Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

Endpoint summary

Description of key information

50 mg/kg body weight/day of FAT 40810/A was established as the no-observed-effect-level (NOEL), and 1000 mg/kg body weight/day of FAT 40810/A as the no-observed-adverse-effect-level (NOAEL) in a subacute oral toxicity study.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

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:

Experiment start date - 25 October 2012; Experiment end date - 17 December 2012; Study completion date - 21 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: FAT 40810/B TE
Description: Black powder (determined at WIL Research Europe B.V.)
Batch: BOP 02-12 (Lot: MHC-1880000)
Content: 100% (w/w)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date 14 September 2017

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany. Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 310 gr (males) or 203 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES
From: 29 October - 17 December 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for at least 14 days.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Two females of Group 1, one of Group 2, one of Group 3 and two females of Group 4 were not dosed during littering.

Frequency of treatment:

Once daily, 7 d/w

Dose / conc.:

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

Remarks:

Group 1 (Control group)

Dose / conc.:

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

Remarks:

Group 2 (Low dose group)

Dose / conc.:

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

Remarks:

Group 3 (Mid dose group)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 - (High dose group)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 28-day toxicity study (RCC Project 847223) in which Wistar rats were dosed at 50, 200 and 1000 mg/kg/day. The mean level of leucocytes (urinalysis) was increased in males treated at 1000 mg/kg. The mean level of sodium was increased in males treated with 200 mg/kg and in both sexes at 1000 mg/kg. Based on the results of this study, 50 mg/kg body weight/day was established as the no-observed-effect-level (NOEL), and 1000 mg/kg of FAT 40'810/A as the no observed-adverse-effect-level (NOAEL).

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from start of treatment onwards, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No.

HAEMATOLOGY
No.

CLINICAL CHEMISTRY
No.

URINALYSIS
No.

NEUROBEHAVIOURAL EXAMINATION
No.

Sacrifice and pathology:

GROSS PATHOLOGY:
- All animals were deeply anaesthetised and subsequently exsanguinated. The animals were not deprived of food overnight.
- According to test guidelines

ORGAN WEIGHTS
- All males: Epididymides and testes

HISTOPATHOLOGY:
- According to test guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity related to test substance treatment were noted during the observation period.
Red faeces was noted for all animals at 300 and 1000 mg/kg. In addition, at 1000 mg/kg all animals showed orange staining of the tail and red discolouration of urine. These findings were caused by the staining properties of the test substance, and not regarded toxicologically relevant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
The statistically significantly decreased body weight gain at 100 mg/kg on Day 4 of lactation was not considered toxicologically relevant as the value was within normal limits and no dose response relationship was apparent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
The statistically significantly increased values noted during the post-coitum phase for treated females were not considered toxicologically relevant as all values were within normal, these concerned slight increases and no dose response relationship was noted.

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy did not reveal any toxicologically relevant alterations.
Reddish or orange discolouration of the testes, epididymides, skin of the tail and/or subcutis (whole body) was noted for all males and eight females treated at 1000 mg/kg. These discolourations were considered due to the staining properties of the test substance and not regarded toxicologically relevant.
A soft yellowish nodule was noted in the epididymides of two males at 100 mg/kg, two males at 300 mg/kg and one male at 1000 mg/kg.
The incidence of other findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation of the kidneys, discolouration of the clitoral glands or thymus, fluid in the uterus, diaphragmatic hernia of the liver, focus on the thymus or clitoral glands, and cyst on the ovaries.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal any toxicologically relevant alterations up to 1000 mg/kg. Phagocytosed yellow-brown granular pigment was recorded at minimal or slight degree in the interstitium of the testes in two males at 300 mg/kg and in all ten males at 1000 mg/kg. Similar pigment also at minimal or slight degree was noted in the epididymides of one male at 100 mg/kg, four males at 300 mg/kg and all ten males at 1000 mg/kg, and in the subcutis of the skin at minimal degree in six males at 1000 mg/kg. This finding was the histologic correlate of the discolouration noted at necropsy in these organs. Similar pigment also at minimal or slight degree was also recorded in the ovaries of all ten females at 1000 mg/kg and in one female at 300 mg/kg.
As phagocytosis of this material was noted only at minor degrees and was not accompanied by any adverse cellular response, it was not considered toxicologically relevant. It was considered most likely to represent the test material which in the dose formulation was dark red in colour. Sperm granuloma were recorded in the epididymides of two animals in each group at 100 mg/kg (moderate and severe), 300 mg/kg (moderate and severe) and 1000 mg/kg (slight and moderate), and were the histologic correlates to the nodules noted at necropsy in this organ. This is a not uncommon finding in male rats of this age and in this type of study and were therefore considered as probably unrelated to treatment.
Animal number 25 (suspected infertile) had a unilateral severe grade of sperm granuloma which may have influenced the apparent lack of reproductive performance. There were no microscopic findings in any of the other animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all males evaluated.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Remarks:

