Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A Combined Repeated Dose Toxicity Study and Reproductive /Developmental Toxicity Screening Study conducted according to OECD TG 422 is available.

Three groups of ten male and ten female rats received Bayscript Magenta BB at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 10 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups. 

Treatment with Bayscript Magenta BB at 1000 mg/kg/day was not tolerated and dosing was suspended early for males and females on Days 11 and 18 of treatment, respectively. There were four premature deaths, three males and one female during the course of the study in the 1000 mg/kg/day group, the precise cause of these deaths were not established due to autolysis however they are considered to be related to treatment. 

Prior to the cessation of dosing at 1000 mg/kg/day the signs observed were related to the colour of the test item, body weight loss was observed in males and females during Week 1 and was still evident in females during Week 2 of dosing (prior to pairing). Mean food consumption was lower for males only. Haematological examinations revealed changes in mean peripheral white blood cell numbers and prothrombin times were slightly prolonged in males only. The biochemical examination of the blood plasma revealed: high bilirubin, bile acids and creatinine concentrations in both males and females given 1000 mg/kg/day. Macroscopic examination of the premature decedents and animals that were terminated early revealed pink colouration affecting numerous tissues. Histopathological evaluation of retained tissues for the animals receiving 1000 mg/kg/day and terminated early revealed treatment related changes in the kidneys, stomach, spleen and testes. In the kidneys, degeneration of cortical tubules was present in all animals and there was regeneration in the cortical tubules in one male and all females given 1000 mg/kg/day. Eosinophilic globules were present in the cytoplasm of epithelial cells in the mucosa of the glandular region of the stomach affecting the majority of animals and this was accompanied by foveolar epithelial hyperplasia in some animals; this was associated with submucosal inflammation in several males. Apoptosis of hepatocytes, hepatocyte vacuolation and prominent mitotic activity in hepatocytes were present in the liver of a few females receiving 1000 mg/kg/day and terminated early. An increase in Periodic acid-Schiff (PAS) staining was present in the interstitial macrophages of the testes in all males given 1000 mg/kg/day. In the spleen, an increase in cellularity of the white pulp was observed in one male and four females that received 1000 mg/kg/day.

Treatment with Bayscript Magenta BB to parental Han Wistar rats at dose levels of 100 and 330 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation was generally well tolerated. There were no premature deaths, no test-item related signs observed during the detailed physical examination and arena observations and no post-dosing observations with the exception of signs related to the colour of the test item. There were no effects on sensory reactivity and grip strength assessments. There was a slight effect observed for motor activity, overall group mean low beam scores were slightly low for both sexes receiving 330 mg/kg/day and overall high beam scores were low for males receiving 330 mg/kg/day.

At the commencement of treatment (prior to pairing), males given 330 mg/kg/day and females given 100 or 330 mg/kg/day showed slightly lower mean body weight gain. Body weight gain of males receiving 330 mg/kg/day for the first week of pairing remained lower than that of Control, subsequent body weight gain for these males were generally similar to Control. After mating, the body weight gain of females receiving 330 mg/kg/day during gestation was lower than that of Control, however following parturition mean body weight gain for females was essentially similar to Control.

Mean food consumption for males receiving 100 and 330 mg/kg/day was generally similar to Controls throughout the dosing period. Mean food consumption for females receiving 330 mg/kg/day was similar to the Control during the pre-pairing period, however lower mean food intake periods were observed during the gestation and lactation periods for these females.  

Estrous cyclicity, pre-coital interval, mating performance and fertility were unaffected by treatment with Bayscript Magenta BB. One Control female (1F No. 86) failed to mate. A slight shift towards longer gestation lengths was apparent for females receiving 330 mg/kg/day in comparison with the concurrent Control.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in total white blood cell count in males given 100 or 330 mg/kg/day. This was a result of lymphocyte, monocyte and neutrophil counts being lower in treated males. White blood cell counts for females on Day 14 of lactation, however, were similar to Control values at the 100 and 330 mg/kg/day dose levels.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Changes in organ weights consisted of slightly lower body weight adjusted epididymides and testes weights in males receiving 330 mg/kg/day. In males, receiving 100 or 330 mg/kg/day slightly high body weight adjusted kidney weights were seen. Slightly high absolute and body weight adjusted kidney and liver weights were evident in females given 330 mg/kg/day. In addition, group mean adjusted ovary and uterus, cervix and oviducts weights were slightly low for females given 100 or 330 mg/kg/day. 

