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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2016 to 22 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)
Cas Number:
75701-36-9
IUPAC Name:
3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)
Test material form:
solid: granular
Specific details on test material used for the study:
grounded to powder before formulation

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: Males: 71 to 78 days;Females: 85 to 92 days
- Weight at study initiation: Males: 338 to 403 g; Females: 248 to 306 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid and a solid bottom (during pairing grid bottomed polypropylene cages)
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter
- Enrichment: Aspen chew block and Plastic shelter (except during pairing and lactation)
- Diet: SDS VRF1 Certified pelleted diet ad libitum
- Water: potable public water ad libitum
- Acclimation period: males 6 days; females 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC:
- Humidity: 40-70 %
- Air changes (per hr): no data: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.

VEHICLE:1% CMC
- Concentration in vehicle: 0, 10, 33 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear as proof of successful mating day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed, under yellow light, to assess the stability and homogeneity of the test item in the liquid matrix.
Formulations were demonstrated to be homogeneous and stable for one day when stored at ambient temperature (15 to 25°C) and for 15 days when stored refrigerated (2 to 8°C).
Samples of each formulation prepared for administration in Week 1 and 4 of treatment and on Day 12 of lactation (females only) were analyzed for achieved concentration of the test item.

Method:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separatio n module and 2487 dual wavelength detector
Column: Phenomenex Synergy Hydro RP, 80Å, 4 µm, 4.6 × 250 mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile Phase: ACN/50 mM Ammonium Acetate in water 7/93 v/v
Flow rate: 1.0 mL/min
Detector wavelength: UV, 336 nm
Injectio n volume: 10 µL
Run time: 9 minutes
Approximate retention time: Peak 1 – 2.7 minutes; Peak 2 – 8.5 minutes

Results methd validation:
Calibration linearity (range 5-25 ug/mL): r > 0.999
Specificity: absence of peak for substance in control sample
Precisions calibration (CV 0.17% 25 ug/mL, 0.83% 5 ug/mL)
Accuracy: procedural recovery value of 98.1% (CV=0.72%, n=5) was obtained for 1 mg/mL and 101.8% (CV=3.84%, n=5) was obtained for 200 mg/mL.
Stability (21 days 2-8 ºC): standard 108-121% of intial; extraction solution 113,8% at 1 mg/L, 100% at 200 mg/L
LOQ: 1.85 µg/mL and 6.18 µg/mL (3 and 10 times baseline noise)

Preparation analyses:
Homogeneity CV 0.36-0.65% at 1 mg/L; 0.35-0.64% at 200 mg/L
Stability over 4 days: 103% of initial at 1 mg/L; 104% of initial at 200 mg/L
Procedural recovery: 98.2-106.3% at 1 and 200 mg/L: during test runs 92.8-104.4% (with the exception of the procedural control in week 1 for samples at 33 mg/L (78.3%)
Accuracy: 87.4-101% of nominal for all concentrations in week 1, week 4 (males) and day 12 of lacatation (females)
Duration of treatment / exposure:
Males Two weeks pre-pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 2-week dose range finding study
In that study (animals dosed at 0, 250, 500 or 1000 mg/kg/day) there were no signs seen at the routine examination that were considered to be related to treatment and there were no toxicologically significant signs associated with dose administration. No animals died during the study. Body weight gain, food and water intake were unaffected by treatment. Kidney weights were slightly high in females given 500 or 1000 mg/kg/day, and spleen weights were increased in females given 1000 mg/kg/day. Macroscopic findings in Direct Blue 279-treated males and females included abnormally dark coloration of the kidneys and stomach glandular mucosa, abnormally dark content of the stomach, jejunum, colon, cecum and rectum, and blue coloration of the skin; all of which was considered consistent with the color of the test item.
It was concluded that, in the absence of any adverse test item related effects, a dose level of 1000 mg/kg/day (the limit dose for the OECD 422 test guideline) was suitable for use as the high dose level in this study.
Positive control:
NA

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-dose, post-dose and at the end of the day
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in a standard arena before treatment commenced and during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day prior to necropsy.
On the day of necrospy.
F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day prior to necropsy.
On the day of necropsy.

