3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic acid] hexasodium salt AND 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)

Endpoint summary

Description of key information

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, 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.

Four animals died prematurely; all deaths were considered incidental and not related to treatment.

There were no signs seen in association with the dosing procedure. At the routine weekly physical examinations the following signs were recorded: dark eyes, tail and pinnae were seen in males, predominantly at 1000 mg/kg/day, and dark eyes and dark colouration of the whole body were seen in females receiving 1000 mg/kg/day. On the day of the scheduled kill, all females receiving 1000 mg/kg/day were hunched and had piloerection.

The behaviour of the animals in the arena and their sensory reactivity and grip strength responses were all unaffected by treatment.

The assessment of motor activity scores indicated that females receiving 1000 mg/kg/day were slightly less active than the control females.

When compared with the controls, the bodyweight gains of all treated female groups in the pre-pairing period were high and, during the lactation phase, females receiving 330 or 1000 mg/kg/day had high weight gain in the first week but low weight gain in the second week. None of these variations in body weight were considered adverse. Male body weight gains were not affected by treatment.

The food consumption of males receiving the substance at all doses was slightly higher than controls in all weeks of treatment with a similar effect seen in females, but only prior to pairing.   These minor variations in food consumption were considered non-adverse. 

Estrous cyclicity was unaffected by the administration of the substance at all dose levels. 

Haematological investigations revealed slightly low haematocrit and haemoglobin concentration in males receiving 1000 mg/kg/day., high neutrophil and low lymphocyte counts in females receiving 1000 mg/kg/day. The biochemical examination of the blood plasma revealed a slight decrease in alanine amino-transferase activity and a slight increase in triglyceride concentrations in males receiving 1000 mg/kg/day. Aspartate amino-transferase activity was high for females receiving 1000 mg/kg/day. Albumin : globulin ratios were slightly low in all Direct Blue treated female groups, and in the high dose animals this associated with a reduction in plasma albumin concentration. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

High kidney weights were seen in animals given 1000 mg/kg/day.

At the macroscopic examination of the adult animals the predominant findings were abnormal colouration (dark/blue) of internal tissues/organs and/or abnormal contents of the gastro-intestinal tract which were reported in the majority of animals given 1000 mg/kg/day and in some males given 330 mg/kg/day. However, abnormal colouration of the kidneys was seen in all males and females given 330 or 1000 mg/kg/day and also in a few animals given 100 mg/kg/day and dark livers were only seen in females given 1000 mg/kg/day.

At the microscopic examination of the adult animals changes related to treatment with the substance were seen in the kidney. An increase of a brown coloured pigment was seen in the cortical tubules of 6/10 females treated at 1000 mg/kg/day. This pigment was identified as lipofuscin together with test item pigment. The finding was considered not adverse.

Oral 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.

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 was considered to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

repeated dose reproduction study OECD 422

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

grounded to powder prior to formluation

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

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

Route of administration:

oral: gavage

Details on route of administration:

using a suitably graduated syringe and a rubber catheter inserted via the mouth (dosing volume adjusted to latest measured body weight)

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral 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

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

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 males and 10 females

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.

Observations and examinations performed and frequency:

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)

ESTOUS CYCLE: Yes
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.

Sacrifice and pathology:

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.

Other examinations:

F0 Females:
Each ovary:Number of corpora lutea
Each uterine horn: Number of uterine implantation sites were recorded.

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify 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. Any cell- or stage-specificity of testicular findings was noted

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, clinical pathology, organ weight data:
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. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.
For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

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 both for i) values c, as applicable.

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.

