2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Endpoint summary

Description of key information

No data is available for the target substance. Thus, available data from the structural analogue (chloride salt) of the target substance was used in a read-across approach. Details on the read-across rational are provided in section 13.

The source substance was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL for general toxicity could not be determined. Thus, the LOAEL for systemic effects can be considered to be 40 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
Clinical signs mainly observed in the treated animals were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. Other clinical signs such as piloerection, damaged ear and cyphosis were generally observed in a limited number of animals during the study.
Recovery groups:
Clinical signs observed during the treatment period were blue staining on the dorsum/tail in all treated animals and salivation in one treated male (no. 96) and three treated females (nos. 91, 93 and 99). Hairloss on the head was observed in one control female (no. 87). During the recovery phase, blue staining of the tail was still observed in all treated animals, in the first week of the recovery period. Hairloss on the head was observed in two males (nos. 84 and 86) and two females (nos. 81 and 87) of the control group. In addition, control females showed brown staining on the head (animal no. 81) and scab on the head ( animal no. 87).
Recovery groups:
Clinical signs observed during the treatment period were blue staining on the dorsum/tail in all treated animals and salivation in one treated male (no. 96) and three treated females (nos. 91, 93 and 99). Hairloss on the head was observed in one control female (no. 87). During the recovery phase, blue staining of the tail was still observed in all treated animals, in the first week of the recovery period. Hairloss on the head was observed in two males (nos.
84 and 86) and two females (nos. 81 and 87) of the control group. In addition, control females showed brown staining on the head (animal no. 81) and scab on the head ( animal no. 87).

Observations of the cage tray
– Blue staining on the cage tray, slight to marked, was observed during the pre-mating
and mating periods in animals of all main groups of both sexes.
– Blue staining on the cage tray, slight to marked, was observed after mating phase in all
treated males (main groups) up to the end of treatment.
– Blue staining on the cage tray, slight to moderate, was seen during the gestation and
post partum periods in females receiving dose levels of 80 and 120 mg/kg body weight/day.
– During the dosing phase, recovery group (males and females) showed blue staining
on the cage tray, slight to moderate. This sign disappeared from Day 2 of the recovery
phase.
The above mentioned findings were considered related to the colour of the test item which was eliminated by the animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

Thirteen high dose group animals died or were sacrificed for humane reasons during the treatment period. Four males (nos. 70, 74, 76 and 80) and 7 females (nos. 61, 65, 69, 73, 75, 77 and 79) of Group 4 died during the treatment phases (premating and mating phase for males and premating, gestation and post partum period for females). In addition, two females were sacrificed for humane reasons, one (no. 63) on Day 22 of the gestation period, due to a prolapse of the uterus occurred and the second one (and its litter) on Day 1 of the post partum period, due to a poor health condition (no. 71). In the high dose group, starting from Day 17 of treatment, the dose level was reduced from 160 mg/kg body weight/day to 120 mg/kg body weight/day. However, the animals continued to die although the dose level was reduced. Five mid-dose group animals died during the treatment period. One male (no. 46) which died on Day 4 mating phase and 4 females died on Days 21 and 22 of the gestation period (nos. 57 and 59, respectively) and on Day 1 of the post partum period (nos. 47 and 51). Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as: hairloss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. The changes observed at post mortem examination were represented by single instances of multiple/single red or dark depressed areas in the stomach (glandular region) and reduced size of the thymus. Blue staining of head, salivary gland, tail or blue content of the stomach were seen and considered related to the test item. Moreover, animal no. 74 (Group 4) showed red fluid in the thoracic cavity related to a possible misdosing, while animal no. 75 (Group 4) showed a subcutaneous mass in the skin, microscopically recognised as adenocarcinoma in the mammary glands. This latter finding was considered an incidental pathology rather than treatment-related.
The histopathological evaluation identified the factors contributory to the death of these animals in the cardiomyopathy, represented by mild to marked multifocal mononuclear cell infiltrations, fibrosis, cardiomyocytes necrosis, associated or not with multifocal mild necrosis in the liver and/or adrenals and/or kidneys. Immunodepression was also reported in some instances in the thymus.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Main groups
No differences of toxicological significance were noted throughout the study in the body
weight of treated males, when compared to the control group. On Day 8 of the study, during the mating phase, body weight and body weight gain were significantly decreased (at statistical analysis) in males of the low dose group (-7 and -82%, respectively). Changes in body weight and body weight gain observed at the end of the mating period were not significant, at statistical analysis, in any treated groups, not dose related and therefore, not considered treatment related. Before pairing, body weight of treated females was comparable to the control group. At the end of the post coitum period (Day 20), body weight of females of the high dose group was slightly lower than controls (-6%) but statistically significant. At post partum period (Day 4), body weight of females of the mid- and high dose levels was lower than controls (-12% and -11%, respectively), statistically significant in the mid-dose group. Significant reduction in body weight gain was observed in all groups of females on Day 7 of the post coitum period (ranging from -27% to -33%). In addition, a statistically significant reduction in body weight gain was noted in the mid-dose group at the end of the post partum period. Reduced body weight gain at the end of the post partum period was also observed in the unique female of the high dose group, which survived treatment and reached the end of the study.

