Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

FAT 21036 did not cause any adverse effects when administered though oral route in a 14 days range finding study as well as during the main reproductive and developmental screening study. However, read across substance, FAT 20297 was found to have a NOAEL of 282 mg/kg bw/day for females on oral administration for 28 days. Hence, FAT 21036 is also considered to have a NOAEL of 282 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Sep 1983 to 11 July 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

"See additional information"

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Code no: FAT 20297/C
Batch no: HS 11029/16
Description: Blue powder
Received: January 25, 1983
Validity: about 30 years

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Details on species / strain selection:

Recommended by the guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY LTD., 4332 Stein / Switzerland
- Age at study initiation: approx. 4-5 weeks
- Weight at study initiation: 105-111 g in males; 105-107 g in females
- Housing: In group of 5 in macrolon cages type 4 with standardised granulated soft wood bedding (Société Parisienne des sciures Pantin).
- Diet: Pelleted, certified standard diet Nafag No. 890 Tox, ad libitum
- Water: Tap water ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
Food: All batches of diet were assayed for composition and contaminant levels by the manufacturer. Analytical results are available at the animal supply office (CIBA-GEIGY LTD., Pharmaceuticals Division PH 2.162).
Water: Results of the routine chemical examination of water at source (Grundwasserfassung Stein) as conducted periodically by the water authority (Baudepartement des Kantons Aargau, Abteilung Gewaesserschutz) are available to CIBA-GEIGY LTD., as well as the results of inhouse chemical analysis by the analytical laboratories of the Pharmaceuticals Division, CIBA-GEIGY LTD.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 16-20 air changes/hour
- Photoperiod:12 hrs light per day

IN-LIFE DATES: From: August 30, 1983; To: October 5 - 6 , 1983

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
FAT 20297/C was weighed on a calibrated Mettler balance. The pulverised diet was then homogeneously mixed with the appropriate concentrations of the compound and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried. The animals in the control group (group 1) were fed with similarly pelleted feed without the test substance..PREPARATION OF DOSING SOLUTIONS:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Prior to the initiation of the study, pretest feed samples were analysed for concentration, homogeneity and stability of FAT 20297/C.
- The mean concentration of active ingredient during the study according to the chemical analysis * was 96.6 - 104.6 % of the added amount.
- These analysis were carried out in the Analytical Laboratories of CIBA-GEIGY Ltd., Basier / Switzerland.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group Males

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group (females)

Dose / conc.:

104 mg/kg bw/day (actual dose received)

Remarks:

low dose for males

Dose / conc.:

297 mg/kg bw/day (actual dose received)

Remarks:

mid dose for males

Dose / conc.:

1 050 mg/kg bw/day (actual dose received)

Remarks:

high dose for males

Dose / conc.:

102 mg/kg bw/day (actual dose received)

Remarks:

low dose for females

Dose / conc.:

282 mg/kg bw/day (actual dose received)

Remarks:

mid dose for females

Dose / conc.:

1 090 mg/kg bw/day (actual dose received)

Remarks:

high dose for females

Dose / conc.:

1 000 ppm

Remarks:

low dose for males and females

Dose / conc.:

3 000 ppm

Remarks:

mid dose for males and females

Dose / conc.:

10 000 ppm

Remarks:

high dose for males and females

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
Animals of the highest dosage group and of the control group were examined before (day -6) and towards the end (day 27) of the treatment period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Hearing test: Animals of the highest dosage group and of the control group were examined before (day -6) and towards the end (day 27) of the treatment period.

Sacrifice and pathology:

At the end of the test period all control and test animals which survived were bled under ether anaesthesia and subjected to detailed autopsy.
Besides the weight of the exsanguinated body the organs weights were also recorded.
After the fixation organ samples from each control and test rat were taken, embedded in paraplast, sectionned at 3-5 micron, stained with haematoxylin and eosin and subjected to microscopic examination.

Statistics:

For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system, parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement. In addition a trend test was applied considering all groups.
Statistical analysis is performed to draw attention to distinct values. A statistically significant difference between two values does not necessarily imply biological relevance of that deviation and is not conclusive for a treatment related effect.
Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may comment on statistically not significant values, which differ substantially from the expected normal values.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical symptoms and no signs of local and/or systemic toxicity were observed.

