disodium 7-{[4-chloro-6-(dodecylamino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[{4-[(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Toxicity to reproduction

Administrative data

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:

from February 10 to April 4, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(2015)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

(2000)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Red GS 3848

IUPAC Name:

Red GS 3848

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALSSource: Toxi-Coop Zrt.Age at study initiation: 105-110 daysWeight at study initiation: 357-433 g for males, 210-255 g for femalesHousing: before mating 2 animals of same sex/cage, at mating 1 male and 1 female/cage, pregnant females individually, males afetr mating 2 animals/cageDiet: ad libitumAcclimation period: 35 daysENVIRONMENTAL CONDITIONSTemperature: 22 ± 3 °CHumidity: 30-70 %Air changes: above 10 per hour by a central air-condition systemPhotoperiod: artificial light from 6 a.m. to 6 p.m

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was formulated in vehicle (distilled water) not longer than three days beforehand.VEHICLE: distilled waterConcentration in vehicle: 200, 60, 20 mg/mlAmount of vehicle: 5 ml/kg bwLot/batch no.: 1509-5529, 1511-5519, 1512- 5503, 1511-5526

Details on mating procedure:

M/F ratio per cage: 1 male and 1 female of the smale dose groulLength of cohabitation: until copulation occurredProof of pregnancy: vaginal plug and 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:

Concentration in dosing formulation was checked two times during the study and found to vary between 97 % and 107 % of nominal values. Suitability of chosen vehicle (recovery and stability) for test item at intended concentrations was analytically verified up front.

Duration of treatment / exposure:

Test item and vehicle were orally administered daily (7 days per week) for a whole period of 51-54 days. Dosing begun after acclimatisation (35 days) and included: -males: 14 days pre-mating period, 1-4 days mating period, 33-36 days post-mating period. -females: 14 days pre-mating period, 1-4 days mating period, 22-23 days gestation period, 13-15 days lactation period.

Frequency of treatment:

Daily (7 days per week).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:100 mg/kg bw/dayBasis:nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:300 mg/kg bw/dayBasis:nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:1000 mg/kg bw/dayBasis:nominal in water

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: chosen on the basis of the results of 28-day repeated dose oral toxicity test with test substance in rats, where dose of 1000 mg/kg bw/day was well tolerated. High dose chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. Low dose chosen to induce no toxic effect. Mid dose was interpolated geometrically.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONSGeneral observation, once a day after administration; detailed observation, at times of weekly weighing.Detailed examinations were carried out on skin, fur, eyes and mucous membranes, autonomic activity, circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. In addition, also tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.BODY WEIGHTParental males weighed on first day of dosing (day 0) and weekly thereafter and on day of necropsy; parental females weighed on the firts day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 3 post-partum.FOOD CONSUMPTIONFood consumption for each animal determined weekly by reweighing non-consumed diet during treatment period except mating phase. For females food consumption was determined for pre-mating days 7, 13, gestation days 0, 7, 14, 21 and lactation days 0,4, 13.

Oestrous cyclicity (parental animals):

Examination of vaginal smears for 2 weeks before treatment initiation. Animals with 4-5 days cycles were included preferably in the study.

Litter observations:

PARAMETERS EXAMINED IN F1 OFFSPRINGNumber and sex of pups, stillbirths, live births, runts (pups significantly smaller than normal pups), presence of gross anomalies.Live pups weights on day 0, 4 and 13 post-partum. Any abnormal behaviour of offspring.Anogenital distance of each pup on day 4. Blood samples for possible determination of serum levels of thyroid hormones (T4): from at least 2 pups per litter on post-natal day 4 and day 13 (termination day).GROSS EXAMINATION OF DEAD PUPsOn day 0, lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after birth (dead pups).In following days, necropsy by macroscopic examination of dead pups.

Postmortem examinations (parental animals):

SACRIFICEMales: day 51Females mated but non-pregnant: day 51Maternal animals: day 51-54GROSS NECROPSYAfter examination of external appearance, cranial, thoracic and abdominal cavities were opened and appearance of tissues and organs was observed, and any abnormality was recorded including details of location, color, shape and size. Special attention was paid to organs of the reproductive system. Number of implantation sites was recorded.ORGAN WEIGHTSBody weight, brain weight and weight of the testes and epididymides of adult animals; absolute organ weight and relative organ weight (to body and brain weight). Paired organs weighed together.HISTOPATHOLOGYFull histopathology examinations on preserved organs and tissues of the animals in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Detailed histological examination on ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland.Fixed tissues trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Statistics:

The statistical evaluation 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 mateMales = (no. of males with confirmed mating / total no. of males cohabited) × 100Females = (no. of sperm positive females / total number of femals cohabited) × 100 Fertility index: measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.Males = (no. of males impregnating a female / total no. of males with confirmed mating) × 100 Females = (no. of pregnant females / no. of sperm positive females) × 100 Gestation index: measure of pregnancy that provides at least one live pup(no. of females with live born pups / no. of pregnant females) × 100

Offspring viability indices:

Post-implantation/ pre-natal mortality (intrauterine mortality)100 × (no. of implantations - no. of liveborns) / no. of implantation Post-natal mortality /Extra-uterine mortality100 × (no. of liveborns - no. of live pups on PN13) / no. of livebornsSurvival Index 100 × no. of live pups on postnatal day 13 / no. of pups born Sex ratio100 × (no. of pups examined - no. of males (females)) / no. of pups examined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

test item or metabolites detected in intestines

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: oral gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: gonad function, mating behaviour, conception

3

Dose descriptor:

NOAEL

Remarks:

raised post-implantation loss

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: gonad function, mating behaviour, conception, parturition, post-natal loss

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING) There was no test item related effect on offspring’s extra uterine mortality. The mean number of dead and missing offspring per litter was similar in all groups. There were no significant differences between the control and test item treated groups (1000, 300 or 100 mg/kg bw/day) in the survival indices.CLINICAL SIGNS (OFFSPRING)Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13. The percentage of pups with findings was slightly higher in the test item treated groups than in the control group. However there was no dose relevance in the percentage of pups with findings (51, 31 and 43 %, respectively to doses of 100, 300 and 1000 mg/kg bw/day). The percentage of cold offspring was higher in all test item treated groups than in the control group. The percentage of cold offspring in the control group of this study was lower than the historical control data. In the lack of any dose relevancy and because of the transient occurrence (it was mainly detected on the day of birth) as well as there were no related changes in the litter/body weight parameters or in the mortality rate, it was not considered to be toxicologically relevant (related to the test item).BODY WEIGHT (OFFSPRING)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 litter weight gain and mean pup weight gain were similar in the control and in all test item treated groups (1000, 300 and 100 mg/kg bw/day) on postnatal days 0, 4 and 13.SEXUAL MATURATION (OFFSPRING)The anogenital distances (absolute and normalized) were similar in the control and test item treated groups (1000, 300 and 100 mg/kg bw/day). Nipples/areoles were not visible in any of the examined male offspring in the control or 1000, 300 or 100 mg/kg bw/day groups on postnatal day 13.GROSS PATHOLOGY (OFFSPRING)Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: development

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under test conditions, the substance administered at 1000, 300 or 100 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behaviour, conception, parturition) in parental male and female rats.Prenatal loss (post-implantation loss, percentage and mean) was slightly higher than in control group in dams at 1000 mg/kg bw/day. Although the slight difference with respect to control, a test item influence cannot be excluded. Development of F1 offspring was not impaired at any dose after repeated oral administration of dams from birth to post-natal day 13. Thus, NOAEL = 1000 mg/kg bw/day for systemic toxicity, reproductive performance of male, F1 offsprings; NOAEL = 300 mg/kg bw/day in females for post-implantation losses.

Executive summary:

Method

Study on toxic potential of test item and its possible effects on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 1000, 300 and 100 mg/kg bw/day compared to control animals. As a screening test, it considered reproductive parameters such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as development of F1 offspring from conception to day 13 post-partum, associated with administration of repeated maternal doses.

Groups of 12/sex animals, three dose groups and one control (only distilled water as vehicle).

Animals of parent (P) generation were dosed prior to mating (14 days) and throughout mating. After mating, males dosed up to 51 days and females up to 51 -54 days, including gestation period and lactation.

Results

At doses up to 1000 mg/kg bw/day, there was no substance-related mortality.; no clinical sign of systemic toxicity, no changes in body weight and body weight gain and no changes in food consumption were noted. No specific macroscopic alterations and no changes in weights of brain, testes and epididymes of males were seen at necropsy. No changes were noted after histopathological examinations of ovaries, uterus, vagina, pituitary, testes, epididymes, prostate and seminal vesicles with coagulating gland.

As for reproductive performance, no substance related effects on gonadal function, mating behaviour, conception, pregnancy, parturition up to 1000 mg/kg bw/day. Higher post implantation loss in comparison with control and historical control was noted at dose of 1000 mg/kg bw/day.

Based upon these results, no-observed-adverse-effect level was considered to be 300 mg/kg bw/day in rats when administered orally by gavage.