FOOD CONSUMPTION : Yes
- Time schedule for examinations:
Weekly, from the day that treatment commenced until animals paired for mating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: 5/sex/group
- Parameters checked: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count:, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: 5/sex/group
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males in week 5, females at day 7-9 of lactation
- Dose groups that were examined: 5/sex/group
- Battery of functions tested: sensory activity (includes: approach response, pinna reflex, auditory startle reflex, tail pinch)/ grip strength (fore- and hindlimb) / motor activity(beam crossing over 6 min intervals for 1 hour)

IMMUNOLOGY: Yes
- Time schedule for examinations: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all adult males
- Dose groups that were examined: all
- Parameters checked: serum samples of the adult males for thyroxine (T4) levels (additional taken samples from all adult females were not further examined)

OTHER
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Pre-coital interval
Percentage mating
Conception rate (%)



Oestrous cyclicity (parental animals):
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
-For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
-After pairing until mating.
-For four days before scheduled termination.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, detailed qualitative examination of the testes, taking into account the tubular stages of the spermatogenic cycle (assessment for treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen)
Litter observations:
Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring
Blood sampling
Day 4 of age F1 offspring, two females per litter (where possible, ensuring that the number of female offspring did not fall below three).
- one for T4 (serum)#
- one for TSH (plasma)
# priority was given to serum sample
Day 13 of age F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority was given to serum sample


Serum samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation at 2000g for ten minutes at 4°C.
All evaluated serum was transferred to appropriately labelled polypropylene "cryo" tubes using micropipettes and kept deep frozen (-60 to -90ºC). Only serum samples of the Day 13 offspring were examined for thyroxine (T4) levels. These examinations were undertaken and no effect of treatment was evident on the circulating levels of this hormone. It was therefore not necessary to analyze any further samples for either T4 or TSH (thyroid stimulating hormone).
Postmortem examinations (parental animals):
ORGAN WEIGHTS: Yes (see tables)

GROSS PATHOLOGY: Yes (see tables)

HISTOPATHOLOGY: Yes (see tables)
Premature deaths All F0 animals from all groups.
Scheduled kill F0 animals in Groups 1 and 4:
All F0 animals. Abnormalities only.

Postmortem examinations (offspring):
Gross pathology:
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Particular attention was paid to the external genitalia; examined for signs of altered development.
F1 offspring selected thyroid hormone analysis on Day 4 of age: Externally normal offspring were discarded without examination; Externally abnormal offspring were examined, and retained pending possible future examination.
F1 offspring on Day 13 of age; All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, clinical pathology, organ weight data, litter size and survival indices.
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level.
For all other comparisons the F1 approximate test was applied (Williams 1971, 1972). If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied.
For grip strength, motor activity, clinical pathology, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control.
Sex ratio was analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test.
For gestation length, an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level. All statistical analyses were carried out separately for males and females. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
Fertility index (%)
Gestation length index (%)