Mortality:

no mortality observed

Description (incidence):

No death occurred during the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of all treated male and female groups was similar to that of the respective controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption was slightly higher in both treated male and female animals of group 4 (10000 ppm) compared to the respective control. The mean food consumption of the other treated male and female groups was similar to that of the respective control groups.

Food efficiency:

no effects observed

Description (incidence and severity):

The mean food conversion of female group 4 (10000 ppm) was higher than the respective control ratio, thus indicating spillage of food due to reduced palatability of the medicated diet.
Specific food consumption in relation to body weight - addressed as food conversion ratio - of the other treated animals was similar to the control ratios.

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

effects observed, non-treatment-related

Description (incidence and severity):

The mean water consumption showed a tendency to increased intake in both treated male and female groups 3 and 4 (3000 and 10000 ppm). However, since water consumption was measured only by cage, the number of values (n = 2) does not allow for a definite judgement about the relevance of this finding. The toxicological significance of this deflection is however in doubt.
The mean water consumption of treated male and female group 2 (1000 ppm) was similar to that of the respective control groups (the low value of the female control at week 4 is considered unreliable).

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic inspections and hearing examinations performed before (day -6) and towards the end (day 27) of the application period revealed no evidence of a reaction to the treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

No difference, which could be related to the test compound was found between treated and control animals in the hematological values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Throughout the treated groups, no changes were observed, which could be related to the substance administration.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative mean liver weight of both treated male and female groups 4 (10000 ppm) were significantly depressed. Additional statistically significant differences in organ weights, attributed to spontaneous variation rather than to the treatment were also observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related gross pathological findings were observed. All other gross and histopathological lesions seen in some control and test animals were only incidental in nature and not due to the application of the tested compound.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopical examination revealed slightly more pronounced fatty changes of the hepatocytes in female and male animals of group 4 (10000 ppm).

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Effect level:

297 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Effect level:

282 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

no

Dosage Levels: The amount of test material in the diet was determined analytically during the study. The results of these analyses revealed a concentration of 96.6 - 104.6% of the nominal value.

According to the analytical results the calculated mean daily intake of FAT 20297/C was approximately 104, 297 and 1050 mg/kg bw. in males and 102, 282 and 1090 mg/kg bw in females.

Conclusions:

"No observed effect level" for FAT 20297/C when offiered to rats continuously in their feed over a period of 28 days is 3000 ppm, corresponding to a mean daily intake of FAT 20297/C of 297 mg/kg body weight for males and 282 mg/kg bw for females.

Executive summary:

The repeated dose toxicity of Acid Blue 344 was investigated in this study conducted according to OECD Guideline 407. In this study a total of 80 RAIf (SPF) rats, 10 males and 10 females per dose group were used. The test article FAT 20297/C was administered in the diet for 28 days at dosages of 1000, 3000 and 10000 ppm ( = mg/kg feed). According to the analytical results the calculated mean daily intake of FAT 20297/C was approximately 104, 297 and 1050 mg/kg bw in males and 102, 282 and 1090 mg/kg bw in females. After 28 days of daily dose administration, no mortality or clinical symptoms/ systemic toxicity were observed at any dose levels. No treatment related effect on body weights, food and water consumption, food conversion, eyes, hearing capability, hematology and blood chemistry was observed. However, absolute and relative mean liver weights of both treated male and female groups 4 (10000 ppm) were significantly depressed. Microscopical examination revealed slightly more pronounced fatty changes of the hepatocytes in female and male animals of group 4 (10000 ppm). Based on these findings, "No observed effect level" for FAT 20297/C is 3000 ppm, corresponding to a mean daily intake of 297 mg/kg bw for males and 282 mg/kg bw for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