Recovery groups
Means of body weight and body weight gain were comparable between controls and the treated group both in males and females throughout the treatment phase. During the recovery period, no differences in body weight and body weight gain were recorded in animals of both sexes, when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
Food consumption measured in treated males and females, before pairing, was comparable to the control group. On Day 7 post coitum, food consumption was significantly lower (at statistical analysis) than the control group in females of the mid- and high dose groups (-11% and -12%, respectively).
Recovery groups:
During treatment and recovery periods, no differences from controls were noted in food consumption in the treated groups in both sexes receiving 80 mg/kg body weight/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
Leucocytes were lower than controls in a number of males treated at 40 and 80 mg/kg body weight/day. Mean group values were 40% and 32% below controls, respectively. Eosinophils were also decreased in males receiving 160/120 mg/kg body weight/day (77%). In addition, some females from all treated groups (nos. 21, 41, 55 and 67) showed lymphocytes higher than controls: the increments were approximately 2.1 fold, with no dose-relation. Moreover, eosinophils were decreased in all treated females (83% to 100%). Due to the absence of dose-relation, the above white blood cell findings were not conclusively attributed to treatment. Concerning the red blood cell parameters, erythrocytes, haemoglobin, haematocrit and
mean corpuscular volume were lower than controls and mean corpuscular haemoglobin concentration was higher than controls and mean corpuscular haemoglobin concentration was higher than controls in some males from all treated groups, without dose-dependency. Changes were approximately 20% for haematocrit and approximately 10% for the other parameters. Changes were insufficient in severity to represent an adverse anaemia. The statistically significant decrease of reticulocytes recorded in males receiving 40 mg/kg body weight/day was not dose-related, therefore it was considered incidental.

Coagulation
Prolonged prothrombin time was recorded in males dosed at 80 and 160/120 mg/kg bodyweight/day. Changes were 16% and 18% above mean control data, respectively.

Recovery phase - Recovery groups
Changes recorded during the dosing phase were no longer observed. The statistically significant differences between control and treated animals (slight increases of lymphocytes in males and erythrocytes in females) were not observed during the dosing phase, therefore they were considered unrelated to treatment.
Coagulation
No changes were recorded, confirming complete reversibility.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Most of the statistically significant differences between control and treated males (alanine aminotransferase, aspartate aminotransferase, cholesterol, triglycerides, phosphorus and potassium) were mainly due to the changes recorded in some control males (nos. 4, 10 and 14), which showed increases of many biochemical parameters. By the contrary, the decreases of urea, creatinine, protein, albumin, globulin and calcium were not associated with the control animal changes. However, due to the absence of dose relation, these findings cannot be definitively attributed to treatment. One female receiving 80 mg/kg/body weight/day (no. 49) showed moderate increases of both transaminases enzymes, total bilirubin and urea. Female no. 67 (160/120 mg/kg body weight/day) showed reduced triglycerides and increased glucose. Due to the limited incidence and the reduced number of animals dosed at 160/120 mg/kg body weight/day, no conclusions could be made for such changes.

