2,7-NAPHTHALENEDISULFONIC ACID 5-[[4-CHLORO-6-(ETHYLPHENYLAMINO)-1,3,5-TRIAZIN-2-YL]AMINO]-3-[[5-[(2,3-DIBROMO-1-OXOPROPYL)AMINO]-2-SULFOPHENYL]AZO]-4-HYDROXY-,SODIUM SALT

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 October - 10 December 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.

Cross-reference

Reason / purpose for cross-reference:

reference to same 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 40508/C TE
- Substance type: Red-brown powder
- Physical state: Powder
- Content: 100% (w/w)
- Lot/batch No.: DER 7520 / BOP 01-12 (Lot: 1110669)
- Expiration date of the lot/batch: 20 February 2017
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store in well-sealed container
- Volatile No
- pH: 4.98 at concentration of 2%
- Solubility in water: Approximately 86.3 g/L

Test animals

Species:

rat

Strain:

other: Wistar (Han)

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 319 gr (males) or 206 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: 22 October - 10 December 2012

Administration / exposure

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: Day 1 to 9 of study: 10 mL/kg body weight. From Day 10 of study onwards: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight. One male of Group 4 expelled formulation during the dosing procedure on Day 9 of study and showed laboured respiration; it was decided not to dose the remaining 1 mL of formulation on this day for this animal. One female of Group 4 expelled a small amount of formulation during the dosing procedure on Day 10 of study and showed laboured respiration.

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 13 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. 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 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, two of Group two, three of Group 3 and one female of Group 4 were not dosed during littering. Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 28 days
Females: 42-49 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

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 albino Sprague-Dawley derived rats (CIBAGEIGY Limited Test no. 945093) in which 50, 200 and 1000 mg/kg were tested. Under the conditions of this test, treatment with FAT 40508/A did not provoke overt signs of toxicity. A reversible nephrotropic effect occurred, which was revealed by water consumption data, laboratory investigations, organ weight analysis and microscopical finding. A "No Observable Effect Level" (NOEL) cannot be defined in this study. The 200 mg/kg bodyweight dose level is regarded to be a "No Observable Adverse Effect Level" (NOAEL), since the effects noted up to this dose level were not accompanied by any overt signs of toxicity or corroborative microscopical finding. Moreover, all effects were reversible within the 2-weeks recovery period at all dose levels. Based on these findings, kidney weight and histopathological examination was added to this reproduction/developmental toxicity screening study.
- 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.

Examinations

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 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.

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
- Kidneys, epididymides and testes

HISTOPATHOLOGY:
- According to test guidelines and kidneys

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 abnomalies, 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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 200 mg/kg showed a total litter loss, and was therefore terminated early on Day 1 of lactation.

CLINICAL SIGNS
No clinical signs of toxicity related to test substance treatment were noted during the observation period. One male at 1000 mg/kg showed hunched posture on Days 8 and 26-28 of treatment. As this was not noted for the other animals at the high dose group, it was considered a chance finding and not toxicologically significant. Salivation seen after dosing for all animals at 1000 mg/kg 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). Red staining of the tail and red faeces noted for all animals treated at 1000 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. Incidental findings that were noted included alopecia, rales, laboured respiration and scabs. 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
Reduced body weights and body weight gain were noted for males treated at 1000 mg/kg. This was statistically significant for body weights on Days 22 and 29 of treatment and for body weight gain on Days 8, 22 and 29 of treatment. One male of Group 4 was most affected; showing only 5% body weight gain over the complete study period. Body weights and body weight gain of treated females remained in the same range as controls over the treatment period. The statistically significantly increased body weight gain noted for high dose females on Day 7 postcoitum was not considered toxicologically relevant as the increase was very slight and occurred only on one occasion.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. The statistically significantly increased relative food consumption noted for females at 1000 mg/kg on two occasions during post-coitum was not considered toxicologically relevant as these increases were slight and inconsistent over time.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, reddish or dark red discolouration of the kidneys was noted for all males and seven females. One of these females showed additional abnormalities of the kidneys, consisting of several nodules, pale pelvis, and enlarged right kidney, and related other findings (dilation of the right ureter, enlarged urinary bladder and enlarged iliac lymph nodes). Several animals at 1000 mg/kg showed red discolouration of the tail subcutis, testes, and epididymides, and three females at 200 mg/kg showed reddish discolouration of the kidneys. These discolourations were considered due to the staining properties of the test substance and not regarded toxicologically relevant. The uterus of one female of the control group contained one fetus. Delivery was probably not triggered for this female due to the small litter size. One female at 200 mg/kg that showed a total litter loss on Day 1 of lactation showed a distended right uterus horn containing reddish fluid. These findings were not considered treatment related and to have occurred by chance. 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, foci at the preputial glands or thymus, nodule at the epididymides, tail of the epididymides reduced in size, alopecia, uterus containing fluid, and a skin wound.