Offspring viability indices:
Post-implantation survival index (%)
Live birth index (%)
Viability index (%)
Lactation index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no signs seen in association with the dosing procedure.
males: dark eyes, tail and pinnae, predominantly at 1000 mg/kg/day.
females: dark eyes (and some females completely brown discoloured) during gestation and lactation
at 1000 mg/kg bw, on day 14 of lactation hunched posture and piloerection at 1000 mg/kg bw
Mortality:
mortality observed, non-treatment-related
Description (incidence):
2 control females: on day 4 and 13 of lactation (undetermined)
1 female at 100 mg/kg bw: on day 13 of treatment (abnormal behaviour)
1 male at 1000 mg/k bw:week 2 (dosing error)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
males: no treatment related effects
females: no treatment related effects (bodyweight and bodyweight gains for females prior to pairing
were +50, +42 and +67% of controls for females receiving 100, 300 or 1000 mg/kg/day, respectively).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
males: increased at all dose groups (no DR)
females: increased at all dose groups during pre-mating (no DR); no treatment related effects
thereafter
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
males: increased at all dose groups (no DR)
females: increased at all dose groups during pre-mating (no DR); no treatment related effects thereafter
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
males: at 1000 mg/kg bw: decrease in ALAT activity (X 0.76 of control) and increase in triglyceride concentrations (X 1.64 of control).
females: all groups A/G ratio sign decreased when compared with the controls (X 0.87, X 0.90 and X 0.85 of control for females receiving 100, 330 or 1000 mg/kg/day, respectively), but only in the high dose females did this associate with a reduction in plasma albumin concentration ((X 0.88 of control).

1000 mg/kg bw: ASAT activity was sign increased (X 2.05 of control --> 2 females); Plasma creatinine, urea, triglyceride and phosphorus concentrations (X 1.5, X 1.4, X 1.3 and X 1.3 of control, respectively) --> attributed to 1 female
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations and grip strength: no treatment related effects
Motor activity: in females receiving 1000 mg/kg/day and to a lesser extent for females receiving 100 or 330 mg/kg/day, was low when compared with the controls. Statistical significances were attained at several of the 6-minute intervals and total scores for both high and low beams for females receiving 1000 mg/kg/day and there were also a few sporadic statistical significances in females receiving 100 or 330 mg/kg/day.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Increased brownish pigment was seen in the cortical tubules of 6/10 females treated at 1000 mg/kg/day
Schmorls positive staining was present at a low level in the cortical tubules of controls, with an increase in positive staining present in the treated females. Additionally in treated females, the Schmorls positive staining featured more irregularly-shaped and often smaller granules, of a blue-dark colour, compared with the positive staining in Controls, which featured more rounded droplets of a blue-green colour. Therefore it is considered that test item pigment was additionally present with lipofuscin in the cortical tubules of the treated animals. The presence of pigment in the cortical tubules was not related to the presence of mineralisation

incidental mineralisation was seen in kidneys, stomach and heart of several control and treated females. For the kidneys the severity was increased compared to controls (cortical tubular degeneration/necrosis and dilatation, occasionally correlated with macroscopically pale kidneys). For the stomach the incidence in females treated at 1000 mg/kg bw wa 71.4% (above historical control values (max 60%)). For the heart incidence and severeity was comparable to contol values (see tables)

The treatment related increased pigment in female kidney cortical tubules was seen independently of cortical tubule mineralisation and associated findings; 3/6 females treated at 1000 mg/kg/day with increased pigment did not feature mineralisation, and 3/6 females treated at 1000 mg/kg/day with mineralisation did not feature increased pigment. This indicates that the increased pigment presence was not related to mineralisation and was an integral effect of treatment
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females allocated to the study showed normal four/five day estrous cycles during the acclimatization period.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment for all remaining females. There was a very slight shift in gestation length with fewer females showing a 22 day gestation length and more females showing a 23 day gestation length in animals receiving 1000 mg/kg/day compared with the controls. However, this was marginal and the difference did not attain statistical significance, hence no effect of treatment is inferred.
Fertility index (%) No treatment related effecst
Gestation length index (%) No treatnment related effects

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
one litter in the control group was killed for animal welfare reasons related to the dam (day 4)
In all other litters few incidental death of pups (total 13 in treatment groups)
most deaths were found on day 1
see table
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
at 330 and 1000 mg/kg bw slightly higher compared to controls (see table)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Ano-genital distance: no tretament related effects
Nipple count (males): no nipples found in any animal
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
in offspring that died in most cases no milk was present in their stomach
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Post-implantation survival index (%) No treatment related effecst (see table)
Live birth index (%) No treatment related effecst (see table)
Viability index (%) No treatment related effecst (see table)
Lactation index (%) No treatment related effecst (see table)

