER: Haematology, clinical chemistry, urinalysis, neurobehavioural examination

Oestrous cyclicity (parental animals):

The number of mating days until vaginal sperm were detected for F0 females.

Sperm parameters (parental animals):

Parameters examined in all F0 male parental generations: testis weight, epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births (on PND 0), postnatal mortality (on PND 0 and between PND 1-4), sex ration (on PND 0 and PND 4), clinical symptoms including gross-morphological findings, body weight gain (on PND 1 and PND 4)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- All parental animals were sacrificed by decapitation under isoflurane anesthesia. Parental males were sacrificed on study day 29 and parental females were sacrificed on study day 53.

GROSS NECROPSY
- The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were prepared for microscopic examination: adrenal glands, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lung, mesenteric and axillary lymph nodes, ovaries, oviducts, peyer's patches, prostate, rectum, sciatic nerve, seminal vesicles, spinal cords, spleen, forestomach and glandular stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
- The following organ weights were determined in all animal sacrificed on schedule: epididymides and testes.
- The following organ weights were determined in 5 animals/sex and test group: adrenal glands, brain, heart, kidneys, liver, spleen and thymus.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on PND 4 under isoflurane anestesia with CO2.

GROSS NECROPSY
- All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

- Statistics of the clinical examinations: DUNNETT test (two-sided), FISCHER'S EXACT test (one-sided), WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment, KRUSKALL-WALLIS test (two sided)
- Statistic of clinical pathology: KRUSKAL-WALLIS (bidrectional changes), WILCOXON-test with Bonferroni-Holm adjustment (undirectional changes), WILCOXON-test, KRUSKALL-WALLIS test (one-way analysis), WILCOXON-test (two-sided)
- Statistics of pathology: KRUSKALL-WALLIS (two-sided), WILCOXON-test (two-sided)

Reproductive indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, postimplantation index

Offspring viability indices:

viability index, live birth index

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No parental animal died prematurely in the present study.
- Discolored feces from red (test groups 2 [60 mg/kg bw/d] and 3 [300 mg/kg bw/d]) and dark brown (test group 3; females only) to black (test group 3) were observed in both sexes during premating, mating and post mating periods, starting on study day 1. These observations were also made for females during gestation and lactation. The effects were related to the test substance but assessed as being non-adverse. The absence of an open vagina was observed in animal No. 140 during mating. This finding was neither test substance- nor treatment-related but spontaneous in nature.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- No test substance-related changes in mean body weights were observed for male and female animals of test groups 1-3 (12, 60 and 300 mg/kg bw/d) when compared to control groups. Temporarily, mean body weight gain was significantly slowed down in female animals of test group 3 (300 mg/kg bw/d) during the premating phase, with a body weight loss between study days 0 to 7. As the finding was the limited to the premating phase and as no further findings occurred it was assessed to be non-adverse.
- Referring to food consumption, no test substance-related findings were observed.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): The mean duration until sperm was detected (GD 0) was 2.0 days for test group 0, 1.3 days for test group 1, 2.2 days for test group 2 and 2.6 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No treatment-related, adverse effects were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- The significantly increased absolute weight of the spleen in males (0.714 g) was within the range of the historical control values (0.502-0.728 g). However, the relative weight (0.216%) was above the historical control range (0.132-0.196%) and correlated with increased extramedullary hematopoiesis. Therefore, it was considered treatment-related. In females of test group 3, although the absolute spleen weight (0.510 g) was marginally above the historical control range (0.382-0.502 g) neither a dose-dependency nor a histopathological correlate were evident. Therefore, the weight increase in females was regarded as incidental.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- In almost all males and females of test group 3 (300 mg/kg bw/d), the contents of the cecum, colon, glandular stomach and jejunum displayed a red discoloration. In addition, the kidneys of 4 out of 10 males and all females, as well as the mesenteric lymph nodes of all males and females of test group 3 displayed a red discoloration. All of these changes revealed the presence of the test substance and were considered treatment-related, but had no histopathological correlate.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- A slightly increased in the severity of the extramedullary hematopoiesis was seen in the spleen of males of test group 3 (300 mg/kg bw/d), only. The extramedullary hematopoiesis was comparable between control animals and treated animals of test groups 1 and 2.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Effect level:

264 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- The viability index indicating pup mortality during lactation (PND 0-4) was 97.7% in test group 0 (control), 98.8% in test group 1 (12 mg/kg bw/d), 97.5% in test group 2 (60 mg/kg bw/d) and 98.9% in test group 3 (300 mg/kg bw/d). The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100.0%

CLINICAL SIGNS (OFFSPRING)
- No treatment-related, adverse effects were observed.

BODY WEIGHT (OFFSPRING)
- Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
- Each one female runt was seen in 1 litter of the test group 0 (control) and test group 3 (300 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.

GROSS PATHOLOGY (OFFSPRING)
- Each one male and female pup of test group 2 (60 mg/kg bw/d; Nos. 128-4 and 124-11) showed a discolored (pale) liver lobe This finding was assessed as being spontaneous in nature and without toxicological relevance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

264 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD guideline.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD 422 study was conducted with the test item (purity: 88 g/100 g). The test item was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 0, 12, 60 and 300 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (drinking water). Considering the content of the test substance, i.e. 88.0 g/100 g, the definitive dose levels were 0, 10.6, 52.8 and 264.0 mg/kg bw/d. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0-7, 7-14, 14-20) and lactation (days 1-4). In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (12, 60 and 300 mg/kg bw/d) during the entire study period. Fertility indices for male and female animals were not impaired by test substance administration even at a dose level of 300 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered. Concerning clinical pathology, in males of test group 3 (300 mg/kg bw/d) a marginal indication of a regenerative, macrocytic anemia was observed because of decreased red blood cell (RBC) counts, increased mean corpuscular volume (MCV) and increased reticulocyte counts. Regarding pathology, target organ was the spleen of male animals. The absolute and relative weight of the spleen in males of test group 3 (300 mg /kg bw/d) were significatly increased (+25% and +30%, respectively). This finding correlated with minimal to moderate extramedullary hematopoiesis. Both, weight increase and extramedullary hematopoiesis were considered treatment-related and adaptive. However, in combination with the marginal anemia observed in the hematology they can be assessed as adverse. Under the conditions of the present Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening, the NOAEL for reproductive performance and fertility was set to 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d) for male and female Wistar rats. The NOAEL for developmental toxicity was 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d).

Short description of key information:
The NOAEL for reproductive performance and fertility was set to 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d) for male and female Wistar rats. The NOAEL for developmental toxicity was 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d).

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD guideline.

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

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for reproduction/developmental toxicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.