Parental generation

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Critical effects observed:

no

Analysis of dose preparations

No test substance was detected in the Group 1 formulations.

The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for at least 14 days.

Conclusions:

In conclusion, treatment with FAT 40810/B TE by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental, reproduction or developmental toxicity for treatment up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Executive summary:

FAT 40810/B TE was evaluated for repeated dose toxicity according to OECD test guideline 421 in a GLP certified laboratory. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-49 days).

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and

implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Results: Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Conclusion: Treatment with FAT 40810/B TE by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental, reproduction or developmental toxicity for treatment up to 1000 mg/kg.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute oral toxicity

OECD 407: Oral administration of FAT 40810/A to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no effects upon mortality, clinical signs (daily or weekly), parameters of hematology, mean organ weights, macroscopic or microscopic findings. The mean level of leucocytes (urinalysis) was increased in males treated with 1000 mg/kg/day after four weeks of treatment. It could be not excluded that this finding was test item related. The mean level of sodium was increased in males treated with 200 mg/kg/day, and in males and females at 1000 mg/kg/day when compared with controls after four weeks of treatment. This was considered to be test item related because a dose response relationship could be observed and it was seen in both sexes. Based on the results of this study, 50 mg/kg body weight/day of FAT 40810/A was established as the no-observed-effect-level (NOEL), and 1000 mg/kg body weight/day of FAT 40810/A as the no-observed-adverse-effect-level (NOAEL). These findings were supported by the observations made during the reproductive and developmental toxicity screen study.

 

OECD 421: FAT 40810/B was evaluated for repeated dose toxicity according to OECD test guideline 421 in a GLP-certified laboratory. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-49 days).

Results: Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Conclusion: Treatment with FAT 40810/B by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental, reproduction or developmental toxicity for treatment up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

 

Acute inhalation toxicity

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Orange 140 is available. However, the vapour pressure for the substance was found to be 6.6E-25 to 3.8E-26 which is considered to be negligible. 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 224 g/L, hence in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared by the cilia present in the respiratory tract, thereby further limiting the absorption. A repeated dose toxicity via gavage is available with Reactive Orange 140, where the NOAEL was established at 1000 mg/kg bw/day. Taking above information into consideration, no toxicity is expected on repeated exposure of Reactive Orange 140 via inhalation 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 Reactive Orange 140 is considered to be scientifically not necessary.

 

Acute dermal toxicity

Currently no study to assess the repeated dose dermal toxicity of Reactive Orange 140 is available. However, the molecular weight of the chemical is between 888 to 1008 g/mol, indicating it being too large for dermal absorption. The water solubility of 224 g/L and n-octanol/water partition coefficient (log P) of <-4.5, indicates that the substance is too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance will be low. A repeated dose toxicity via gavage is available with Reactive Orange 140, where the NOAEL was established at 1000 mg/kg bw/day. Taking above information into consideration, no toxicity is expected on repeated exposure of Reactive Orange 140 via dermal route. Similarly, absence of systemic toxicity in skin irritation, sensitization and acute dermal toxicity study, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Further, 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. Taking the above arguments into consideration, low toxicity potential is expected on repeated exposure of Reactive Orange 140 via dermal route 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 dermal route for Reactive Orange 140 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the consistent results from a subacute oral toxicity study and the reproductive screening test, the substance did not show any signs of systemic toxicity considered being adverse and thus, the substance is not classified for Specific Target Organ Toxicity, Repeat Exposure (STOT, RE) according to CLP (Regulation EC No 1272/2008).