1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

Endpoint summary

Description of key information

NOAEL = 1000 mg/kg bw/day (OECD 422, GLP, K, rel.1)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Pre-dose trial): 11 April 2017 Experimental completion date: 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed in compliance with OECD TG 422

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Remarks:

No deviations occured that were considered to have affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: n/a
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the 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.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
- Preliminary purification step (if any): n/a
- Final concentration of a dissolved solid, stock liquid or gel: 20, 60 and 200 mg/mL (volume dose 5 mL/kg)

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

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:yes
- Age at study initiation: Males x 71-78; Females x 85-92 days
- Weight at study initiation: Males: 325-415 g; Females: 240-293
- Fasting period before study: none
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage:
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
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum. Bottles were changed at appropriate intervals. Certificates of analysis were routinely provided by the water supplier.
- Acclimation period: Males: 8 days prior to the commencement of treatment. Females: 22 days prior to the commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): n/a. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 June 2017 To: 20 August 2017

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen as it is the preferred route for human risk assessment.
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Volume dose -5 mL/kg body weight.
Individual dose volume - Calculated from the most recently recorded scheduled body weight.
Control (Group 1) - Vehicle at the same volume dose as treated groups.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the 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.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation:
Refrigerated (2 to 8°C) for up to 15 days.
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
The homogeneity and stability was confirmed for Macrolex Grün G in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days.

Achieved concentration
Samples of each formulation prepared for administration in Week 1 (for each treatment group) and on Day 10-12 of lactation (females only) were analyzed for achieved concentration of the test item.
The mean concentrations of Macrolex Grün G in test formulations analyzed for the study were within 3% of nominal concentrations, confirming accurate formulation. The % difference between the samples was within 3%, confirming precise analysis.
Procedural recoveries prepared during the trial and routine occasions were within ±7.5% of the mean found value in the validation, confirming the continued accuracy of the method.

Duration of treatment / exposure:

Males - Two weeks before pairing up to necropsy after minimum of five weeks.
Females - Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected, following the completion of the preliminary study Envigo Study Number: KQ40WR and after consultation with the Sponsor. In that study there were no effects on clinical condition, body weights, food consumption, organ weights or macropathology at doses of 500, 800 or 1000 mg/kg/day.
Therefore, the high dose level for this study was 1000 mg/kg/day with the intermediate and low dose levels (300 and 100 mg/kg/day, respectively) chosen to allow the determination of a dose response.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s)
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
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 observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS AND ARENA OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and
20 after mating and Days 1, 6 and 12 of lactation.
On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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.
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.

WATER CONSUMPTION AND COMPOUND INTAKE: No

SENSORY REACTIVITY AND GRIP STRENGH
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.
The following measurements, reflexes and responses were recorded:
- Approach response
- Pinna reflex
- Auditory startle reflex
- Tail pinch response
- Grip strength

MOTOR ACTIVITY
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).
Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

OESTROUS CYCLICITY
Dry and wet smears were taken as follows:
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 (nominally Days 11-14 of lactation).

SPERM PARAMETERS
During necropsy, for the assessment of the testes, a detailed qualitative examination 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.

Sacrifice and pathology:

Method of Kill
All adult animals: Carbon dioxide asphyxiation.
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. A detailed assessment of any color change in the internal organs, adipose tissue or skin was documented.

Time of Necropsy
F0 males: Week 6.
F0 females: Day 14 of lactation (following terminal blood sampling).
F1 offspring:
Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed in tables in the section 'any other information on materials and methods' for F0 animals.

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection.

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed.

F1 offspring on Day 4 of age: Blood sampling required.
Subjected to an external macroscopic examination; particular attention was paid to the external genitalia.
Externally abnormal offspring examined, and retained pending possible future examination.

F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination
Thyroid glands were preserved from one male and one female in each litter.


Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - Initially in modified Davidson’s fluid.
Eyes - In Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: The five lowest surviving F0 males and the first five surviving lactating females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined as follows:
Scheduled kill:
Groups 1 and 4: Five lowest surviving F0 males and the first five surviving lactating females - All specified in the table in section 'any othe information on materials and methods'
All remaining F0 animals - Tissue with abnormalities only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination 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.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Optional endpoint(s):

Optional endpoints: Yes (combined study - Cf. IUCLID section) 7.8.1)

Other examinations:

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasion:

At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
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)

* Derived value calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.


Blood Chemistry
Blood samples were collected after overnight withdrawal of food at the following occasion:

At termination: The five lowest numbered surviving males per group.
The first five surviving lactating females in each group.


Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
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)
Globulin (Glob)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination: All surviving adult males and females.
Day 4 of age: F1 offspring, two females per litter (where possible, ensuring that the number of female offspring did not fall below three).
No pups were allocated to these procedures if the resultant live litter size would fall below ten pups/litter.
- one for T4 (serum)#
- one for TSH (plasma)
# priority 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 given to serum sample

Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Anesthetic: F0 animals: Isoflurane
F1 offspring : None
Blood sample site: F0 adults: Sublingual vein
F1 offspring : Decapitation
Anticoagulant:
Plasma samples: K2EDTA. Standard tubes used for collection of samples did not contain separator gel.
Serum samples: None (Greiner Minicollect tubes with clotting activators)
Blood volume:
Adults: 2 x 0.5 mL.
Offspring: maximum possible.

Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.
Serum samples: Samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C.
Number of aliquots per sample: All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes.
Final storage conditions: Deep frozen (-60 to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Envigo.
Thyroid hormone analysis Performed by the Department of Bioanalysis, Envigo.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no signs were observed in association with dose administration.
Clinical signs observed at routine examination comprised several incidences of vocalisation and irritable behaviour in males and females at all dose levels, including Controls. One female receiving 1000 mg/kg/day was recorded to have a twisted muzzle on Day 14 of lactation. No test item-related changes in general clinical condition were observed following treatment with Macrolex Grün G.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values (control 111 g, dosed 102-105 g). This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1 following commencement of treatment (control 30 g, 1000 mg/kg 23 g), with statistical significance observed at 1000 mg/kg/day (p=<0.01).

During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration with Macrolex Grün G. For females receiving 300 mg/kg/day, a non-significant reduction in body weight gain was observed between Weeks 0-1 (4 g dosed v. 8 g control, 50% lower), and resolved during weeks 1-2 (14 g dosed v. 9 g control, 55% higher). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a greater body weight gain between weeks 0-1, and a decrease in body weight gain between Weeks 1-2 (5 g v. 9 g, 45% lower). Mean body weight gain was unaffected when the whole premating period (Weeks 0-2) was considered.

During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), although no statistical significance was observed (138 g 1000 mg/kg bw/day v. 154 g control).

Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment.
For females receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day, food consumption values were similar to those of the Controls throughout the treatment period, gestation and lactation phases.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination of peripheral blood performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly low neutrophil and eosinophil concentrations in males receiving 100, 300 or 1000 mg/kg/day, low prothrombin times in males at 100, 300 and 1000 mg/kg/day, high platelet count in males at 1000 mg/kg/day and high reticulocyte count in females at 100, 300 and 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of plasma performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly, but statistically significantly low alkaline phosphatase in males at 300 and 1000 mg/kg/day and alanine aminotransferase in males at 100, 300 and 1000 mg/kg/day and the slightly, but statistically significantly high cholesterol levels in females at 1000 mg/kg/day. Low creatinine concentration was evident in males at 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The evaluation of organ weights of males killed after 5 weeks of treatment and of females killed on Day 13 of lactation revealed marginally low absolute and adjusted thymus weights in females treated at 100 and 1000 mg/kg/day, as well as in males given 1000 mg/kg/day. No microscopic correlates were identified, therefore these differences are of doubtful relationship to treatment. No other treatment-related changes in organ weights were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 5 weeks of treatment revealed the following changes in the caecum, colon, ileum, jejunum, rectum, stomach and general comments.

Gastrointestinal Tract
Green content, representing the colour of the test item, was seen in the caecum, colon, ileum, jejunum, rectum and stomach in treated animals with a dose dependent increase in incidence.