The mean number of implantations in females treated at 1000 mg/kg/day was slightly low (90%) but this did not affect litter size and there was also no effect of treatment on post implantation survival, live birth or viability indices.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
serum T4 concentrations in offspring day 13: no effects (see table)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects found

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

Parental effects (see details under repeated dose toxicity)

Dose

0

 

100

 

330

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

0/10

2/10

1/10

0/10

0/10

0/10

1/10

0/10

No

Clinical signs

-vocalisation

-irritable

-dark eyes

-piloerection/hunched posture

-brown discoloration upper dorsal thorax

 

 

 

 

+

+

 

 

 

 

 

 

 

 

+ (lact)

 

+

+

+

 

 

 

+ (PM + lact)

+ (lact)

+ (PM + lact)

 

Yes (non-adverse)

Body weight (gain)

NTRE*

No

Food consumption

NTRE

No

Behavioral effects (wk 5 +lact)

NTRE

No

Motoractivity (wk 5 + lact)

 

 

 

 

 

 

 

(lact)

Yes (non-adverse)

Estrus cycle (pre-test +PM)#

NTRE (4-5 days)

 

Pre-coital interval

NTRE (1-4 days)

 

Conception rate

NTRE (100%)

 

Fertility index

NTRE (100%)

 

Gestation length

NTRE (22-23 days)

 

Gestation index

NTRE (100%)

 

Implementation sites

 

16.6

 

16.0

 

15.7

 

15.1

 

Litter size

 

14.8

 

14.6

 

15.3

 

14.8

 

Haematology

 

 

 

 

 

 

Hct↓ (9.5%)

Pt ↓ (25%)

MCH↑(5%)

Neut ↑ (83%)

Lymph ↓ (54%)

No

Clinical biochemistry

 

 

phos↑ (17%)

 

 

 

ALAT↓ (24%)

triglyc↑ (64%)

ASAT↑(105%)

Alb ↓(12%)

Yes (non-adverse)

Organ weights

 

 

 

 

 

 

Kidney (rel)↑ (18%)

Kidney (rel)↑ (19%)

Yes (non-adverse)

Marcoscopy-GI tract dark/blue(dark/blue contents)

- kidneys dark/blue (lipofuscin)

- liver dark

- lymphnodes (mes) dark/blue

- urinary bladder dark/blue

- thymus dark/blue

- uterus/ovaries/vagina dark/blue

- testes dark/blue

- dark tail

 

 

 

 

 

1/10 (1-6/10)

 

 

10/10

 

 

6-8/9 (3-9/9)

9/9

 

4/9

 

 

 

4/9

9/9

 

6-10/10 (5-7/10)

10/10

7/10

8/10

6/10

5/10

5-7/10

 

Yes (non-adverse)

Histopathology

- Kidneys Cortical Tubular basophilia

-kidneys Cortical Tubular other effects $

- heart mineralization (myocord, vasc)

-AdrenalsCortical Vacuolation

-Prostate Inflammatory cell infiltr

-Ax lymphnodes sinus erythro- cytosis/Erythrophagocytosis

- Ax lymphnodes Plasmacytosis

- Gland stomach mineralization

- Gland stomach necrosis

 

0/5

 

 

 

 

 

2/5

3/5

 

0/5

 

 

 

2-3/5

 

 

1/5

 

 

 

 

1/5

1/5

1/5

 

0/2

 

 

 

2/4

 

 

2/10

 

 

 

 

 

3-4/8

 

 

 

 

 

 

 

 

0/4

2/4

 

2/9

 

 

 

 

 

3/5

1/5

 

2/5

 

2/10

 

5-6/10

 

 

2/5

 

 

 

 

2/5

5/7

0/7

 

