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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Due to unexpected toxicity in the high dose group, a new low dose group was started with a time delay.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)
EC Number:
403-720-7
EC Name:
A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)
Cas Number:
117527-94-3
Molecular formula:
C21H21N304.C16H11N304.Cr unspecified.C12-C14 chain unspec.
IUPAC Name:
hexachromium(3+) hexakis(2-methyltridecan-2-aminium) tetrakis(1-[(1E)-2-(4-nitro-2-oxidophenyl)diazen-1-yl]naphthalen-2-olate) tetrakis(1-[(1E)-2-(5-nitro-2-oxidophenyl)diazen-1-yl]naphthalen-2-olate) tetrakis(1-[(1E)-2-[5-(2-methylbutan-2-yl)-3-nitro-2-oxidophenyl]diazen-1-yl]naphthalen-2-olate)
Details on test material:
- CAS no 117527-94-3
- Substance type: Black powder
- Physical state: solid
- Analytical purity: 100 % UVCB
- Purity testing date: March - May 2011
- Lot/batch No.:0004856991
- Stability under test conditions: stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: (P): See table 1
- Fasting period before study: no
- Housing: Males were housed in fives, in grid bottomed cages until pairing and after pairing. Females were housed in fives in grid bottomed cages until pairing. Mated females were housed individually in solid bottomed cages throughout gestation and with litter during lactation.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 °C to 21 °C (target range 21 ° C ± 2 °C),
- Humidity (%): 45 % to 67 % (target range 55 % ± 15 %)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
1 % aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Separate formulations were prepared for each dose level. The
appropriate weight of test item was added to a pestle and mortar and the vehicle was
gradually added to produce a smooth paste. Additional vehicle was then added under
continuous gentle mixing. Suspension was then transferred to a container and the remaining
vehicle added. This was then stirred until a visibly homogeneous suspension was obtained.
Formulations were prepared daily and were used within 4 hours of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Addition to 1% methylcellulose allows forming forming a stable suspension of the poorly water soluble substance.
- Concentration in vehicle:
Details on mating procedure:
From Day 15 of dosing, each female was paired with a male from the same dose group for up to 14 days. Vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smear. The smears were examined under light microscopy and the stage of the oestrous cycle was recorded. On confirmation of mating, the males were returned to the group cages and the females were housed individually.
The day on which sperm was detected is referred to as Day 0 of gestation and the following day as Day 1 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from formulations once a week, but only those detailed below were submitted for analysis. All samples from formulation preparations not analysed were designated as frozen reserves and stored in the freezer at approximately -18 °C.On Day 1 of dosing, formulations for Groups 1 to 6 were analysed to assess their achieved concentration and homogeneity. As Group 6 formulations were found to be outside the
specified range for homogeneity, additional samples from Group 6 were taken on Day 2 of dosing, analysed and found to be accurate and homogenous. Once homogeneity had been established, samples from formulations prepared towards the end of the dosing period were taken from Groups 1 to 3 and Groups 5 and 6 and analysed for achieved concentration only. As Group 2 and 3 formulations were found to be below the
specified range for concentration, additional samples for these groups were taken from subsequent formulations, analysed for achieved concentration and found to be accurate. One set of triplicate samples was analysed by the department of analytical chemistry and the
other sets were designated as frozen reserves and stored in the freezer at approximately -18 °C.
Duration of treatment / exposure:
With the exception of Group 4,
main study males were dosed daily for 14 days prior to pairing, during pairing and until the
day prior to necropsy. With the exception of Group 4, main study females were dosed daily
for 14 days prior to pairing, during pairing and until Day 3 of lactation. Control animals
received the vehicle only, according to the same regimen. Group 4 animals given
1000 mg/kg bw/day were dosed for 6 days prior to termination of the group on Day 7 of
dosing.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 12.5, 500 mg/kg bw
Basis:
actual ingested
additional dose levels after mortality/morbidity at 1000 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of existing OECD 407 study
- Rationale for animal assignment (if not random): Allocation to main study groups was performed using a stratified randomisation procedure
based on body weight ranges recorded during the acclimatisation period.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All main study animals were examined daily from the start of treatment for clinical signs of toxicity or changes in behaviour and appearance

BODY WEIGHT: Yes
- Time schedule for examinations: Main study male body weights were recorded weekly throughout the study and at
termination. Main study female body weights were recorded weekly during their pre-pairing
and pairing dosing periods, and on Days 0, 7, 14 and 20 of gestation, and then on Days 0 (if required), 1 and 4 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE:
The amount of food consumed by the main study animals in each cage was recorded at weekly intervals for males and females during their pre-pairing dosing period. Food consumption of the females was also recorded over Days 0 to 7, 7 to 14 and 14 to 21 of gestation and Days 0 to 4 of lactation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER: Females were observed from Day 21 of gestation until all pregnant females had littered or at the start of the working day when the last pregnant female was on Day 25 of gestation or 26 days after mating, whichever was the earliest. Following completion of parturition, pups were designated as Day 0 of age, with the next day classified as Day 1 of age. No dead offspring were removed from the litter until parturition had been completed.
Mated females that failed to litter were subject to necropsy (see Section 3.9.1) on Day 26 after mating.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:no / not applicable


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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals after lactation day 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examination

HISTOPATHOLOGY / ORGAN WEIGHTS
Reproductive organs were prepared for microscopic examination and weighed, respectively.