Recovery groups
Most of the findings recorded during the dosing phase were completely recovered with few exceptions, which showed partial reversibility. In particular, protein, albumin and calcium were still slightly lower than controls in males (-6%, -9% and -4%, respectively), and globulin was slightly higher in females (7%). In addition, statistically significant differences of sodium (-1%) in treated females and phosphorus (13%) in treated males were recorded. Due to the minimal severity, the above changes
were considered of no toxicological relevance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item. Sensory reaction to stimuli and motor activity did not show relevant differences between control and treated groups in both sexes. Significant reduction, at statistical analysis, in grip
strength (second trial) and the relative mean data was noted in all treated male groups, while no relevant differences were observed in female animals.

Recovery groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item. No significant alterations in sensory reaction to stimuli were observed in the treated group at the examination performed at the end of treatment. A statistically significant decrease in grip strength (first, second trial and mean) was noted in treated males. In addition, a statistically significant increase in motor activity measurement was noted in treated females. For males, due to the low control values measured (when compared to all other control values in the study), no relevance was attributed at the statistically significant increase measured. At the end of the recovery period, no variations in motor activity, grip strength or sensory reaction to stimuli were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease was observed in the terminal body weight of mid-dose females (-11%), when compared to the controls. Slight reduction, not statistically significant, was noted in terminal body weight of low and high dose males (-6% and -4%, respectively). The reduction observed in males and females was not considered toxicologically relevant, due to the very slight severity (ranging from -4% to -11%), even if statistically significant for females. A statistically significant increase in absolute and relative heart weight was noted in females receiving 80 mg/kg body weight/day. No relevant changes were observed in the remaining absolute and relative organ weights of treatment groups of both sexes, when compared to control data, with the exception of the decreased absolute and relative liver weight of low and high dose male groups, absolute and relative thymus weight in low dose males. However, since histomorphologies of these organs were comparable to control animals, no clear toxicological significance could be attributed to these findings.

Recovery sacrifice
After 2 weeks of recovery period, no relevant changes were observed on terminal body weight, absolute and relative organ weights in the recovery phase animals previously dosed
at 80 mg/kg body weight/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The changes observed at post mortem as blue staining of tail, head, clitoral or salivary glands were attributed to the colour of the test item. Reduced size of the thymus seen in control and treated females was considered related to physiological changes, often seen in this kind of study in some treated Sprague Dawley rats of the same age.

Recovery sacrifice
No remarkable differences were noted at post mortem examination in treated animals, when compared with controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were seen in the heart of all high dose males and the remaining surviving high dose female, consisting in moderate to marked cardiomyopathy, represented by multiple mononuclear cell infiltrations, fibrosis and cardiomyocyte necrosis. Cardiomyopathy was also observed, with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group. Increased incidence or presence of minimal inflammatory cell foci was seen in the heart of low dose males and females, respectively, when compared with controls. This finding consisted mainly in mononuclear cells such as macrophages and lymphocytes in some cases accompanied by necrosis of single cardiomyocyte and fibrosis. The remaining lesions reported in control and/or treated animals such as nephropathy of kidneys were considered to be an expression of spontaneous and/or incidental pathology commonly seen in this species and age under our experimental conditions. Following the extension of the histopathological evaluation of the spleen to all treated males and females, this organ was considered with normal morphological aspect, when compared with all control animals.

Recovery sacrifice
At the histopathology investigations performed on the heart, cardiomyopathy was still observed in most treated rats of both sexes with minimal to moderate degree in males and minimal to mild degree in females, after a wash-out period of two weeks.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), adverse effects were found after oral administration of the test item in male and female rats. Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL (No Observed Adverse Effect Level) can not be determined and thus the LOAEL for systemic toxicity is considered to be 40 mg/kg bw/day.