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: decreased PT time at all dose groups (control values were very high)
1000 mg/kg bw: sign decreased Hct (slightly lower Hb)
females: at 1000 mg/kg bw sign increased MCV, neutrophils (1 female) and sign. increased lymphocytes

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 this was associated 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.

see table

Immunological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males (see table)
The microscopic examination of thymus, thyroid, adrenal and pituitary glands and the reproductive organs was also unremarkable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

males: 1000 mg/kg bw sign increased kidney weight (X 1.18 of control)
females: 1000 mg/kg bw sign increased kidney weight (X 1.18 of control)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal colouration (dark/blue) of internal tissues/organs and/or abnormal contents of the gastro-intestinal tract. These findings were reported in the majority of animals given 1000 mg/kg/day and in some males given 330 mg/kg/day. However, abnormal colouration of the kidneys was seen in all males and females given 330 or 1000 mg/kg/day and also in a few animals given 100 mg/kg/day and dark livers were only seen in females given 1000 mg/kg/day (see table)

Neuropathological findings:

not examined

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

Other effects:

no effects observed

Description (incidence and severity):

no treatment related effect (oestrus cycle 4-5 days pre-treatment, at termination all females were in diestrous phase)

Details on results:

No adverse effects were seen on body weight or food consumption.
The behavior of the animals in the arena was not affected by treatment. Sensory reactivity and grip strength were also unaffected by treatment but females receiving 1000 mg/kg/day were less active than the controls.
The haematological investigation revealed slightly low haematocrit and haemoglobin concentration in males receiving 1000 mg/kg/day and high neutrophil and low lymphocytes counts in females receiving 1000 mg/kg/day. These effects were considered not related to treatment. Clinical biochemistry showed decrease in alanine amino-transferase activity and increase in triglyceride concentrations in males at 1000 mg/kg/day. Aspartate amino-transferase activity was high for females receiving 1000 mg/kg/day. A/G ratios were low in all female groups and in the high dose females this associated with a reduction in plasma albumin concentration. In the absence of any effects seen on liver weights and no remarkable histopathological findings reported in the liver the small variations in the hepatic enzymes and triglyceride concentrations were considered non-adverse. The profile of proteins in the plasma may also be influenced by changes in hepatic metabolism (influencing the production) but also by kidney function (controlling the excretion). The mineralization seen in the kidneys, which was considered incidental, may however have contributed to a disturbance in renal metabolism and therefore, the variations in the plasma proteins may also be incidental and not directly related to treatment.
The microscopic evaluation of the tissues revealed an increased presence of brownish pigment in the cortical tubules of the kidneys of 6/10 females treated at 1000 mg/kg/day, identified by special staining as lipofuscin (considered of limited or no functional significance, (Frazier et al, 2012)), but it was considered that test item material was also present. Due to the minimal severity and the lack of associated kidney pathology, the presence of lipofuscin and test item material in the cortical tubules was considered non-adverse.
Incidences and severities of mineralization observed in kidney, stomach and heart are considered incidental and unrelated to treatment. The increased pigment presence was not related to mineralisation and was an integral effect of treatment, but as stated above considered non-adverse.
Kidney weights were increased in males and females treated at 1000 mg/kg/day. There was no clear histopathological correlate for this.
Frequent macroscopic observations of abnormal colour (blue or dark) and/or abnormal content (blue or dark) in several tissues were not correlated histopathologically. These changes are considered to represent the colour of the test item, which was likely removed during fixation or processing.

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

Remarks on result:

other: absence of adverse effects

Critical effects observed:

no

Functional observations

Group/sex:		Statistical	1M	2M	3M	4M	1F	2F	3F	4F
Number of animals:		test (M)	5	5	5	5	5	5	5	5
Parameter	Grade
Approach response (1-3)	1		0	0	0	1	0	0	0	1
	2		5	5	5	4	5	5	5	4
Pinna reflex (1-3)	2		5	5	5	5	5	5	5	5
Auditory startle reflex (1-4)	2		1	2	0	0
	3		4	3	5	5	5	5	5	5
Tail pinch response (1-4)	3		3	4	3	5	0	0	0	2
	4		2	1	2	0	5	5	5	3
Forelimb grip strength (kg)	Mean	Williams’ test	1.22	1.32	1.21	1.28	1.15	1.20	1.19	1.22
	SD		0.03	0.08	0.08	0.06	0.08	0.07	0.09	0.08
Hindlimb grip strength (kg)	Mean	Williams’ test	0.70	0.80	0.73	0.68	0.48	0.50	0.49	0.55
	SD		0.06	0.09	0.06	0.08	0.06	0.08	0.02	0.07

