trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The NOAEL for fertility was determined to be 1000 mg/kg bw/day (the highest dose tested).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2012 to 24 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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 40826/B TE
Description Orange powder
Batch TZ 5604-BS-BOP 01-12
Purity 53.4 % Main constituent
74.4 % all coloured constituents
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 28 January 2017

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rationale This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

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 317 g (males) or 212 g (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: 31 July - 24 September 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on 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.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

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 19 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 41-55 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 3 and two females of Group 4 were not dosed during littering. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the animals in the study: Approximately 13 weeks

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Middle dose

Dose / conc.:

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

Remarks:

High dose

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 in which FAT 40826/A was dosed to Wistar rats at doses of 50, 200 and 1000 mg/kg. Test item-related findings were generally restricted to minimal to slight and reversible focal spongiosis of the stratified squamous epithelium in the forestomach of all animals treated with 1000 mg/kg. Based on the results of this study, 200 mg/kg of FAT 40826/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg of FAT 40826/A as the noobserved-adverse-effect-level (NOAEL).
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Positive control:

no

Parental animals: Observations and examinations:

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 post-coitum 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.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis for all males of the control and high dose group and animals suspected to be infertile.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnormalities, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

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).

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Clinical signs:

no effects observed

Description (incidence and severity):

At 1000 mg/kg, orange discolouration of the faeces (all animals) and tail (two males) was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Salivation seen after dosing among all animals of the 1000 mg/kg dose group was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included alopecia and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period. As female no. 77 (1000 mg/kg) was pregnant with only one pup, a lower body weight gain was noted during post-coitum. At this single occurrence it was not considered toxicologically significant. The statistically significantly increased body weight gain noted on Day 4 post-coitum for high dose females was not considered toxicologically significant as it was a slight increase and occurred only on one occasion.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Orange brown pigment was present in the stratum corneum of the skin for three males treated at 1000 mg/kg. This finding was considered to be due to the deposition of the (orange) test item onto the skin excreted in the feces. There were two Group 1 (male no. 7 and female no. 47) and two Group 3 animals (male 27 and female 67) of which the females were not pregnant and were therefore selected for histopathological examination of the reproductive organs. Their infertility could not be attributed to treatment. Spermatogenic staging profiles were normal for all males examined. All microscopic findings recorded in animals surviving to the end of the assigned study period were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The slightly lower mean number of corpora lutea and implantation sites (not statistically significant) at 1000 mg/kg were due to one female which had only two corpora lutea and implantation sites. At this single occurrence it was not considered toxicologically significant.

Dose descriptor:

NOAEL

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

Clinical signs:

no effects observed

Description (incidence and severity):

One pup (litter 43) at the control group showed a lean appearance on Day 4 of lactation (which concurred with a lower body weight) and the pup at 100 mg/kg (litter 60) that was found dead at first litter check showed absence of milk in the stomach. The nature and incidence of these signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

One pup (litter 60) at 100 mg/kg was found dead at first litter check and two pups (litter 59 at 100 mg/kg and litter 76 at 1000 mg/kg) were missing on Day 2 of lactation. The pups that were missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

DEVELOPMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. At 1000 mg/kg, one female had only one pup. At this single occurrence it was not considered toxicologically significant.

Dose descriptor:

NOAEL

Generation:

F1

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

Reproductive effects observed:

no

Conclusions:

The parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of FAT 40826/B at least 1000 mg/kg was derived.

Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40826/B TE in rats by oral gavage was carried out according to OECD guideline 421. Based on the results of a 28-day toxicity study, the dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. The observations and examinations evaluated were 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. There was no adverse effects on 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. However, at 1000 mg/kg, orange discolouration of the faeces (all animals) and tail (two males) was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Orange brown pigment was present in the stratum corneum of the skin for three males treated at 1000 mg/kg. This finding was considered to be due to the deposition of the (orange) test item onto the skin excreted in the feces. No parental, reproduction and developmental toxicity was observed 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40826/B in rats by oral gavage was carried out according to OECD guideline 421. Based on the results of a 28-day toxicity study, the dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.The observations and examinations evaluated were 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. There was no adverse effects on 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. However, at 1000 mg/kg, orange discolouration of the faeces (all animals) and tail (two males) was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Orange brown pigment was present in the stratum corneum of the skin for three males treated at 1000 mg/kg. This finding was considered to be due to the deposition of the (orange) test item onto the skin excreted in the feces. No parental, reproduction and developmental toxicity was observed up to 1000 mg/kg.Based on these results, a parental, reproduction and developmental No Observed Adverse EffectLevel (NOAEL) of at least 1000 mg/kg was derived.