Stomach
Green colour of the stomach was seen in treated animals with a dose dependent increase in incidence.

General comments
Green staining of the fur was seen in males treated at 1000 mg/kg/day and in one female treated at 100 mg/kg/day.
Green staining of the tail was seen in males treated at 1000 mg/kg/day and females treated at 100, 300 and 1000 mg/kg/day.

Summary tables of the findings can be found in section 'any other information on results'

The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment Related Findings
There were no treatment related changes.

Incidental Findings
Mineralisation was seen in the kidneys (cortex, sometimes correlated with macroscopically pale kidneys) and the glandular mucosa of the stomach of control and treated females. In the stomach; minimal mineralisation was seen in controls and treated females at a similar incidence and was considered unrelated to treatment. In the kidneys; histopathological findings associated with the observed distribution of mineralisation included minimal to slight degeneration/necrosis and minimal dilatation of cortical tubules. The incidence and/or severity of the kidney mineralisation and associated findings appeared slightly increased in treated females compared with controls.
A compilation of background control data (BCD) from similar recent studies was performed for the all above findings. In this BCD, mineralisation of the kidney cortex and cortical tubular degeneration/necrosis each have an incidence range of 0 – 100% and a severity range of minimal to moderate. Cortical tubular dilatation has a BCD incidence range of 0 – 83.3%, with a severity range of minimal to moderate. Mineralisation of the glandular stomach mucosa has a BCD incidence range of 0 – 60%, with a severity range of minimal to moderate. All these findings within the current study are well within these ranges.
Therefore, the discussed findings are considered to be incidental, with the apparent slight increase in treated females due to chance and not related to treatment.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

Sensory Reactivity Observations and Grip Strength
The sensory reactivity observations conducted during Week 5 of treatment for males and Day 7-9 of lactation for females revealed no findings which were considered treatment related in animals receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day.

Motor Activity
The motor activity assessment conducted during Week 5 of treatment revealed no test-item related effects in male or female animals receiving Macrolex Grün G at all dose levels. It was noted that in male animals receiving 1000 mg/kg/day, a lower than Control statistical significance was seen in the low beam break at the 6 minute testing point, as well as a higher than Control statistical significance seen in the low beam break at 60 minutes. These inconsistent, isolated findings were considered not to be associated with administration of Macrolex Grün G.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Thyroid Hormone Analysis

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

Group	Treatment	Dose		Adult Male at Termination (pg/mL)	Day 13 of age Offspring (pg/mL)
		(mg/kg/day)			Male	Female
1	Corn oil	0	Mean	28500	29700	33400
			SD	9340	6580	13400
			CV	32.8	22.2	40.1
			N	10	10	10
2	Macrolex Grün G	100	Mean	27600	37700	37000
			SD	11000	7420	5300
			CV	39.9	19.7	14.3
			N	10	10	10
3	Macrolex Grün G	300	Mean	26700	36000	38500
			SD	12500	6540	7660
			CV	46.8	18.2	19.9
			N	10	10	10
4	Macrolex Grün G	1000	Mean	32000	28800	37400
			SD	8570	5390	7890
			CV	26.8	18.7	21.1
			N	10	10	10

 

Summary of findings in the gastrointestinal tract for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	500	0	100	300	500
Finding	Abnormal Contents, Green
Caecum	0	3	6	9	0	1	1	4
Colon	0	3	5	9	0	0	1	2
Ileum	0	0	1	2	0	0	1	1
Jejunum	0	0	2	3	0	0	0	0
Rectum	0	0	3	6	0	0	0	2
Stomach	0	1	4	4	0	2	2	6
Number of tissues examined	10	10	10	10	10	10	10	10

Summary of findings in the stomach for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Abnormal colour, Green	0	2	5	9	0	8	7	9
Number of tissues examined	10	10	10	10	10	10	10	10

Summary of general comments for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Fur stained, Green	0	0	0	8	0	1	0	0
Tail stained, Green	0	0	0	5	0	1	2	6
Number of tissues examined	10	10	10	10	10	10	10	10

Conclusions:

It was concluded that the oral administration of Macrolex Grün G to parental Sprague-dawley rats at dose levels of 100, 300 or 1000 mg/kg/day administered for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was well tolerated.
Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Macrolex Grün G showed no evidence of being an endocrine disruptor.
The no-observed adverse-effect level (NOAEL) of Macrolex Grün G for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Macrolex Grün G for reproductive/developmental toxicity was also considered to be
1000 mg/kg/day.

