A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL reproductive toxicity: 1000 mg/kg/day for males and females

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

Additional information

In order to provide information on toxicity as well as any possible effects of the test item on male and female reproductive performance, a study was performed according to the OECD Guideline 421 (2016).

Four groups containing 10 male and 10 female rats each were administered the test item in water softened by reverse osmosis (10 mL/kg of body weight) for 30 days (males) and at least 51 days (females). The test item was given to Sprague Dawley rats by oral administration (gavage) at dosages of 100, 300 and 1000 mg/kg/day. The control group received only the vehicle.

Dose levels were selected based on information from a preliminary non-GLP compliant study.

The dosing period for females included (a) a pre-pairing period of two weeks; (b) a pairing phase which lasted between 1 and 4 days; (c) a gestation period of 22 days; and (d) a lactation period of 14 days. Males were dosed for the two-week pre-mating period and pairing phase, then a post-pairing phase; up to a total of 30 days, after which they were sacrificed. All surviving dams with live litters were sacrificed at the end of the study period; females which did not give birth after 25 days of gestation were sacrificed shortly after; dams with total post-natal litter loss were sacrificed shortly after the loss of their litter. At necropsy, all animals were subjected to clinical pathology investigation; gross examination for external/internal abnormalities; number of visible implantation sites (pregnant animals); number of corpora lutea (pregnant animals); thyroid hormone determination (males); terminal body weight; organ weights; histopathological examination; and the copulatory indices and fertility indices were calculated. Litters were adjusted on day 4 post-partum to yield 4 male and 4 female pups per litter; and blood was collected for hormone determination on at least one culled male and one culled female. All surviving pups of the adjusted litters were monitored for sex (at birth), weight (days 1, 4 and 13), anogenital distance (day 1) and nipple retention (day 14) during the post-partum period then were sacrificed on day 14 post-partum. At necropsy, pups found dead were examined for internal/external abnormalities; culled pups were examined externally only; thyroid of one male and one female per dose group was weighed; any abnormalities were preserved.

The following results for reproduction were obtained.

Mortality and fate of females

No mortality occurred during the study. One female receiving 300 mg/kg/day and one receiving 1000 mg/kg/day were proved not pregnant at necropsy. A total of 3 females, one control, one receiving 100 and one receiving 300 mg/kg/day had total litter loss on Days 1, 7 and 5 post partum, respectively. The control females with total litter loss showed also unilateral implantation at necropsy. The number of females with live pups on Day 14 post partum was: 9 each in the control, low dose (100 mg/kg/day) and high dose (1000 mg/kg/day) group and 8 in the mid-dose dose group (300 mg/kg/day).

Clinical signs

The only clinical sign recorded during the treatment period was black staining on the tail (slight to marked) in all treated males and in females (slight to moderate) receiving the dose level ≥300 mg/kg/day.

Body weight and body weight gain

No adverse effects were observed in body weight or body weight gain of treated animals compared to control group.

Food consumption

Food consumption of parental animals of both sexes was unaffected by treatment.

Thyroid hormones

Parental males

Statistically significant increases of triiodothyronine and thyroxine were recorded in males dosed at 1000 mg/kg/day (46 % and 12 %, respectively). Similar increases were also recorded in some animals receiving 100 and 300 mg/kg/day, with no statistical significance nor clear dose-relation. Since changes were of low severity, with no dose-relation nor concurrent alteration of thyroid stimulating hormone, the above increases were considered to be of no toxicological relevance.

Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data and sex ratio were unaffected by treatment, at birth, on Days 1, 4 and 14 postpartum.

Terminal body weight and organ weights

No relevant changes were observed on terminal body weight, absolute and relative organ weights of treated animals, when compared to the controls.

Macroscopic observations

An increased incidence of dark colour and/or areas of cervical and mesenteric lymph nodes, lungs and intestinal tract (caecum, colon, ileum and rectum) were noted in treated animals of both sexes. An increased incidence of reduced size of thymus was observed in high dose treated females, when compared to the controls.

Microscopic observations and spermatogenic cycle

Treatment-related lesions were noted in mesenteric lymph nodes of males and females receiving test item at 1000 mg/kg/day, in the lungs of males dosed at 1000 mg/kg/day and in females dosed at ≥300 mg/kg/day.

The findings consisted in the presence of black pigment laden macrophages in the mesenteric lymph nodes in all high dose males and females, and intracytoplasmic black granular pigment in the alveolar macrophages, associated or not with chronic inflammatory reactions in the lungs reported in males dosed at 1000 mg/kg/day and females dosed at ≥300 mg/kg/day.

Spermatogenic cycle

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males.

Effects on developmental toxicity

Description of key information

NOAEL developmental toxicity: 1000 mg/kg/day for males and females

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000

Species:

rat

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

In order to provide information on toxicity as well as any possible effects of the test item on male and female reproductive performance, a study was performed according to the OECD Guideline 421 (2016).

Four groups containing 10 male and 10 female rats each were administered the test item in water softened by reverse osmosis (10 mL/kg of body weight) for 30 days (males) and at least 51 days (females). The test item was given to Sprague Dawley rats by oral administration (gavage) at dosages of 100, 300 and 1000 mg/kg/day. The control group received only the vehicle.

Dose levels were selected based on information from a preliminary non-GLP compliant study.

The dosing period for females included (a) a pre-pairing period of two weeks; (b) a pairing phase which lasted between 1 and 4 days; (c) a gestation period of 22 days; and (d) a lactation period of 14 days. Males were dosed for the two-week pre-mating period and pairing phase, then a post-pairing phase; up to a total of 30 days, after which they were sacrificed. All surviving dams with live litters were sacrificed at the end of the study period; females which did not give birth after 25 days of gestation were sacrificed shortly after; dams with total post-natal litter loss were sacrificed shortly after the loss of their litter. At necropsy, all animals were subjected to clinical pathology investigation; gross examination for external/internal abnormalities; number of visible implantation sites (pregnant animals); number of corpora lutea (pregnant animals); thyroid hormone determination (males); terminal body weight; organ weights; histopathological examination; and the copulatory indices and fertility indices were calculated. Litters were adjusted on day 4post-partumto yield 4 male and 4 female pups per litter; and blood was collected for hormone determination on at least one culled male and one culled female. All surviving pups of the adjusted litters were monitored for sex (at birth), weight (days 1, 4 and 13), anogenital distance (day 1) and nipple retention (day 14) during the post-partum period then were sacrificed on day 14post-partum. At necropsy, pups found dead were examined for internal/external abnormalities; culled pups were examined externally only; thyroid of one male and one female per dose group was weighed; any abnormalities were preserved.

The following results for devolopment were obtained.

Pups - Day 14 post partum

No changes were observed between the control and treated pups

Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females

No significant differences were observed for these parameters between the treated groups and controls. All pregnant dams gave birth on Day 22 post coitum (mean value).

Clinical signs of pups

No compound-related effects were observed. Pre-weaning clinical signs were comparable between treated and control groups or considered incidental.

Anogenital distance and nipple count

No treatment-related effects were seen both in males and in female pups compared to controls.

No nipples were present in males on Day 13 post partum.

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Pups thyroid weight on Day 14 post partum

No treatment-related changes were seen between control and treated groups.

Justification for classification or non-classification

According to CLP Regulation (EC) No 1272/2008, Reproductive toxicity section, 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 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.

Based on the available information, no significat effect on fertility and development were observed. Therofore, the substance does not meet the criteria to be classified for reproductive toxicity, according to CLP Regulation (EC) No 1272/2008.