282 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

Currently no 28- or 90-days repeated dose toxicity study is available to assess the toxicity potential of FAT 21036/G. A 14 days range finding study, conducted to decide dose range for the reproductive and developmental screening is available. Further, the reproductive and developmental screening study conducted according to OECD Guideline 421, can also help in this assessment. However, to fulfil the study requirements at this tonnage level and to complete the assessment, a 28-days repeated dose toxicity study on read across substance, was used. The repeated dose toxicity potential of the read across substance, FAT 20297 was investigated in a study conducted according to OECD Guideline 407. In this study a total of 80 RAIf (SPF) rats, 10 males and 10 females per dose group were used. The test article FAT 20297/C was administered in the diet for 28 days at dosages of 1000, 3000 and 10 000 ppm ( = mg/kg feed). According to the analytical results the calculated mean daily intake of FAT 20297/C was approximately 104, 297 and 1050 mg/kg bw in males and 102, 282 and 1090 mg/kg bw in females. No mortality or clinical symptoms/ systemic toxicity were observed at any dose levels. The repeated intake of FAT 20297/C had no treatment related effect on body weights, food and water consumption, food conversion, eyes, hearing capability, hematology and blood chemistry. However, absolute and relative mean liver weights of both treated male and female groups 4 (10000 ppm) were significantly depressed. Microscopical examination revealed slightly more pronounced fatty changes of the hepatocytes in female and male animals of group 4 (10000 ppm). Hence, based on these findings, it can be inferred that a "No observed effect level" for FAT 20297/C when offered to rats continuously in their feed over a period of 28 days is 3000 ppm, corresponding to a mean daily intake of FAT 20297/C of 297 mg/kg bodyweight for males and 282 mg/kg bw for females.

 

FAT 21036 was assessed for the toxicity potential on repeated exposure in a 14 days range finding study. The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for fourteen consecutive days, at dose levels of 500, 750 and 1000 mg/kg bw/day (incorporating a correction factor for 63.5% purity). A control group of three males and three females was dosed with vehicle alone (distilled water) over the same treatment period. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination. There were no unscheduled deaths observed during the study. Clinical signs, body weight changes, intergroup differences for food consumption or food conversion efficiency and water consumption as well as necropsy did not indicate any obvious systemic toxicity for either sex at 500, 750 or 1000 mg/kg bw/day. Hence, the NOAEL was determined to be 1000 mg/kg bw/day in this study. Further, no clinical symptoms or systemic effects were seen with the repeated administration of FAT 21036/G over a duration of 6 weeks in a reproductive and developmental screening study conducted according to OECD Guideline 407. The NOAEL in this study was determined to be 1000 mg/kg bw/day.

Hence, based on the available data from a 28-days dietary toxicity study with a read across substance, along with a 14-days range finding study and a reproductive and developmental screening study with the target substance, the substance is considered to have a NOAEL of 282 mg/kg bw/day on repeated exposure through oral route.

 

Repeated dose toxicity: Inhalation

Currently no study to assess the repeated dose inhalation toxicity potential of Acid Blue 225 is available. However, the vapour pressure for the substance can be considered low owing to the high melting point (>300 °C). Hence the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Further the chemical is found to have water solubility of 64.3 g/L, hence in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. No systemic toxicity was observed when Acid Blue 225 was administered upto 1000 mg/kg bw/day via gavage in a reproductive/developmental toxicity screening study. Further, a 28-day repeated dose toxicity study with source substance Acid Blue 344 is available. No elevated toxicity other than seen with the source substance are expected from repeated dose inhalation exposure of Acid Blue 225 and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Acid Blue 225 is considered to be scientifically not necessary.

 

Repeated dose toxicity: Dermal

Currently no study to assess repeated dose dermal toxicity study of Acid Blue 225 is available. However, the molecular weight of the substance is 669.5 g/mol, which indicates substance is too large for dermal absorption. Further, high water solubility (64.3 g/L) and low partition coefficient (log Pow: 0.58), indicate the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance is expected to be low. Further, production and spray drying is performed in closed processes without isolation of reaction products. Isolated product is present in dust free granules (non-dusty solid). In addition, the use of this substance will not result in aerosols, particles or droplets, so exposure to humans via the dermal route will be unlikely to occur. Risk management measures established for workers and professionals are considered sufficient to enable safe handling and use of the final products containing the formulated dye. Acid Blue 225 when applied to skin of the rabbits, did not induce any local or systemic effects in the skin irritation study. No systemic toxicity was seen in three different skin sensitization studies. Further exposure to test animals via the oral route in an acute toxicity study as well as 14-days range finding study and a reproductive/developmental screening study did not result to systemic toxicity. Further results of repeated dose exposure of the source substance, Acid Blue 225 are available and hence no elevated toxicity other than seen with the source substance are expected via the inhalation route and safety for human health can be estimated via route to route extrapolation. Hence, based on the above discussion, the conduct of repeated dose dermal toxicity study with Acid Blue 225 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the available data, Acid Blue 225 does not warrant classification for specific target organ toxicity- repeated exposure as per the CLP (Regulation 1272/2008) criteria.