N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Endpoint summary

Description of key information

Based upon the results obtained in the repeated dose toxicity study with reproduction and developmental screening, the NOAEL was set at 100 mg/kg bw/day for male and females rats when administered orally by gavage.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Few deviation from the study plan regarding hisptopathology and clinical pathology. However, these deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Q30450ADZX
- Expiration date of the lot/batch: 08/08/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: In water- 3.26 mg/l

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 9-10 weeks old, females: 9-10 weeks old
- Weight at study initiation: males: 253 - 281 g and females: 171 - 191 g
- Housing: 2 animals/ sex/ cage in IVC cages (type III H, polysulphone cages)
- Diet :ad libitum
- Water: ad libitum
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc).

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer).
The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine.

VEHICLE

- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): MKBP7039V

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

54 days

Frequency of treatment:

Once daily (7 days per week)

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

300 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

Control (C): 0 mg/kg/d
Low Dose (LD): 100 mg/kg/d
Medium Dose (MD): 300 mg/kg/d
High Dose (HD): 1000 mg/kg/d

The test item formulation was prepared once in every ten days and stored at 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups as well as all gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: terminal sacrifice from five males and five randomly selected females from each group
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5/sex

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals: 5/sex

URINALYSIS: Yes
- Time schedule for collection of urine: Terminal sacrifice
- Metabolism cages used for collection of urine: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

One-way ANOVA and a post-hoc Dunnett Test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered due to local effect of test item. There were other clinical signs including alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation in a few animals of control and/ or dose groups. These were considered incidental. During the weekly detailed clinical observation, no changes or differences between the groups and the baseline values.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males and female, there were no adverse changes of toxicological relevance on body weight and body weight gain. However, In males there was statistically significantly lower weight gain in HD groups after premating days 7-14. There was also statistically significantly lower weight gain in MD group after mating/ post matings days 7-14. There were also lower weight gain in LD and HD groups after the 1st and last week of mating/postmating period. These changes were either transient and/ or without dose response relation. Therefore, the findings was not considered to be an adverse effect of treatment. In females, there was statistically significantly higher weight gain after lactation days 0-4 in HD group. This higher weight gain was within the range of historical control data and was not considered likely to be an effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were no adverse changes of toxicological relevance for food consumption in dose groups when compared to control. There were no statistically significant differences between the dose and control group values. However, in males there was very marginal decrease in food consumption after premating period 7-14 in HD group and in females the food consumption was higher in HD group after lactation days 0-4. These changes in food consumption in males or females correlated with changes in weight gain. In the absence of statistically significant differences, the lower or higher food intake in males and females were not considered an adverse effect of treatment.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were findings of toxicological relevance in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV in MD and HD group, MCH and reticulocyte count in HD group. There findings were associated with microscopically observed erythrocytic extramedullary hemopoiesis in the spleen of both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. There was also statistically significantly lower MCHC value in male MD group and statistically significantly higher Eosinophil and statistically significantly lower LUC count in female MD group. These were not in dose relation and were not considered related to treatment. In males and females, there were no effect on the blood coagulation parameters.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups. These findings were associated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings were considered to be adverse event attributable to treatment with the test item. There was also statistically significantly lower absolute and relative (to terminal body weight) pituitary gland weight in MD group of male animals. There was also statistically significantly higher relative kidney weight in HD group. In the absence of dose response relation, the finding was not considered likely to be related to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged of spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in three males of MD and 4 males and one female of HD group. This was considered to be treatment-related change. “Discoloured pale” or “Discoloured” of the kidney was recorded in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. The sperm stages of testes were checked on completeness of cell populations, completeness of stages and degenerative changes. Nothing particular was observed in testes of animal no. 2 (control), which was mated with female no. 42 not giving birth. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2. However, similar minimum to slight lesions are often observed in male animals that can produce pregnancy to the pairing females, and therefore, it is unlikely that these findings were related to the infertility. In male no. 31 (HD group), marked tubular atrophy (ie. so-called sertoli cell-only tubules) in testes and marked oligospermia (ie. azoospermia) in epididymides were observed, and this was considered to be the cause why the paired female no. 71 did not give birth. Complete tubular atrophy sometimes happens spontaneously in the male rats of this strain and age, and sporadic occurrence with only one case is unlikely to be related to treatment with the test item. Nothing particular was observed in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42 (control) and 71 (HD group) which did not give birth. As a result of the detailed examination of the testis, there were no abnormalities on the completeness of stages and cell populations, except for male no. 31 (HD group). In the testis of male no. 31, it was impossible to evaluate the stages and cell population because of complete tubular atrophy. However, this was judged to be spontaneous change as described above. There was no dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age, or were considered to be accidental changes which can occur in the toxicology studies using the rodent models.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/d for systemic toxicity in males and females.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw (total dose)

