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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:
12 April 2017 to 19 August 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
Chemical structure
Reference substance name:
Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate
EC Number:
228-327-6
EC Name:
Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate
Cas Number:
6227-20-9
Molecular formula:
C34H27N5O11S3.3Na
IUPAC Name:
trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate
Test material form:
solid: flakes
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: ca 72 days; Females: ca 86 days
- Weight at study initiation: Males: 345 to 415 g; Females: 236 to 298 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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was ground before weighing out the required amount. Approximately 50% of the required volume of vehicle was added to the test item and it was magnetically stirred for a minimum of one hour to ensure accurate mixing. The remaining amount of vehicle was added and it was magnetically stirred for a minimum of 20 minutes.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE:water
- Concentration in vehicle: 0, 1, 3 and 8 mg/mL
- Amount of vehicle (if gavage): 5 mL
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the 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:
Samples of the formulations (1 mL, accurately weighed) were dissolved using ultrasonic vibration in a suitable volume of water and refrigirated for 1 day to allow full dissolution of substance before re-equilibration to ambient temperature prior to dilution. Once at room temperature the extract was diluted to provide a solution containing the substance at an expected concentration within the range 2 µg/mL to 4 µg/mL. These solutions were anaylsed by HPLC/UV.

HPLC: Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Poroshell C18-SB, 2.7 µm, 100 × 4.6 mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile Phase A: ACN/10mM ammonium formate 10/90 v/v
Mobile Phase B: ACN/10mM ammonium formate 90/10 v/v
Gradient: Time (min) A(%) B(%)
0.0 95 5
0.5 95 5
5.0 20 80
5.2 20 80
5.3 95 5
8.0 95 5

Flow rate: 1.0 mL/min
Detector wavelength: UV, 560 nm
Injection volume: 10 µL
Run time: 8 minutes
Approximate retention time:3.7 minutes

Calibration (1.0 to 5.01 ug/mL): y= 41812x + 257 (R2=0.99980) (standards 1 and 5 ug/ml CV% 0.86 and 0,53)
Procedural recovery (validation) at 1 and 100 mg/mL: 100.5-102.5% of nominal
Procedural recovery (test solutions) at 1 and 100 mg/L on day 0,1 and 15: 94.9-104.4% of nominal; on day 8 90-100.9% of nominal
Stability at 21˚C over 15 days; at 1 and 100 mg/L: 108 and 114% of nominal
Stability (refrigerated) over 8 dys: at 100 mg/L congealed on Day 8
Homogeneity at 1 and 100 mg/L: CV% <0.87 and <1.44
Analyzed Concentrations: 85.5- 100.5 % of nominal (see attached table under repeated dose toxicity)
Duration of treatment / exposure:
Males Two weeks pre-pairing up to necropsy after a minimum of five weeks of treatment
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.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
40 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, treatment at 100, 250 or 500 mg/kg/day was not tolerated and resulted in one or more animals either found dead without any prior warning of a decline in clinical condition, or killed for reasons of animal welfare. Treatment at 50 mg/kg/day was tolerated, but body weight losses were recorded for one out of three males and one out of three females (19g and 6g respectively) during the second week of treatment and thus suggests an effect of treatment. Taking into consideration the much larger group size, the much longer duration of treatment and the increased physiological demand on the females during pregnancy and lactation on this main OCED 422 study; the high dose was limited to 40 mg/kg/day as the dose was expected to be tolerated but may elicit signs of toxicity manifesting as initial body weight loss in some animals. The intermediate and low doses were set at 15 and 5 mg/kg/day to permit evaluation of any dose related trends over an approximate geometric progression of doses.
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 start of treatment until start of 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 (in all males that survived or mated successfully):
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.
females: dark brown discoloration of dorsal surface iat controls, 5 and 15 mg/kg bw
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Incidental
1 female at 15 mg/kg bw: on day 14 of gestation (rupture of a mass on the upper ventral surface; this mass was found to correlate with a mammary adenocarcinoma)
1 male at 40 mg/k bw:day 27(dosing error)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
males: no treatment related effects
females: BW increased during gestation and/or lactation at 5, 15 and 40 mg/kg bw (can be attributed to low control values)
BW gain was decreased at 40 mg/kg bw during gestation and lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects
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: slightly low hematocrit counts attaining statistical significance at 15 or 40 mg/kg/day ( both -9.6%)
females: at 40 mg/kg bw sign increased MCV (+5%) and sign decreasd PT (-20%); increase in WBC with significant increase of lymphocytes (+75%)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For females on Day 14 of lactation, data were variable with atypically high results observed in many parameters for the control females, such as Urea (13.05 - 35.53 mmol/L) or Creatinine (Mean: 38 - 112 μmol/L). These findings were consistent with the observed mineralization identified at histopathological examination (See Section 6.3.12) across all study groups, predominantly in control females.
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 males receiving 40 mg/kg/day was low when compared with the controls. Statistical significances were attained at the first of the 6-minute intervals and total scores for high beams
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):
Microscopic examination performed after 5 weeks of treatment revealed minimal to slight degeneration/atrophy in the testes in 4 / 9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day, but at the incidence and severity were within historical control ranges (0-20%). In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate (see table)
Microscopic examination performed on Day 14 of lactation revealed lesions in the heart (myocardial/vascular mineralization), kidneys (cortical tubular degeneration/necrosis, mineralization) and stomach (mucosal necrosis, mineralization) were seen in several females, including in control animals with no relationship to test item. The microscopic findings in the heart of myocardial/vascular mineralisation correlated with the pale areas seen macroscopically. Cortical tubular degeneration/necrosis and mineralization seen in the kidneys correlated with pale areas seen at necropsy. Mucosal necrosis and mineralization were also seen in the glandular stomach. Dark areas seen in the stomach macroscopically correlated with the mucosal erosion/congestion seen microscopically.
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 in diestrous stage at termination.
Reproductive function: sperm measures:
effects observed, treatment-related
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.
Microscopic examination performed after 5 weeks of treatment revealed minimal to slight degeneration/atrophy in the testes in 4 / 9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day, but at the incidence and severity were within historical control ranges (0-20%). In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate (see table)
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The number of females raising their litters to weaning was 10, 10, 9 and 4 for 0, 5, 15 and 40 mg/kg/day, respectively. Five females receiving 40 mg/kg/day were not pregnant and one female of this group did not litter; one dead autolysed pup was found in the uterus.