 

Motoractivity

Group /Sex	Number of animals	Beam level		Group /Sex	Number of animals	Beam level
			Total				Total
Statistical test: Williams’ test
1M	5	High	216.6	1F	5	High	356.8
		SD	103.0			SD	64.1
2M	5	High	225.0	2F	5	High	288.8
		SD	81.5			SD	109.9
3M	5	High	346.2	3F	5	High	313.2
		SD	182.8			SD	159.1
4M	5	High	235.8	4F	5	High	145.4**
		SD	134.7			SD	62.8
Statistical test: Williams’ test
1M	5	Low	601.6	1F	5	Low	858.2
		SD	251.4			SD	188.2
2M	5	Low	586.6	2F	5	Low	715.8
		SD	195.9			SD	265.3
3M	5	Low	819.8	3F	5	Low	664.2
		SD	324.2			SD	149.0
4M	5	Low	649.4	4F	5	Low	467.0**
		SD	196.3			SD	203.3

** p<0.01

Mean serum T4 concentrations (pg/mL)

Group	Dose (mg/kg/day)		Adult terminal males
1	0	Mean	43390
		SD	6279
		CV %	14.5
		N	10
2	100	Mean	38120
		SD	5153
		CV %	13.5
		N	10
3	330	Mean	48040
		SD	9312
		CV %	19.4
		N	10
4	1000	Mean	46489
		SD	5611
		CV %	12.1
		N	9

 

Summary of findings of abnormal colour and/or content in all affected tissues in animals killed after 5 weeks of treatment(males) or on Day 14 of lactation (females)