System:

haematopoietic

Organ:

bone marrow

erythrocyte development

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/d for systemic toxicity in males and females.

Executive summary:

On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d conducted as per OECD 422 guideline (GLP compliance), the adverse effects found such as haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/d for systemic toxicity in males and females.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer chapter 13 for detailed read across justification.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

300 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw (total dose)

System:

haematopoietic

Organ:

bone marrow

erythrocyte development

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Based on the read across data generated from the combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/d for systemic toxicity in males and females.

Executive summary:

Data on repeat dose toxicity study was not available for the target substance. To fill the data gaps, read across approach is adapted using similar substance FAT 36034/W. Read-across is claimed basis of structural relationship of the target and the source chemicals. Read across substance is FAT 36034/W and have been investigated for repeat dose toxicity. On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d conducted as per OECD 422 guideline (GLP compliance), the adverse effects found such as haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/d for systemic toxicity in males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality GLP study

System:

haematopoietic

Organ:

bone marrow

erythrocyte development

spleen

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

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

On the basis of a read across study of FAT 36034/W, reproduction/developmental toxicity screening test in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d was performed. Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d. There were no effects on reproduction/ developmental parameters. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/Z, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental Toxicity.

Inhalation:

Currently no study to assess the repeated dose inhalation toxicity potential of Disperse Red 86:1 is available. However, the vapour pressure for the substance can be considered low (3.69E-11 Pa at 25 °C). Hence, the substance is considered to have low volatility.Based on column 2, ‘Specific rules for adaptation from column 1’ of the table given in REACH Annex VII, the study on repeated dose inhalation toxicity only needs to be conducted if exposure via inhalation is to be expected, based on vapour pressure and/or the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the substance is imported into the EU in a formulated form as a dust-free powder or as a granulate, the exposure via inhalation is considered to be unlikely. Further, results of exposure to test animals from the repeated dose toxicity study with reproductive/developmental screening via oral route with Disperse Red 86 (source substance) are available. Taking into consideration the above arguments, no elevated toxicity other than already seen in repeated dose oral toxicity study with source substance, are expected via the inhalation route and safety for human health can be estimated using the principles of read across and route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Disperse Red 86:1 is considered to be scientifically not necessary.

Dermal:

Currently no study to assess the repeated dose dermal toxicity of Disperse Red 86:1 is available. However, the molecular weight of the chemical is 408.43 g/mol, indicating it being large for dermal absorption. Hence, the dermal uptake for the chemical is expected to be limited. Absence of systemic toxicity in skin irritation studies, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon dermal exposure and not after systemic application. Further, results of exposure to test animals from the repeated dose toxicity study with reproductive and developmental screening via oral route with Disperse Red 86 (source substance) are available. Taking into consideration the above arguments, no elevated toxicity other than already seen in repeated dose oral toxicity study with source substance, are expected via the dermal route and safety for human health can be estimated using the principles of read across and route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for Disperse Red 86:1 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 as a Specific Target Organ Toxicity - Repeated Exposure (STOT-RE) category 2 for toxic effects on the blood following oral exposure.