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/day (the highest dose tested).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

OECD 421 study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2012 to 24 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

reference to same 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 (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification FAT 40826/B TE
Description Orange powder
Batch TZ 5604-BS-BOP 01-12
Purity 53.4 % Main constituent
74.4 % all coloured constituents
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 28 January 2017

Species:

rat

Strain:

Wistar

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 317 g (males) or 212 g (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: 31 July - 24 September 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on 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 19 days.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Age at mating of the animals in the study: Approximately 13 weeks
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

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 41-55 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 3 and two females of Group 4 were not dosed during littering.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 28 days
Females: 41-55 days

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Middle dose

Dose / conc.:

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

Remarks:

High dose

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 in which FAT 40826/A was dosed to Wistar rats at doses of 50, 200 and 1000 mg/kg. Test item-related findings were generally restricted to minimal to slight and reversible focal spongiosis of the stratified squamous epithelium in the forestomach of all animals treated with 1000 mg/kg. Based on the results of this study, 200 mg/kg of FAT 40826/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg of FAT 40826/A as the no observed-adverse-effect-level (NOAEL).
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Maternal examinations:

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 post-coitum 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.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

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: Epididymites and testes

HISTOPATHOLOGY:
- According to test guidelines

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnormalities, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

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).

Indices:

Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. At 1000 mg/kg, orange discolouration of the faeces (all animals) and tail (two males) was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Salivation seen after dosing among all animals of the 1000 mg/kg dose group was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included alopecia and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period. As one female at 1000 mg/kg was pregnant with only one pup, a lower body weight gain was noted during post-coitum. At this single occurrence it was not considered toxicologically significant. The statistically significantly increased body weight gain noted on Day 4 post-coitum for high dose females was not considered toxicologically significant as it was a slight increase and occurred only on one occasion.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

MACROSCOPIC EXAMINATION
Necropsy did not reveal any toxicologically relevant alterations. Three males at 1000 mg/kg showed orange discolouration of the tail, which was considered due to the staining properties of the test substance and not regarded toxicologically relevant. The incidence of other incidental 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 nodule at the epididymides or uterine adipose tissue, pelvic dilation of the kidney, reddish discolouration of the thymus, foci on the thymus or clitoral glands, and enlarged spleen.

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

MICROSCOPIC EXAMINATION
There were no treatment-related microscopic findings in the reproductive organs. Orange brown pigment was present in the stratum corneum of the skin for three males treated at 1000 mg/kg. This finding was considered to be due to the deposition of the (orange) test item onto the skin excreted in the feces. There were two Group 1 and two Group 3 animals of which the females were not pregnant and were therefore selected for histopathological examination of the reproductive organs. Their infertility could not be attributed to treatment. Spermatogenic staging profiles were normal for all males examined. All microscopic findings recorded in animals surviving to the end of the assigned study period were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The slightly lower mean number of corpora lutea and implantation sites (not statistically significant) at 1000 mg/kg were due to one female which had only two corpora lutea and implantation sites. At this single occurrence it was not considered toxicologically significant.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall study findings

Abnormalities:

no effects observed

Developmental effects observed:

no

DEVELOPMENTAL DATA No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. At 1000 mg/kg, one female had only one pup. At this single occurrence it was not considered toxicologically significant. MORTALITY PUPS One pup at 100 mg/kg was found dead at first litter check and one pup at 100 mg/kg and one pup at 1000 mg/kg were missing on Day 2 of lactation. The pups that were missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. OBSERVATIONS One pup at the control group showed a lean appearance on Day 4 of lactation (which concurred with a lower body weight) and the pup at 100 mg/kg that was found dead at first litter check showed absence of milk in the stomach. The nature and incidence of these signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of FAT 40826/B for the developmental toxicity was at least 1000 mg/kg.

Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 40826/B in rats by oral gavage was carried out according to OECD guideline 421, in which four groups of ten male and ten female Wistar rats received the test substance at 0, 100, 300 and 1000 mg/kg via oral gavage. Apart from investigations into reproductive parameters, the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment-related effects on mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the reproduction/developmental toxicity screening test discussed under 'Reproductive toxicity section', the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment related effects on mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse EffectLevel (NOAEL) of at least 1000 mg/kg was derived.

Justification for classification or non-classification

Based on the absence of adverse effect in the reproduction/developmental toxicity screening test, Reactive Yellow 214 does not need to be classified according to Regulation (EC) No. 1272/2008 (CLP).