Yes (non-adverse)

testes

NTRE

no

NTRE= no treatment related effects

*↑during pre-mating at 1000 mg/kg bw

# at termination all females were in diestrous phase

↑/↓= significantly increased/decreased at 1% or 5% level (Dunnet test)

% compared to controls

$ includes: Cortical Tubular Degeneration/Necrosis; Cortical Tubular Dilatation; Increased Cortical Tubular Pigment; Mineralisation, Cortex

Effects on offspring

Dose

0

 

100

 

330

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Litter size day 1 (day 13)

14.4 (13.3)

14.2 (12.4)

15.1 (13.1)

15.6 (12.6)

No

BW gain (day 1-13)

20.7

20.5

21.8

22.0

23.5

22.5

23.4

23.1

 

Sex ratio (% M)

55.1

43.4

38.8

47.1

 

Live birth index (%)

97.1

97.6

98.7

98.7

 

Viability index (%)

99.4

100

98.8

100

 

Lactation index (% day 13)

100

100

100

100

 

Anogenital distance (mm)

4.3

2.2

4.2

2.1

4.1

2.0

4.2

2.1

 

↑/↓= significantly increased/decreased BW Williams’ test/sex ratio Wald’s test

 

Litter size

 

Total litter size

Live litter size on Day

 

 

 

Day

Before blood sampling

After blood sampling

Group

 

Implantations

1‡

1

4

4

13

Statistical test:

Wi

Wi

Wi

Wi

 

 

1

Mean

16.6

14.8

14.4

14.3

13.3

13.3

 

SD

1.6

2.6

3.0

2.9

2.1

2.1

 

N

10

10

10

10

9

9

2

Mean

16.0

14.6

14.2

14.2

12.4

12.4

 

SD

1.9

1.4

1.9

1.9

1.5

1.5

 

N

9

9

9

9

9

9

3

Mean

15.7

15.3

15.1

14.9

13.1

13.1

 

SD

2.5

2.6

2.6

2.6

2.3

2.3

 

N

10

10

10

10

10

10

4

Mean

15.1

14.8

14.6

14.6

12.6

12.6

 

SD

2.3

1.9

1.9

1.9

1.9

1.9

 

N

10

10

10

10

10

10

                 Includes offspring that died prior to designated Day 1 of age

Wi              Williams test

 

 

Offspring survival

 

Post implantation

Live birth

Viability

Lactation index (%)

Group

 

survival index (%)

index (%)

index (%)

on Day 13

Statistical test:

Wi

Fe

Fe

 

1

Mean

88.9

97.1

99.4

100.0

 

N

10

10

10

9

2

Mean

91.4

97.6

100.0

100.0

 

N

9

9

9

9

3

Mean

97.5

98.7

98.8

100.0

 

N

10

10

10

10

4

Mean

95.2

98.7

100.0

100.0

 

N

10

10

10

10

Wi              Williams test

Fe               Fisher’s Exact test

 

Offspring body weight (average per litter)

 

Group

 

Day of age
(before blood sampling)

Day of age
(after blood sampling)

 

Change

 

1

4

4

7

13

 

1-4

4-7

7-13

1-13

Statistical test:

Wi

Wi

Wi

Wi

Wi

 

Wi

Wi

Wi

Wi

1

Mean

7.2

9.8

10.3

15.6

28.0

 

3.0

5.3

12.4

20.7

SD

0.7

2.0

1.4

2.0

3.1

 

0.7

0.7

1.4

2.5

 

N

10

10

9

9

9

 

9

9

9

9

2

Mean

7.2

10.4

10.4

15.7

29.0

 

3.2

5.2

13.3

21.8

 M

SD

0.7

1.3

1.3

1.7

2.9

 

0.7

0.5

1.6

2.5

 

N

9

9

9

9

9

 

9

9

9

9

3

Mean

7.5

11.2

11.2

17.2

31.0*

 

3.6

6.0*

13.8*

23.5*

 M

SD

0.8

1.6

1.6

2.3

3.4

 

0.9

0.8

1.5

2.9

 