CLINICAL PATHOLOGY
All surviving males and females in Groups 1 to 6 were sampled during Week 2 (at the end of the pre-pairing period). In addition, the remaining Group 4 animals were sampled on Day 7. Blood samples were taken from the sublingual vein under isoflurane anaesthesia for Groups
1, 2, 3, 5 and 6 and from the tail vein for Group 4. The following parameters were measured:
Haemoglobin concentration (Hb)
Red blood cell count (RBC)
Packed cell volume (PCV)
Mean cell volume (MCV)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Platelet count (Plate)
Total leucocyte count (WBC) and leucocyte differential count :
Neutrophil (Neut),
Lymphocytes (Lymph),
Monocytes (Mono),
Eosinophils (Eosin),
Basophils (Baso),
Large unstained cells (LUC)
Cell Morphology
Reticulocytes (%RBC)

For Groups 1, 2 and 3 approximately 0.5 mL blood samples were taken into citrate tubes (anticoagulant) but due to the dark colouration of the samples no parameters were measured.

Urea
Urease
Creatinine
Glucose
Alkaline phosphatase
Alanine aminotransferase
Total Protein
Aspartate aminotransferase (AST)
Albumin
Globulin
Albumin/Globulin ratio
Total Bilirubin
Cholesterol
Calcium
Sodium
Potassium

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

No effects on mating performance
No effects on gestation index
No effects on gestation length
no effect on testes or epididymis weights and histopathology
No effects on maternal corpus lutea and implantation scars

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality at 500 mg/kg bw and above; effects on body weight at 200 mg/kg bw

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
whole body discolored (black)
Histopathological findings:
not examined

Details on results (F1)

At 200 mg/kg/day there was a significant difference in the mean number of pups born when compared with the mean number of implantation scars per female, suggesting there was a high incidence of pups missing (presumed cannibalised, possibly resorbed) at birth in these litters.
Four females lost their litters by day 4
lower body weights on Day 1 of lactation (m,f) and day 4 (f)
lower body weight gain over Days 1 to 4 of lactation, being statistically significant (p<0.01) for females
Pups show strong blackish discoloration
Whole body stained black upon necropsy


At 50 mg/kg bw all pups were born alive.
Pups show blackish discoloration
Whole body stained black upon necropsy
One female lost 90% of litter (considered incidental)
One female including litter needed to be sacrificed because of dosing error.

NOAEL = 50 mg/kg bw

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced number of pups born at 200 mg/kg bw and reduced pup weights

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weight of males

Day 1 Day 7 Day 8 Day 15 Day 22  Day 29  Day 36 Day 43 Day 47 Day 50 Day 55
control Mean 295.3 0 314.6 327.2 340.3 351.9 363.0 375.2
S.D. 6.5 10.7 12.1 11.9 14.9 20.4 20.7
N 10 10 10 10 10 10 10 0 0
50 mg/kg bw Mean 297.5 0 313.2 328.7 341.8 356.6 367.9 376.7 379.5
S.D. 10.5 11.6 12.6 12.9 14.2 17.4 15.6 15.7
N 10 10 10 10 10 10 10 10 0 0
200 mg/kg bw Mean 301.9 0 315.9 329.5 341.8 351.3 362.2 373.3 372.4
S.D. 18.4 18.3 19.1 23.6 25.3 26.3 28.8 25.8
N 10 10 10 10 10 10 10 10 0 0
1000 mg/kg bw Mean 307.9 302.1
S.D. 13.4 14.3
N 10 9 0 0 0 0 0 0 0 0 0
control (to 12 mg/kg bw) Mean 323.4 0 338.2 350.1 356.7 371.1 383.3 397.4 406.5 409.7
S.D. 12.9 14.3 14.7 20.0 21.5 23.6 23.6 25.6 26.6
N 10 10 10 10 10 10 10 0 10 10
12 mg/kg bw Mean 326.8 0 344.5 360.5 365.6 382.7 393.1 402.7 413.2 417.9
S.D. 14.2 17.0 21.0 22.4 28.9 30.4 30.2 31.1 31.9
N 10 10 10 10 10 10 10 0 10 10

# - statistically analysed: Group 1 vs 2-3 (Williams’/Shirley’s Test)

# - statistically analysed: Group 5 vs 6 (Student’s T-test/Wilcoxon’s Test)

Group 4 terminated on Day 7 of study; excluded from statistical analysis

Table 2: Body weight for females pre-mating

Day 1 Day 7 Day 8 Day 15
control Mean 192.4 0 204.2 212.7
S.D. 8.2 7.4 8.6
N 10 10 10
50 mg/kg bw Mean 194.5 0 203.1 215.1
S.D. 5.3 8.5 9.1
N 10 10 10
200 mg/kg bw Mean 193.9 0 199.6 208.8
S.D. 6.2 7.7 9.4
N 10 10 10
1000 mg/kg bw Mean 197.5 196.8
S.D. 10.1 12.8
N 10 4 0 0
control (to 12 mg/kg bw) Mean 204.2 0 212.7 216.0
S.D. 13.9 11.4 10.2
N 10 10 10
12 mg/kg bw Mean 211.6 0 216.3 222.7
S.D. 8.4 12.1 11.6
N 10 10 10

# - statistically analysed: Group 1 vs 2-3 (Williams’/Shirley’s Test)

# - statistically analysed: Group 5 vs 6 (Student’s T-test/Wilcoxon’s Test)

Group 4 terminated on Day 7 of study; excluded from statistical analysis

Applicant's summary and conclusion

Conclusions:
The substance reduces the number of pups born alive at the high dose group at doses about twofold lower doses that cause morbidity/mortality in parental animals. Adverse effects on fertility were not oserved.