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Adipose Tissue
Abnormal colour, Dark	0	0	0	2	0	0	0	0
Abnormal colour, Blue	0	0	0	1	0	0	0	0
Number of tissues examined	10	10	10	9	9	9	10	10
Caecum
Abnormal colour, Dark	0	0	1	8	0	0	0	5
Abnormal colour, Blue	0	0	0	0	0	0	0	2
Abnormal contents, Dark	0	0	4	9	0	0	0	6
Abnormal contents, Blue	0	0	0	0	0	0	0	1
Number of tissues examined	10	10	10	9	9	9	10	10
Colon
Abnormal colour, Dark	0	0	0	6	0	0	0	5
Abnormal colour, Blue	0	0	0	0	0	0	0	2
Abnormal contents, Dark	0	0	3	5	0	0	0	4
Number of tissues examined	10	10	10	9	9	9	10	10
Duodenum
Abnormal colour, Dark	0	0	0	0	0	0	0	2
Abnormal colour, Blue	0	0	0	0	0	0	0	1
Abnormal contents, Dark	0	0	1	0	0	0	0	0
Number of tissues examined	10	10	10	9	9	9	10	10
Eyes
Dark	0	0	0	0	0	0	0	2
Number of tissues examined	10	10	10	9	9	9	10	10
Ileum
Abnormal colour, Dark	0	0	0	7	0	0	0	4
Abnormal colour, Blue	0	0	0	0	0	0	0	2
Abnormal contents, Dark	0	0	3	3	0	0	0	4
Number of tissues examined	10	10	10	9	9	9	10	10
Jejunum
Abnormal colour, Dark	0	0	1	7	0	0	0	5
Abnormal colour, Blue	0	0	0	0	0	0	0	3
Abnormal contents, Dark	0	0	2	3	0	1	0	5
Number of tissues examined	10	10	10	9	9	9	10	10
Kidneys
Abnormal colour, Dark	0	1	10	9	0	2	8	8
Abnormal colour, Blue	0	0	0	0	0	0	2	2
Number of tissues examined	10	10	10	9	9	9	10	10
Left Axillary Lymph Node
Abnormal colour, Dark	0	0	0	1	0	0	0	2
Number of tissues examined	10	10	10	9	9	9	10	10
Liver
Dark	0	0	0	0	0	0	0	7
Number of tissues examined	10	10	10	9	9	9	10	10
Mesenteric Lymph Node
Abnormal colour, Dark	0	0	0	4	0	0	0	5
Abnormal colour, Blue	0	0	0	0	0	0	0	3
Number of tissues examined	10	10	10	9	9	9	10	10
Ovaries
Abnormal colour, Dark	-	-	-	-	0	0	0	4
Abnormal colour, Blue	-	-	-	-	0	0	0	2
Number of tissues examined	-	-	-	-	9	9	10	10
Rectum
Abnormal colour, Dark	0	0	0	6	0	0	0	5
Abnormal colour, Blue	0	0	0	0	0	0	0	3
Abnormal contents, Dark	0	0	4	4	0	0	1	5
Abnormal contents, Blue	0	0	0	0	0	0	0	1
Number of tissues examined	10	10	10	9	9	9	10	10
Skin
Abnormal colour, Dark	0	0	0	2	0	0	0	1
Abnormal colour, Blue	0	0	0	1	0	0	0	5
Number of tissues examined	10	10	10	9	9	9	10	10
Stomach
Abnormal colour, Dark	0	0	6	7	0	0	0	3
Abnormal colour, Blue	0	0	0	1	0	0	0	7
Abnormal contents, Dark	0	0	4	5	0	0	0	3
Abnormal contents, Blue	0	0	0	0	0	1	0	2
Number of tissues examined	10	10	10	9	9	9	10	10
Trachea
Abnormal colour, Blue	0	0	0	0	0	0	0	1
Number of tissues examined	10	10	10	9	9	9	10	10
Testes
Abnormal colour, Dark	0	0	0	2	-	-	-	-
Abnormal colour, Blue	0	0	0	2	-	-	-	-
Number of tissues examined	10	10	10	9	-	-	-	-
Thymus
Abnormal colour, Dark	0	0	0	1	0	0	0	3
Abnormal colour, Blue	0	0	0	0	0	0	0	2
Number of tissues examined	10	10	10	9	9	9	10	10
Urinary Bladder
Abnormal colour, Dark	0	0	0	0	0	0	0	2
Abnormal colour, Blue	0	0	0	0	0	0	0	4
Number of tissues examined	10	10	10	9	9	9	10	10
Uterus
Abnormal colour, Dark	-	-	-	-	0	0	0	5
Abnormal colour, Blue	-	-	-	-	0	0	0	2
Number of tissues examined	-	-	-	-	9	9	10	10
Uterine Cervix
Abnormal colour, Blue	-	-	-	-	0	0	0	1
Number of tissues examined	-	-	-	-	9	9	10	10
Vagina
Abnormal colour, Dark	-	-	-	-	0	0	0	4
Abnormal colour, Blue	-	-	-	-	0	0	0	1
Number of tissues examined	-	-	-	-	9	9	10	10
General Comments
Fur stained, Dark	0	0	0	0	0	0	0	1
Tail stained, Dark	0	0	0	9	0	0	0	0
Tail stained, Blue	0	0	0	0	0	0	0	1
Number of tissues examined	10	10	10	9	9	9	10	10

 

Summary of treatment related findings in the kidney for animals killed after 5 weeks of treatment (males) or on Day 14 of lactation (females)

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Increased Cortical Tubular Pigment
Minimal	0	0	0	0	0	0	0	6
Total	0	0	0	0	0	0	0	6
Number of tissues examined	5	2	10	9	6	4	7	10

Decedent Control Female No. 67 is included in this incidence table

 

Summary of incidental findings in the kidney after 5 weeks of treatment (males) or on Day 14 of lactation (females)

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Mineralisation, Cortex
Minimal	0	0	0	0	2	3	2	0
Slight	0	0	0	0	1	0	0	3
Moderate	0	0	0	0	1	0	1	3
Total	0	0	0	0	4	3	3	6
Cortical Tubular Degeneration/Necrosis
Minimal	0	0	0	0	1	2	3	0
Slight	0	0	0	0	2	0	0	5
Moderate	0	0	0	0	0	0	1	1
Total	0	0	0	0	3	2	4	6
Cortical Tubular Dilatation
Minimal	0	0	0	0	3	0	0	4
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	0	3	0	0	5
Number of tissues examined	5	2	10	9	6	4	8	10