Fertility index (%) 100, 100, 100 and 50% at 0, 5, 15 and 40 mg/kg bw
Gestation length index (%) No treatment related effects

Details on results (P0)

No relationship between the effects on the male reproductive system and the decreased pregnancy rate was found (see table)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
cauda epididymis
testes
Treatment related:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean numbers of pups was slightly low at 40 mg/kg/day due to one atypically small litter comprising five pups

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 15 in treatment groups + 2 scheduled kills)

see table
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
in males and females at 40 mg/kg bw higher compared to controls (see table)
This is considered to reflect the lower mean litter size in this group.
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 (see table)
Nipple count (males): Nipples were recorded in a few isolated male offspring, including controls, but there was no relationship to treatment.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
in offspring that died the following incidental findings were noted in all groups including controls:
abnormal thin; stomach: no milk present; lungs: dark colour; abdomininal contents autolysed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Post-implantation survival index (%) No treatment related effects (see table)
Live birth index (%) No treatment related effects (see table)
Viability index (%) No treatment related effects(see table)
Lactation index (%) No treatment related effects (see table)

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:
40 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:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects

Any other information on results incl. tables

Parental effects (see details under repeated dose toxicity)

Dose

0

 

5

 

15

 

40

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

0/10

0/10

0/10

0/10

0/10

1/10

1/10

0/10

No

Clinical signs

-brown discoloration upper dorsal surface

 

 

 

+ (lact)

 

 

+ (lact)

 

 

+ (lact)

 

 

 

No

Body weight

 

 

 

(gest)

 

(gest+lact)

 

(gest+lact)

No (low control)

Body weight gain

 

 

 

 

 

 

 

↓(gest+ lact)

Yes

Food consumption

 

 

 

(gest)

 

(gest)

 

(gest)

No (within normal variation)

Behavioral effects (wk 5 +lact)

NTRE

No

Motoractivity (wk 5 + lact)

 

 

 

 

 

 

(wk 5)

 

Yes

Estrus cycle (pre-test +PM)

NTRE (4-5 days)#

 

Thyroxine (T4) (males)

NTRE

Controls atypically low

Pre-coital interval

NTRE (1-5 days)

 

Conception rate (%)

 

100

 

100

 

