1,3'-Bipyridinium, 5'-[[4-[[4-[(1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo[1,3'-bipyridinium]-5'-yl)azo]benzoyl]amino]phenyl]azo]-1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo-, salt with 2-hydroxypropanoic acid (1:2)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL = 1000 mg/kg bw/day (756 mg/kg bw/day as a. i.)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

756 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

Description of key information

NOAEL = 300 mg/kg bw/day (226.8 mg/kg bw/day as a. i.)

LOAEL = 1000 mg/kg bw/day (756 mg/kg bw/day as a. i.), due to effec on body weight development of pups.

Such effects was associated to maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

226.8 mg/kg bw/day

Study duration:

subacute

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

OECD guideline 421 (screening on reproductive toxicity effects).

Rats has been dosed by oral route at doses of 0 (control), 75.6, 226.8 and 756 mg/kg bw/day as a.i., equivalent to 0, 100, 300 and 1000 mg/kg bw/day as test item. Distilled water was used as vehicle. Groups of 12 rats/sex were used. Male rats were dosed 14 days prior to mating, during mating and post mating, for 48 days. Females were exposed 14 days prior to mating, during mating, during gestation and up to lactation days 13 -16, i.e. for 51 -55 days.

Rats were observed for mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored before the treatment for 2 weeks, in 2 weeks pre-mating period and during the mating period until evidence of mating. Serum levels of thyroid hormones (T4 and TSH) were also monitored.

Dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter (at day 13) for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, testes, epididymides in control and high dose groups. These organs were also processed and examined histologically in not mated and non-pregnant females and males these females cohabited with in the low and middle dose groups.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

The results of this study were:

- no test item related mortality at any dose level (75.6, 226.8 and 756 mg/kg bw/day).

- clinical signs of systemic toxicity related to the test item were detected at 226.8 (decreased activity, decreased body tone, diarrhea in single animals) and 756 mg/kg bw/day (decreased activity, decreased body tone, piloerection, hunched back, noisy breathing with low incidence and wet bedding material) dose levels in male and female animals at the daily or at the detailed weekly clinical observations.

- soft and red stool in each animal at 75.6, 226.8 and 756 mg/kg bw/day.

- body weight gain and body weight were reduced in male and female animals at 75.6, 226.8 and 756 mg/kg bw/day. The difference with respect to the control in the mean body weight was low (< 10 %) in male animals at 75.6 mg/kg bw/day and was not related to doses in female animals at 75.6 and 226.8 mg/kg bw/day during the gestation and lactation period. Thus, the toxicological relevance of these findings at 75.6 mg/kg bw/day (male and female) and 226.8 mg/kg bw/day (female) is questionable.

- mean daily food consumption was not adversely affected.

- estrous cycle was not affected.

- no toxicologically relevant effects on the evaluated delivery parameters of dams.

- no test item related differences between the control and test item treated groups in the investigated parameters of reproductive performance of male and female animals.

- no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

- test item related changes in the stomach (thickened wall, erythematous mucous membrane) were observed in some male and female animals at 756 mg/kg bw/day.

- presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in several male (75.6, 226.8 and 756 mg/kg bw/day) and female animals (226.8 and 756 mg/kg bw/day) at the gross macroscopic observation.

- no adverse changes in the organ weights (absolute and relative to body or brain weights) of brain, testes, epididymides and seminal vesicles of male animals at any dose level.

- histopathological examinations of the selected sexual organs (ovaries, testes, epididymides) did not reveal any test item related changes at any dose level.

- focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in three male animals and one female animal at 756 mg/kg bw/day. This lesion was in connection with the local effect of high dose of the test item and might be considered as a slight, reversible lesion.

- test item related reduced body weight development of the offspring was found at 756 mg/kg bw/day.

- no adverse findings on the mortality, clinical signs or at necropsy were detected in the offspring terminated as scheduled.

Conclusion

Basic Orange 64 caused clinical signs (piloerection, decreased activity, decreased body tone, hunched back, noisy breathing with low incidence, soft red stool and wet bedding material), reduced body weight, macroscopic and microscopic local changes in the stomach (thickened and erythematous mucous membrane, focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach) at 756 mg/kg bw/day administered by oral gavage to parental male Han:WIST rats.

At the same dose level, clinical signs, as piloerection, decreased body tone, soft red stool or wet bedding material, were observed in females; macroscopic and microscopic significant changes were not detected.

At 226.8 mg/kg bw/day, soft red stool, piloerection, decreased activity or decreased body tone and changes in the body weight were observed in the male and female animals.

At 75.6 g/kg bw/day, soft red stool and minor changes in the body weight (<10 %) were observed in male and female animals.

The development of the F1 offspring was influenced at 756 mg/kg bw/day dose levels due to the reduced body weight after repeated oral administration of dams from birth to post-natal day 13. Maternal toxicity could be assumed in arising of body weight changes of offspring.

Based on these observations:

NOAEL for systemic toxicity of male/female rats 75.6 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 756 mg/kg bw/day

NOAEL for F1 offspring: 226.8 mg/kg bw/day

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, adverse effects on development of the offspring and adverse effects on or via lactation.

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.

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.

In a screening test on reproductive toxicity, no significant effects on reproduction parameters of male and female rats were reported, thus a NOAEL of 756 mg/kg bw/day was identified.

Effects on general toxicity of parental animals were seen and led to the identification of a NOAEL at 75.6 mg/kg bw/day due to clinical signs and reduced body weights.

A NOAEL of 226.8 mg/kg bw/day for pups development was identified due to effects on pups body weights development; however, such effect was considered as secondary to maternal toxicity.

Based on available data, no classification for reproductive toxicity was applied.