Executive summary:

Summary

The purpose of this study was to assess the potential systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Macrolex Grün G, by oral administration for at least five weeks.

Three groups of ten male and ten female rats received Macrolex Grün G at doses of 100, 300 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, corn oil, at the same volume dose as the treated groups.

 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, 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

F0 responses

Administration with Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to generally be well tolerated throughout the treatment period. There were no premature deaths, no dosing signs and no clinical signs observed in association with dose.

 

For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values (control 111 g, dosed 102-105 g). This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1 following commencement of treatment (control 30 g, 1000 mg/kg 23 g), with statistical significance observed at 1000 mg/kg/day (p=<0.01).

 

During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration with Macrolex Grün G. For females receiving 300 mg/kg/day, a non-significant reduction in body weight gain was observed between Weeks 0-1 (4 g dosed v. 8 g control, 50% lower), and resolved during weeks 1-2 (14 g dosed v. 9 g control, 55% higher). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a greater body weight gain between weeks 0-1, and a decrease in body weight gain between Weeks 1-2 (5 g v. 9 g, 45% lower). Mean body weight gain was unaffected when the whole premating period (Weeks 0-2) was considered.

 

During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), although no statistical significance was observed (138 g 1000 mg/kg bw/day v. 154 g control).

 

Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.

 

Estrous cyclicity, pre-coital interval, gestation length, mating performance, fertility and gestation index were unaffected by treatment.

 

The hematological examination of blood and the biochemical examination of plasma did not reveal any conclusive effect of treatment and there was no effect upon circulating levels of thyroxine (T4) in adult males.

 

Macroscopic findings were limited to green content, representing the colour of the test item, seen in the caecum, colon, ileum, jejunum, rectum and stomach in treated animals with a dose dependent increase in incidence and green colour of the stomach seen in treated animals with a dose dependent increase in incidence. Histopathological findings in the full range of tissues examined were considered to be unrelated to treatment. Organ weights were considered unaffected by treatment.

 

Applicant’s Discussion

A review of findings following conduct of an OECD 422 study with Macrolex Grün G in Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day allows the clear conclusion to be reached of no adverse outomes. For those parameters in which effects were observed, namely bodyweight gain changes, thymus weight, and kidney mineralisation, none may be considered as clearly treatment-related. Bodyweight gain changes were transient in nature and without any clear indications of associated adversity. Changes in the weight of thymus lacked a clear dose-response in female animals, were not significant in either sex, and were without microscopic correlate following histopathological exam. As such, they are not considered to be a treatment-related adverse effect. Furthermore, mineralisation described in the kidney was considered to be incidental following review of historical control data. Finally, the colour changes described during gross pathological evaluation are clearly relatable to the passage of Macrolex Grün G through the gastrointestinal tract, alongside staining of the tail which is very likely to be due to contamination from fecal matter. There is no supporting evidence from this or any other study findings to indicate that the test item was absorbed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP-compliant and of high quality (Klimisch score = 1)

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

Additional information

In an OECD guideline 422 study (Combined Repeated Dose Toxicity Study and Reproductive/ Developmental Toxicity Screening Study in the Crl:CD(SD) Rat by Oral Gavage Administration) Solvent Green 28 (CAS 4851-50-7) was administered to three groups of ten male and ten female rats by oral gavage administration, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day.  A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups.

The no-observed adverse-effect level (NOAEL) of Solvent Green 28 for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Solvent Green 28 for reproductive/developmental toxicity was also considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification:

In a subacute study with Solvent Green 28 (CAS 4851-50-7) a NOAEL of 1000 mg/kg bw/day was found for male and female rats. 

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.