100

 

50

yes

Fertility index (%)

 

100

 

100

 

100

 

50

yes

Gestation length

NTRE (22-23 days)

 

Gestation index (%)

 

100

 

100

 

90

 

80

yes

Live litters

 

10

 

10

 

9

 

4

yes

Implementation sites

 

14.4

 

16.2

 

16.2

 

13.0**

yes

Litter size

 

14.1

 

16.2

 

14.3

 

11.8**

yes

Haematology

 

 

 

 

Hct↓ (9.6%)

WBC/Leuk ↑ (ns)

 

 

Hct↓ (9.6%)

WBC/Leuk ↑ (ns)

 

MCV↑(5%)

Pt ↓ (20%)

WBC/Lymph ↑ (ns/75%)

No

Clinical biochemistry*

 

 

 

 

 

 

ALP↑ (28%)

Bile ac↑ (ns)

ALAT↓ (28%)

Bile ac ↑ (69%)

Urea ↓(62%)

Creat ↓(55%)

No (high control values for urea + creat, within hist controls)

Organ weights

 

 

 

Ovaries (rel) ↑ (14%)

 

Ovaries (rel) ↑ (12%)

brain (rel)↑ (7%)

brain (rel)↑ (7%)

ovaries (rel) ↑ (20%)

uterus (rel) ↑ (27%)

Yes (non-adverse)

Marcoscopy

NTRE

 

Histopathology

- epididymides luminal cell debris

- epididymedes inflame infiltrate, - testes tubular degen/atrophy

- heart myocardial degeneration

- kidney foci inflammatory cells

- kidney tubular basophilia

- kidney tubular degeneration 

- kidney corical miniralization

- kidney tubular dilatation

- liver inflammatory cell infiltrate

- stomach mineralization

- stomach congestion

- stomach necrosis

 

0/10

0/10

0/10

1/5

0/5

1/5

 

 

 

 

3/5

 

 

6/6

5/6

4/6

0/5

3/5

1/5

2/5

 

0/10

0/10

0/10

 

 

 

 

 

 

 

1/1

1/1

1/1

 

1/4

3/4

2/4

 

1/10

0/10

0/10

 

 

 

 

 

 

 

4/4

3/4

3/4

 

0/5

3/5

4/5

 

7/9 (min)

3/9(min-mod)

4/9 (min)

2/5

2/6

2/6

 

 

 

 

0/4

 

 

1/4

0/4

0/4

2/4

0/4

0/4

0/4

 

Yes

NTRE= no treatment related effects

# incidental irregular circle; at termination all surviving females with live litters were in diestrous phase

↑/↓= significantly increased/decreased at 1% or 5% level

*no statistical analysis of data for females due to effects of mineralization (effects considered atypical)

**based on 4 litters with one consisting of only 5 pups

% compared to controls

Effects on offspring

Dose

0

 

5

 

15

 

40

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Litter size day 1 (day 13)

14.1 (12.1)

16.2 (13.3)

14.3 (12.2)

11.8 (9.5)

no

BW gain (day 1-13)

19.6

19.2

19.3

18.6

21.6

20.2

23.1

23.5

no

Sex ratio (% M)

47.4

55.8

52.7

52.5

no

Live birth index (%)

99.4

99.4

98.3

96.9

no

Viability index (% day 4)

100

99.4

98.5

100

no

Lactation index (% day 13)

100

99.4

98.4

95

no

Anogenital distance (mm)

4.2

2.3

4.3

2.4

no

2.2

4.3

2.5

no

Nipple count

NTRE (incidental)

 

Macroscopy

NTRE

 

↑/↓= significantly increased/decreased

NTRE no treatment related effects

Summary of treatment related findings in the epididymides and testes for animals killed after 5

weeks of treatment

Group

 

Control

5 mg/kg bw

15 mg/kg bw

40 mg/kg bw

Epididymides

No. examined

10

10

10

9

    Cell Debris, Luminal

Minimal

0

0

1

7

 

Total

0

0

1

7

    Infiltrate, Inflammatory Cell

Minimal

0

0

0

2

 

Moderate

0

0

0

1

 

Total

0

0

0

3

    Edema

Slight

0

0

0

1

 

Total

0

0

0

1

Testes

No. examined

10

10

10

9

    Degeneration/Atrophy, Tubular

Minimal

0

0

0

2

 