Macroscopic examination of the adult males and females revealed abnormally pink colouration of the kidneys in all males and females given 100 and 330 mg/kg/day. Abnormally pink colouration of multiple organs was recorded in the majority of animals given 330 mg/kg/day. Several males and females at 100 mg/kg/day were noted to have abnormal colour (pink) of the mesenteric lymph nodes. 

Histopathological evaluation of retained tissues revealed degeneration in the cortical tubules of the kidneys in most males and all females given 300 mg/kg/day; this was associated with regeneration in the cortical tubules for females only. An increase in Periodic acid-Schiff (PAS) staining was present in the interstitial macrophages of the testes for males given 330 mg/kg/day.

F1 litter responses

A reduction in mean number of implantation sites of females receiving 330 mg/kg/day resulted in lower mean litter size when compared with Control. The post implantation survival index was also slightly low at 330 mg/kg/day; all values were outside the historical control data. 

The clinical condition, sex ratio, ano-genital distances and body weight of the F1 offspring was unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment. One female given 330 mg/kg/day had a total litter loss on the day of parturition (Day 25 of gestation).

Conclusion

In conclusion, the oral administration of Bayscript Magenta BB at 1000 mg/kg/day was not tolerated and dosing was terminated early for males and females on Days 11 and 18 of dosing, respectively. The oral administration of Bayscript Magenta BB up to and including 330 mg/kg/day for five weeks in males and for two weeks prior to pairing, throughout gestation and up to Day 13 of lactation in females, was generally well tolerated. 

Test item-related histopathological changes were evident in the kidneys, consisted of tubular degeneration in both sexes, tubular regeneration predominately in females and hyaline droplets in males in animals given 330 or 1000 mg/kg/day, associated with increases in adjusted kidney weights in both sexes given 330 mg/kg/day and with gross changes of abnormal coloured kidneys seen at necropsy; these kidney changes were considered to be adverse. Test-item related histopathological changes were also evident in the stomach, spleen and liver of animals given 1000 mg/kg/day and terminated early. These included eosinophilic globules and foveolar hyperplasia in the stomach, with submucosal inflammation observed in males only, increased cellularity of the splenic white pulp and, hepatocellular apoptosis and vacuolation in the liver of females. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 100 mg/kg/day.

There was no evidence of any adverse effects on mating performance of the adult animals or on the development of the F1 offspring. The reduction in the number of uterine implantations at 330 mg/kg/day (and as a consequence litter size) was associated with the slightly low post implantation survival index. This effect, however, was statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be potentially related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 100 mg/kg/day.

Bayscript Magenta BB showed no evidence of being an endocrine disruptor.

Justification for classification or non-classification

A Combined Repeated Dose Toxicity Study and Reproductive /Developmental Toxicity Screening Study conducted according to OECD TG 422 is available. Severe toxicity including deaths is observed at the limit dose of 1000 mg/kg/day. Severe toxicity, although no deaths, is observed at the mid dose of 330 mg/kg/day. At the same dose reduction in the number of uterine implantations was associated with the slightly low post implantation survival index. This effect is considered to be related to treatment and it is assumed likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined. In particular the severe systemic toxicity at 330 mg/kg/day and the high dose, 1000 mg/kg/day, has to be taken into account in the context of classification. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 100 mg/kg/day.

No final conclusion can be drawn on the reduced uterine implantations and litter size in the presence of severe maternal and paternal toxicity observed in the Combined Repeated Dose Toxicity Study and Reproductive /Developmental Toxicity Screening Study. A final EOGRT is proposed to finally decide on potential classification for toxicity to fertility.