Decedent Control Female No. 67 is included in this incidence table

 

Summary of incidental findings in the stomach after 5 weeks of treatment (males) or on Day 14 of lactation (females)

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Mucosal Mineralisation – Glandular Region
Minimal	0	0	-	0	1	0	0	3
Slight	0	0	-	0	1	0	0	2
Total	0	0	-	0	2	0	0	5
Mucosal Haemorrhage – Glandular Region
Minimal	0	0	-	0	3	0	1	0
Slight	0	0	-	0	0	1	3	2
Total	0	0	-	0	3	1	4	2
Mucosal Erosion – Glandular Region
Minimal	0	0	-	0	0	0	0	1
Slight	0	0	-	0	1	0	0	0
Moderate	0	0	-	0	0	0	1	0
Total	0	0	-	0	1	0	1	1
Mucosal Necrosis – Glandular Region
Minimal	0	0	-	0	1	0	2	0
Total	0	0	-	0	1	0	2	0
Number of tissues examined	5	1	0	5	6	1	4	7

Decedent Control Female No. 67 is included in this incidence table

 

Conclusions:

Oral 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.
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 was considered to be 1000 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, 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.

Four animals died prematurely; all deaths were considered incidental and not related to treatment.

There were no signs seen in association with the dosing procedure. At the routine weekly physical examinations the following signs were recorded: dark eyes, tail and pinnae were seen in males, predominantly at 1000 mg/kg/day, and dark eyes and dark colouration of the whole body were seen in females receiving 1000 mg/kg/day. On the day of the scheduled kill, all females receiving 1000 mg/kg/day were hunched and had piloerection.

The behaviour of the animals in the arena and their sensory reactivity and grip strength responses were all unaffected by treatment.

The assessment of motor activity scores indicated that females receiving 1000 mg/kg/day were slightly less active than the control females.

When compared with the controls, the bodyweight gains of all treated female groups in the pre-pairing period were high and, during the lactation phase, females receiving 330 or 1000 mg/kg/day had high weight gain in the first week but low weight gain in the second week. None of these variations in body weight were considered adverse. Male body weight gains were not affected by treatment.

The food consumption of males receiving the substance at all doses was slightly higher than controls in all weeks of treatment with a similar effect seen in females, but only prior to pairing.   These minor variations in food consumption were considered non-adverse. 

Estrous cyclicity was unaffected by the administration of the substance at all dose levels. 

Haematological investigations revealed slightly low haematocrit and haemoglobin concentration in males receiving 1000 mg/kg/day., high neutrophil and low lymphocyte counts in females receiving 1000 mg/kg/day. The biochemical examination of the blood plasma revealeda slight decrease in alanine amino-transferase activity and a slight increase in triglyceride concentrations in males receiving 1000 mg/kg/day. Aspartate amino-transferase activity was high for females receiving 1000 mg/kg/day. Albumin : globulin ratios were slightly low in all Direct Blue treated female groups, and in the high dose animals this associated with a reduction in plasma albumin concentration. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

High kidney weights were seen in animals given 1000 mg/kg/day.

At the macroscopic examination of the adult animals the predominant findings were abnormal colouration (dark/blue) of internal tissues/organs and/or abnormal contents of the gastro-intestinal tract which were reported in the majority of animals given 1000 mg/kg/day and in some males given 330 mg/kg/day. However, abnormal colouration of the kidneys was seen in all males and females given 330 or 1000 mg/kg/day and also in a few animals given 100 mg/kg/day and dark livers were only seen in females given 1000 mg/kg/day.

At the microscopic examination of the adult animals changes related to treatment with the substance were seen in the kidney. An increase of a brown coloured pigment was seen in the cortical tubules of 6/10 females treated at 1000 mg/kg/day. This pigment was identified as lipofuscin together with test item pigment. The finding was considered not adverse.

Oral 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.

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 was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: no adverse effects observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information and in absence of any adverse effects, the substance does not need to be classified according to EC No 1272/2008 (CLP Regulation).