Slight

0

0

0

2

 

Total

0

0

0

4

 

Group 4 (40 mg/kg bw) Relationship between pregnancy rate and effects on testes and epididymides in males

male

female

epididymides

testes

pregnancy

1

41

+

+

 

2

42

 

 

 

3

43

 

 

+

4

44

+

+

+

5

45

+

+

+

6

46

+

 

+

7

47

 

 

 

8

48

+

 

 

9

49

+

+

 

10

50

+

 

+*

* one autolysed fetus was found

Litter size

Group

 

Implantations

Total @

Live on Day

 

 

/Sex

 

 

 

Before bleed

After bleed

 

 

 

 

 

1

4

4

13

Statistics

test

Wi

Sh

Wi

Wi

 

 

1F

Mean

14.4

14.1

14.0

14.0

12.1

12.1

 

SD

1.84

1.85

1.76

1.76

1.60

1.60

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

2F

Mean

16.2

16.2

16.1

16.0

14.1

13.3

 

SD

2.15

1.81

1.79

1.76

1.73

2.75

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3F

Mean

16.2

14.3

14.1

13.9

12.4

12.2

 

SD

2.05

1.58

1.76

1.69

1.33

1.20

 

N

9

9

9

9

9

9

 

 

 

 

 

 

 

 

4F

Mean

13.0

11.8

11.3

11.3

10.3

9.5

 

SD

5.66

5.74

5.32

5.32

4.27

3.32

 

N

4

4

4

4

4

4

 

 

 

 

 

 

 

 

Wi= Williams test

Sh= Shirley’s test

 

Offspring survival

Group

 

Post implantation

Live birth

Viability index (%)

Lactation index (%)

/Sex

 

survival index (%)

index (%)

Day 4

Day 13

Statistics

test

Sh

Ch

Ch

 

1F

Mean

97.1

99.4

100.0

100.0

 

SD

3.72

1.98

0.00

0.00

 

N

10

10

10

10

 

N<100%

4

1

0

0

 

 

 

 

 

 

2F

Mean

98.8

99.4

99.4

94.4

 

SD

2.44

1.86

1.86

15.71

 

N

10

10

10

10

 

N<100%

2

1

1

2

 

 

 

 

 

 

3F

Mean

89.7

98.3

98.5

98.4

 

SD

14.17

3.46

2.94

4.67

 

N

9

9

9

9

 

N<100%

8

2

2

1

 

 

 

 

 

 

4F

Mean

90.8

96.9

100.0

95.0

 

SD

14.67

6.25

0.00

10.00

 

N

4

4

4

4

 

N<100%

2

1

0

1

 

 

 

 

 

 

Ch= Chi-squared test

Sh= Shirley’s test

 

Pup body weight and body weight change - group mean values (g)

 

Group

 

Day of age (before bleed)

Day of age (after bleed)

 

 

Change

Change

Change

Change

 

/Sex

 

1

1 @

4

4

7

13

 

1-4

4-7

7-13

1-13

Statistics

test

Wi

Wi

Wi

Wi

Wi

Wi

 

Sh

Wi

Wi

Wi

1M

Mean

6.6

6.6

9.2

9.2

13.9

26.2

 

2.6

4.7

12.3

19.6

 

SD

0.55

0.55

0.88

0.88

1.25

2.00

 

0.38

0.48

0.97

1.60

 

N

10

10

10

10

10

10

 

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

2M

Mean

6.5

6.5

8.8

8.8

13.6

25.9

 

2.3

4.7

12.1

19.3

 

SD

0.50

0.50

1.14

1.14

1.69

2.47

 

0.79

0.68

1.80

2.07

 

N

10

10

10

10

10

10

 

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

3M

Mean

7.2

7.2

10.3

10.3

15.6

28.7

 

3.2

5.2

13.2

21.6

 

SD

0.57

0.57

0.94

0.94

1.73

2.59

 

0.53

0.94

1.35

2.34

 

N

9

9

9

9

9

9

 

9

9

9

9

 

 

 

 

 

 

 

 

 

 

 

 

 

4M

Mean

7.0

7.0

10.8

10.8

16.6*

30.2*

 

3.8

5.8*

13.6

23.1*

 

SD

1.19

1.19

2.88

2.88

4.28

6.68

 

1.70

1.53

2.59

5.59

 

N

4

4

4

4

4

4

 