ORGAN WEIGHTS
Kidneys weights (absolute and relative) were increased for animals at 1000 mg/kg. This reached statistical significance for the relative kidneys weights. In addition, terminal body weights for males were decreased at the high dose level. It should be noted that one female of Group 4 showed a much higher kidneys weight which correlated to the macroscopic findings for the kidneys of this animal. Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.

MICROSCOPIC EXAMINATION
Treatment-related microscopic findings were present in kidneys and consisted of:
- Tubular eosinophilic granules (pigment in vacuoles) in all males (slight to moderate) and all females (moderate to marked) treated at 1000 mg/kg. In one of the females this accumulation of granular material was associated with moderate to marked changes that are indicative for a significant ongoing degenerative and inflammatory process. (e.g. necrosis, inflammation/cellular debris, fibrosis).
- An increased incidence and severity in tubular hyaline droplets (minimal to slight hyaline droplets is considered to be within back ground severity) in all males treated at 1000 mg/kg (2 minimal, 7 slight, 1 moderate) compared to 1/10 males of the control group (minimal), 4/10 males at 50 mg/kg (3 minimal, 1 slight) and 4/10 males at 200 mg/kg (all minimal).
Cortical hyaline droplets representing alpha2-µglobulins are male rodent specific and considered to be not adverse when this finding is not accompanied with any indications of tubular damage. Therefore, in the present study, presence of hyaline droplets was only considered adverse at 1000 mg/kg. All remaining microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain. There were no treatment-related microscopic findings in both male and female reproductive organs. No cause of infertility for the selected pairs could be established (spermatogenic staging profiles were normal in the male testes and characteristics of normal cycle in the female reproductive organs or characteristics of a normal preceding pregnancy period).

REPRODUCTIVE DATA
At 1000 mg/kg, fertility and conception indices were decreased when compared to the control group (70 and 78%, respectively, compared to 100% for both). This was due to one female that did not mate during the 14-day mating period and two females that were not pregnant. Mating index, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 200 - < 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

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

Results (fetuses)

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.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

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.   MORTALITY PUPS Two pups of the control group, two pups at 50 mg/kg and four pups at 1000 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.   OBSERVATIONS All pups of one litter of the control group showed necrosis of the tail apex on Day 5 of lactation (last litter check) and during macroscopic examination. Most of the pups also showed this on Day 4 of lactation; two pups showed a blue spot on the tail for that day. As it concerned a control litter it was not treatment related and considered a chance finding. Necrosis of the tail apex was also observed for one high dose pup. Other incidental findings consisted of absence of milk in the stomach, blue spot on the back, and moderate autolysis. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with FAT 40508/C TE by oral gavage in male and female Wistar Han rats at dose levels of 50, 200 and 1000 mg/kg revealed parental toxicity at 1000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 200 mg/kg was derived. The reproduction and developmental NOAEL was determined to be at least 1000 mg/kg.

Executive summary:

In the reproduction/developmental toxicity screening test (OECD 421, GLP), four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 200 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 42-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. 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). Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Parental toxicity was observed at 1000 mg/kg. This consisted of reduced body weights and body weight gain for males and kidneys abnormalities for both sexes. In the kidneys, treatment-related changes consisted of tubular eosinophilic granules (pigment in vacuoles) in all males and females treated at 1000 mg/kg (macroscopically seen as red discolouration of the kidneys), in one of the females this was accompanied by tubular necrosis, granulocytic inflammation with necrosis/debris, papillary necrosis and interstitial fibrosis. Increased kidneys weights were noted for both sexes at 1000 mg/kg. No reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). Therefore, a parental No Observed Adverse Effect Level (NOAEL) of 200 mg/kg was derived. The reproduction and developmental NOAEL was determined to be at least 1000 mg/kg.