Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000
GLP compliance:
yes
Remarks:
NOTOX B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene
EC Number:
408-210-8
EC Name:
Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene
Cas Number:
124605-82-9
Molecular formula:
C33 H26 N8 O14 S4 .x Li .x Na
IUPAC Name:
dilithium(1+) disodium 5-amino-3-{2-[4-(2-{4-[2-(8-amino-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazen-1-yl]-2-methylphenyl}diazen-1-yl)phenyl]diazen-1-yl}-4-hydroxynaphthalene-2,7-disulfonate
Details on test material:
- Analytical purity: 78%
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) Approximately 12 weeks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
- 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 prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for purity of the test substance (78%).

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX.
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (08 September 2010), according to a validated method (NOTOX Project 495053; BASF Project 05Y0361/10X076). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 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 6 hours.
Duration of treatment / exposure:
Males were exposed for 29 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. Females 46 (Group 1), 56, 57 (Group 2), 61, 66, 70 (Group 3), 76 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 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 the results of a 10-day dose range finding study (NOTOX Project 495052; BASF Project 10R0361/10X075) in which 100, 300, 500 and 1000 mg/kg bw/day were tested.

Examinations

Parental animals: Observations and examinations:
MORTALITY / VIABILITY: Yes
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

CLINICAL SIGNS: Yes
Daily, detailed clinical observations were conducted in all animals immediately after dosing. The time of onset, grade and duration of any observed sign was recorded. All symptoms were recorded and graded according to fixed scales.

BODY WEIGHT: Yes
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 AND COMPOUND INTAKE (if feeding study): Yes
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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight
Litter observations:
Mortality / Viability:
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 for 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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration.
- Maternal animals: Females which delivered were sacrificed on Lactation Days 5-7; females with total litter loss (no. 74) were sacrificed within 24 hours of litter loss.

GROSS NECROPSY
- All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes, Epididymides, Uterus, Ovaries, Vagina, Preputial gland, all gross lesions.
Of the males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The preserved organs, tissues and reproductive organs* of female no. 78 (euthanized in extremis on Day 22 post-coitum, no live offspring; implantation sites only) and the cohabitated male no. 38.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate
gland, seminal vesicles, testes, uterus, and vagina.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. 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.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto- 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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group. 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.
Reproductive indices:
Mating Index, Fertility Index, Conception Index, Gestation Index, Duration of Gestation
Offspring viability indices:
Percentage live males/females, percentage post natal Loss, viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
One female at 300 mg/kg bw/day (no. 78) was killed in extremis on Day 22 post-coitum due to delivery difficulties. Additionally, one female at 300 mg/kg bw/day (no. 74) was necropsied on Day 2 of lactation due to total litter loss. Both deaths were considered to have occurred by chance and not to
be related to treatment. No other unscheduled mortalities occurred during the study period.

CLINICAL SIGNS (PARENTAL ANIMALS)
No toxicologically relevant clinical signs were noted during the observation period.
One female (animal no. 78) treated at 300 mg/kg bw/day, showed piloerection on Day 22 post-coitum and was killed on that day due to delivery difficulties.
Salivation seen after dosing for all animals treated at 300 mg/kg bw/day 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.
Bluish staining of the tail, blue feces, blue urine and/or general blue discoloration of the skin was noted for animals treated at 100 and 300 mg/kg bw/day. This finding was attributed to the dark blue/black staining property of the test substance. As such it was not considered a change of toxicological significance.
Incidental findings that were noted included rales, alopecia and scabs at the right ear. 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 WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
The statistically significantly increased body weight noted for males at 30 mg/kg bw/day on Day 15 of the mating period was considered unrelated to treatment as no dose response relationship was apparent.

FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
The statistically significantly increased food consumption (absolute and relative to body weight) noted for females at 300 mg/kg bw/day during gestation was not considered toxicologically relevant as changes were relatively small and a decrease rather than increase would have been expected in case of toxicity.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males. The remainder of the microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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.
Most animals mated within the first four days of pairing. For three females of the control group and one female of the low dose group it took 13-14 days to mate. As no dose response relationship was noted, this was considered to have occurred by chance.
Female number 53 of the low dose group showed a high value for corpora lutea (CL) with a normal number of implantation sites. This high value was considered to be caused by counting of CL of pregnancy together with CL derived from post partum ovulation, and not to be treatment related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios (testes and epididymides) up to 300 mg/kg bw/day.
The statistically significantly increased terminal body weight and concurrent decreased relative epididymides weight noted for males at 30 mg/kg bw/day, were considered unrelated to treatment as no dose response relationship was apparent, the values were within normal limits and no microscopic correlate was observed.
The testes of male number 34 (treated at 300 mg/kg bw/day) were reduced in size macroscopically; this was confirmed by a lower testes weight and minimal unilateral seminiferoustubular atrophy at microscopic examination. At this single occurrence, it was not regarded toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations that were considered to have arisen as a result of treatment.
Female number 74 (300 mg/kg bw/day) that was euthanized on Day 2 of lactation due to total litter loss, showed black-brown discoloration of the kidneys. The microscopic correlate consisted of bilateral moderate progressive nephropathy. Whilst this finding is unusual in a rat of this age it was regarded as not treatment-related as no other animals at this dose level showed any kidney abnormality.
Female number 78 (300 mg/kg bw/day) was killed in extremis due to delivery difficulties showed slimy contents of the uterus. The microscopic correlate consisted of fetal/maternal membranes in the lumen of the uterus.
There were macroscopic findings in two females treated at 300 mg/kg bw/day which were considered to be due to complications with the gavage procedure. Animal no. 71 showed isolated bluish foci in the lungs and animal no. 77 showed bluish discoloration of the lungs.
Bluish contents and/or discoloration were recorded in/at the GI-tract, urinary bladder, skin of the tail, iliac and mandibular lymph nodes, subcutis, the testes and the uterus of most rats treated at 100 and/or 300 mg/kg bw/day. The test item was a dark blue/black coloured compound which was assumed to have led to the observed discolorations. These findings were not associated with any pathological alteration.
Incidental findings included pelvic dilation of the kidneys, greenish or yellowish soft nodule at the tail of the epididymides, testes reduced in size, red-brown or tan foci at the clitoral glands, and alopecia of several body parts. The incidence of these findings 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No toxicologically relevant treatment related microscopic findings were noted up to 300 mg/kg bw/day.
Microscopic findings of note were recorded in the lungs of two Group 4 females (nos. 71 and 77) and the kidneys of a Group 4 female (no. 74), but were not regarded treatment related. No cause of moribundity or parturition problems could be established from the sections examined of Group 4 female no.78.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related parental toxicity was observed at any dose level.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
One, four, three or seven pups of the control, low, mid or high dose group, respectively, were found dead or missing during the first days of lactation. Pups missing were most likely cannibalized. 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.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of small size, blue or red spot on the head, swelling of the left eye and pale appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
One pup of the control group (litter 46, pup 12) showed abnormal posture of the hindlegs. As this was a pup of the control group, it was not considered treatment related.
Blue discoloration of several parts of the body was noted for several pups of the high dose group. This finding was attributed to the dark blue/black staining property of the test substance. As such it was not considered a change of toxicological significance.
Clinical signs noted for pups of litter 74 (total litter loss on Day 2 of lactation) consisted of absence of milk in the stomach and cold, lean and pale appearance and blue discoloration.

BODY WEIGHT (OFFSPRING)
Body weights of pups were comparable among groups, including controls, and considered to be unaffected by treatment.
Pups of litter 74 (total litter loss on Day 2 of lactation) showed slightly lower body weights compared to controls.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings included small size, absence of milk in the stomach and abnormal posture of the hindlegs. 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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related reproduction toxicity was observed at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg bw/day revealed no parental, reproduction and developmental toxicity up to 300 mg/kg bw/day. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 300 mg/kg bw/day was derived.
Executive summary:

In a GLP-compliant reproduction/developmental toxicity screening test according to OECD guideline 421 the test substance was administered by daily oral gavage to four groups of 10 male and female Wistar Han rats at dose levels of 0, 30, 100 and 300 mg/kg body weight/day. Rats of the control group received the vehicle water alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during gestation, and at least 4 days of lactation (for 41-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. The following parameters were evaluated: mortality / viability, clinical signs, body weights, food consumption, reproduction/developmental parameters, observations pups, macroscopy, limited organ weights and histopathology. No treatment related parental toxicity was observed at any dose level. No treatment-related changes were noted in any of the parental parameters investigated in this study. Based on macroscopic and microscopic findings there were two females treated at 300 mg/kg body weight/day which showed signs that were considered to be related to the gavage procedure. These females did not show any clinical signs or effects of body weight and food consumption. As such it can be concluded that these incidents did not affect the study integrity. These findings were considered to be unrelated to the test substance. No treatment related reproduction toxicity was observed at any dose level. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites). In conclusion, treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg bw/day revealed no parental and reproduction toxicity up to 300 mg/kg body weight/day. Based on these results, a parental and reproduction No Observed Adverse Effect Level (NOAEL) of at least 300 mg/kg boy weight/day was derived.