4

4

4

4

1F

Mean

6.3

6.3

8.8

8.8

13.6

25.5

 

2.5

4.8

11.9

19.2

 

 

SD

0.52

0.53

0.87

0.89

1.21

1.90

 

0.44

0.51

1.06

1.57

 

 

N

10

10

10

10

10

10

 

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2F

Mean

6.2

6.2

8.4

8.5

13.2

24.9

 

2.2

4.7

11.7

18.6

 

 

SD

0.48

0.47

0.83

0.74

1.26

1.79

 

0.49

0.63

1.83

1.66

 

 

N

10

10

10

10

10

10

 

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3F

Mean

6.6

6.5

9.6

9.5

14.4

26.7

 

3.0

4.9

12.3

20.2

 

 

SD

0.55

0.54

0.99

1.09

1.95

3.21

 

0.68

0.92

1.60

2.89

 

 

N

9

9

9

9

9

9

 

9

9

9

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4F

Mean

7.0

7.1*

10.6*

10.6**

18.0**

30.5**

 

3.6**

7.3

12.4

23.5**

 

 

SD

1.12

1.11

2.32

2.30

4.61

6.25

 

1.22

3.01

2.48

5.14

 

 

N

4

4

4

4

4

4

 

4

4

4

4

 

Wi= Williams test

@ - Includes only those pups surviving after the bleed

 

Mean serum T4 concentrations (pg/mL)

Group

Dose (mg/kg/day)

 

F1 female offspring on

Day 13 of age

F1 male offspring on

Day 13 of age

1

Control

Mean

38200

35400

 

 

SD

7270

5310

 

 

CV%

19.0

15.0

 

 

N

10

10

2

5

Mean

37600

35500

 

 

SD

4770

4330

 

 

CV%

12.7

12.2

 

 

N

10

10

3

15

Mean

36400

36200

 

 

SD

5890

5490

 

 

CV%

16.2

15.2

 

 

N

9

9

4

40

Mean

35900

33100

 

 

SD

8770

6740

 

 

CV%

24.4

20.4

 

 

N

4

4

 

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 testes/epididymides of adult males treated at 40 mg/kg/day. A decreased pregnancy rate was seen at 40 mg/kg bw, but this could not be related to the effects on the male reproductive system or any effects in females.
There were no adverse effects on offspring.
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 15 mg/kg/day.
Executive summary:

Three groups of ten male and ten female rats 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 necropsy 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, 1water, 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

There were no treatment related adverse effects of treatment on clinical condition, sensory reactivity, grip strength, body weight gain, food intake, haematology, blood chemistry or organ weight measurements in males and females. 

The assessment of motor activity scores indicated that males receiving 40 mg/kg/day were slightly less active than the control males.

Estrous cyclicity was unaffected by the administration of the substance at all dose levels. Out of 10 females that mated in Group 4 (40 mg/kg/day), only five achieved pregnancy

Hematological examination for males revealed, when compared with controls, slightly low hematocrit counts attaining statistical significance at 15 or 40 mg/kg/day. A slight increase in mean cell volume (attaining statistical significance) was observed in females treated at 40 mg/kg/day. In addition a decrease of the number of lymphocytes was seen in females at 40 mg/kg bw.

Biochemical evaluation revealed no treatment-related findings.

At the macroscopic examination of the adult animals no treatment related effects were observed. Pale areas in heart and kidneys could be related to mineralization as observed during histopathological examination.

At the microscopic examination of the adult animals changes related to treatment with the substance were seen in males at 40 mg/kg bw. These findings included minimal to slight degeneration/atrophy in the testes in 4/9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day,but the effect remained within historical control ranges (0 -20%).  In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate. 

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 pregnancy rate in females at 40 mg/kg bw was 50% (leading to 4/10 viable litters). This reduction in fertility at 40 mg/kg/day did not show a correlation between the findings in the testes and those females which were found not pregnant. Therefore it remains unclear if the fertility was attributed to the males or the females treated with the substance. Apart from the reduction in fertility, there was no systemic toxicity detected in females treated with the substance.

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 40 mg/kg/day were slightly high when compared with the controls (this could be attributed to to one small litter).

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 and fertility seem to have been affected by treatment at 40 mg/kg bw. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive toxicity was considered to be 15 mg/kg/day. The NOAEL for developmental toxicity is 40 mg/kg bw.