Executive summary:

The test item (90.6% purity) was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) at dose levels of 0, 40, 80 and 160 mg/kg bw/day in 10 animals per sex/dose. Due to the toxicity observed in the high dose group, the high dose level was reduced to 120 mg/kg bw/day from day 17 on. Males were treated for a total of 28 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post-partum, for at least 41 days. During the study, a total of 13 high dose animals died or were sacrificed for humane reasons during the treatment period. Five mod-dose group animals died during the treatment period. Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as hairloss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. No toxicological significant effects on body weight were noted throughout the study in males. Before pairing, body weight of treated females was comparable to the control. At the end of the post coitum period, the body weight of females of the high dose group was only slightly lower than controls, but statistically significant. At post-partum period, body weight of females in the mid and high dose levels were lower than the control group with statistical significance in the mid-dose group. In the recovery groups no effect on body weight were seen. Food consumption in treated males and females, before pairing, was comparable to the control group. On day 7 post coitum, food consumption was slightly but statistically significant lower than the control in females of the mid-and high dose groups. In the recovery groups, no differences from controls were noted in food consumption. A prolonged prothrombin time was recorded in mid and high dose male animals. In the recovery groups this effect was complete reversible. Decreases of urea, creatinine, protein, albumin, globulin and calcium were observed in treated males. However, due to the absence of a dose-relationship, these findings cannot be definitively attributed to the treatment.

A statistically significant decrease was observed in terminal body weight of mid-dose females when compared to controls. A significant increase in absolute and relative heart weight was noted in females of the mid-dose group. Treatment related changes were seen in the heart of all high dose males and one surviving female, consisting in moderate to marked cardiomyopathy, which was also observed with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group, while mononuclear cell infiltration associated or not with single necrosis of cardiomyocytes was also reported in low dose males and females.

Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL for general toxicity cannot be determined. Thus, based on the findings from the study, the LOAEL is considered to be 40 mg/kg bw/day for systemic toxicity.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

System:

cardiovascular

Organ:

heart

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data is available for the target substance. Thus, available data from the structural analogue (chloride salt) of the target substance was used in a read-across approach. Details on the read-across rational are provided in section 13.

The source substance was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) at dose levels of 0, 40, 80 and 160 mg/kg bw/day in 10 animals per sex. Due to the toxicity observed in the high dose group, the high dose level was reduced to 120 mg/kg bw/day from day 17 on. Males were treated for a total of 28 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post-partum, for at least 41 days. During the study, a total of 13 high dose animals died or were sacrificed for humane reasons during the treatment period. Five mod-dose group animals died during the treatment period. Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as hairloss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. No toxicological significant effects on body weight were noted throughout the study in males. Before pairing, body weight of treated females was comparable to the control. At the end of the post coitum period, the body weight of females of the high dose group was only slightly lower than controls, but statistically significant. At post-partum period, body weight of females in the mid and high dose levels were lower than the control group with statistical significance in the mid-dose group. In the recovery groups no effect on body weight were seen. Food consumption in treated males and females, before pairing, was comparable to the control group. On day 7 post coitum, food consumption was slightly but statistically significant lower than the control in females of the mid-and high dose groups. In the recovery groups, no differences from controls were noted in food consumption. A prolonged prothrombin time was recorded in mid and high dose male animals. In the recovery groups this effect was complete reversible. Decreases of urea, creatinine, protein, albumin, globulin and calcium were observed in treated males. However, due to the absence of a dose-relationship, these findings cannot be definitively attributed to the treatment.

A statistically significant decrease was observed in terminal body weight of mid-dose females when compared to controls. A significant increase in absolute and relative heart weight was noted in females of the mid-dose group. Treatment related changes were seen in the heart of all high dose males and one surviving female, consisting in moderate to marked cardiomyopathy, which was also observed with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group, while mononuclear cell infiltration associated or not with single necrosis of cardiomyocytes was also reported in low dose males and females.

Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL for general toxicity cannot be determined. Thus, based on the findings from the study, the LOAEL is considered to be 40 mg/kg bw/day for sytemic toxicity.

Justification for classification or non-classification

Marked cardiomyopathy was observed in the high and mid dose groups, while mononuclear cell infiltration associated or not with single necrosis of cardiomyocytes was reported in low dose (40 mg/kg bw/d) males and females in an OECD 422 study. According to CLP table 3.9.2.9.7, the guidance value for classification as STOT RE 2 is a LOAEL between 10 and 100 mg/kg/day reported in a 90d study (or 30-300 mg/kg/d based on a sub-acute study). Therefore, the organ specific adverse effects at 40 mg/kg/day reported in the present study warrant classification for specific target organ toxicity (heart), STOT RE 2 (H373) in accordance with CLP regulation (EC) No 1272/2008.