N

10

10

10

10

10

 

10

10

10

10

4

Mean

7.6

11.1

11.1

17.1

31.1*

 

3.5

6.0*

14.0*

23.4*

 M

SD

0.7

1.0

1.0

1.6

2.6

 

0.5

0.9

1.1

2.3

 

N

10

10

10

10

10

 

10

10

10

10

 

Group

 

Day of age
(before blood sampling)

Day of age
(after blood sampling)

 

Change

 

1

4

4

7

13

 

1-4

4-7

7-13

1-13

Statistical test:

Wi

Wi

Wi

Wi

Wi

 

Wi

Wi

Wi

Wi

1

Mean

6.9

9.5

10.0

15.2

27.4

 

3.1

5.2

12.3

20.5

 F

SD

0.7

1.9

1.4

1.9

3.3

 

0.7

0.6

1.7

2.8

 

N

10

10

9

9

9

 

9

9

9

9

2

Mean

7.0

10.1

10.1

15.4

29.0

 

3.1

5.3

13.5

22.0

SD

0.8

1.4

1.4

1.9

2.9

 

0.8

0.7

1.5

2.5

 

N

9

9

9

9

9

 

9

9

9

9

3

Mean

7.1

10.5

10.5

16.3

29.6

 

3.5

5.7

13.3

22.5

SD

0.7

1.5

1.5

2.0

3.2

 

0.8

0.7

1.7

2.7

 

N

10

10

10

10

10

 

10

10

10

10

4

Mean

7.2

10.6

10.7

16.4

30.3

 

3.4

5.7

13.9*

23.1

SD

0.5

1.0

1.0

1.7

3.3

 

0.5

0.8

1.7

2.9

 

N

10

10

10

10

10

 

10

10

10

10

Wi              Williams test

 

Mean serum T4 concentrations (pg/mL)

 

Group

Treatment

Dose

(mg/kg/day)

Male

 offspring on Day 13 of age

Female offspring on Day 13 of age

1

Control

0

Mean

40411

43322

SD

5342

5343

CV %

13.2

12.3

N

9

9

2

 

Direct Blue 279

100

Mean

43000

43244

SD

4504

9736

CV %

10.5

22.5

N

9

9

3

Direct Blue 279

330

Mean

42700

41350

SD

6108

8774

CV %

14.3

21.2

N

10

10

4

Direct Blue 279

1000

Mean

43760

42790

SD

5761

5191

CV %

13.2

12.2

N

10

10

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Repeated administration of the substance was well tolerated in the adult animals but was associated with changes in the kidneys of females treated at 1000 mg/kg/day. An increased presence of brownish pigment in the cortical tubules of 6/10 animals, identified as lipofuscin and considered to be accompanied by test item material, was identified. This finding was considered non-adverse.
Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.
In the context of this study,the substance showed no evidence of being an endocrine disruptor.
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.
Executive summary:

Three groups of ten male and ten femalerats received the substance at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to n ecropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, 1% w/v methylcellulose, at the same volume-dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results

Repeated administration of the substance was well tolerated in the adult animals but was associated with changes in the kidneys of females treated at 1000 mg/kg/day.  An increased presence of brownish pigment in the cortical tubules of 6/10 animals, identified as lipofuscin and considered to be accompanied by test item material, was identified.  This finding was considered non-adverse (details and complete summary can be found under repeated dose)

Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by the administration of Direct Blue at all dose levels. The mean number of implantations in females treated at 1000 mg/kg/day was slightly low but this did not affect litter size.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Ano-genital distances of both male and female offspring on Day 1 of age and nipple counts of male offspring on Day 13 of age showed no adverse effects from parental treatment. 

Male and female offspring body weight and body weight change for F0 males and females receiving 330 or 1000 mg/kg/day were slightly high when compared with the controls throughout the lactation period. 

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Conclusion

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.

In the context of this study the substance showed no evidence of being an endocrine disruptor.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.