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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40810/B TE
- Substance type: Black powder
- Physical state: Powder
- Content: 100% (w/w)
- Lot/batch No.: BOP 02-12 (Lot: MHC-1880000)
- Expiration date of the lot/batch: 14 September 2017
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required: No
- Volatile No
- pH: 4.98 at concentration of 2%

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 (Control group)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2 (Low dose group)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 (Mid dose group)

Doses / concentrations 4

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.

Examinations

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).

Results and discussion

Results of examinations

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

Effect levels

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.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.