Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Model considered reiliable by OECD. Read-across from experimental results for the same organic structure. This substance has an identical structure of CAS 1195028-55-7 in respect of the anionic components, but containing Na cations instead of a mixture of Na+ and Li+.

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: PAL-022588
Orange coloured powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Wistar Han™:RccHan™:WIST strain rats

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for four hours. Formulations were therefore prepared daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for four hours. Formulations were therefore prepared daily.
Duration of treatment / exposure:
42 days (including 15 days before mating)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: nominal conc.
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
other: nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
other: nominal conc.
Remarks:
Doses / Concentrations:
750 mg/kg bw/day
Basis:
other: nominal conc.
No. of animals per sex per dose:
12 males per dose and 12 females per dose including control group (total 48 males and 48 females)
Control animals:
yes
Details on study design:
) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Examinations

Observations and examinations performed and frequency:
1. Clinical observations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

2. Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

2.1.Behavioural Assessments (Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation)
2.2. Functional Performance tests: Motor activiyt, Forelimb/Hindlimb Grip Strength
2.3. Sensory Reactivity ( Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch Blink reflex, Tail pinch Startle reflex, Finger approach)

3. Body Weight: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

4. Food Consumption: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

5. Water consumption: daily

6. Reproduction screening:
6.1.Mating behaviour
6.2. Pregnancy and Parturition
6.3 Litter data including number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on days 1 and 4 post partum, clinical condition of offspring from birth to day 5 post partum, individual offspring weights on days 1 and 4 post partum;
6. 4 Physical Development of offspring



Sacrifice and pathology:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals Prostate, Brain Seminal vesicles, Epididymides Spleen, Heart Testes, Kidneys Thymus, Liver Thyroid (weighed post-fixation with Parathyroid), Ovaries Uterus (weighed with Cervix), Pituitary (post fixation)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining anmals:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus,Caecum Rectum,Coagulating gland Salivary glands (submaxillary),Colon Sciatic nerve,Duodenum, Seminal vesicles, Epididymides , Skin (hind limb), Eyes* , Spinal cord (cervical, mid-thoracic and Gross lesions lumbar),Heart Spleen,Ileum (including peyer’s patches) Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea,Liver Testes, Lungs (with bronchi) # , Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder,Mammary gland, Uterus/Cervix, Muscle (skeletal), Vagina.

Tissues were despatched to the histology processing Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU, UK) for processing (Principal Investigator: N Fower). The tissues from five selected control and 750 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 750 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 750 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Other examinations:
Laboratory investigations: Haematology and Blood analysis
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and StatisticsModule

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths which were related to test item toxicity.One female treated with 300 mg/kg bw/day was killed in extremis on Day 8 following the presence of an open wound to the left forelimb. One male treated with 750 mg/kg bw/day was killed in extremis on Day 15 following signs of emaciation. Isolated instances of noisy respiration, pilo-erection and hunched posture were noted for males treated with 750 and 300 mg/kg bw/day. Isolated instances of noisy respiration were also noted for three females treated with 750 mg/kg bw/day and also for females treated with 300 mg/kg bw/day. Isolated instances were also evident at 30 mg/kg bw/day. Orange stained bedding and faeces were also recorded for animals treated with 750 and 300 mg/kg bw/day from Day 7 onwards. This was attributable to the coloured test item formulations and did not represent an adverse effect of treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths which were related to test item toxicity.One female treated with 300 mg/kg bw/day was killed in extremis on Day 8 following the presence of an open wound to the left forelimb. One male treated with 750 mg/kg bw/day was killed in extremis on Day 15 following signs of emaciation. Isolated instances of noisy respiration, pilo-erection and hunched posture were noted for males treated with 750 and 300 mg/kg bw/day. Isolated instances of noisy respiration were also noted for three females treated with 750 mg/kg bw/day and also for females treated with 300 mg/kg bw/day. Isolated instances were also evident at 30 mg/kg bw/day. Orange stained bedding and faeces were also recorded for animals treated with 750 and 300 mg/kg bw/day from Day 7 onwards. This was attributable to the coloured test item formulations and did not represent an adverse effect of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 750 mg/kg bw/day compared to controls: Lower overall body weight gains were detected. Females treated with 750 mg/kg bw/day: body weight losses were evident during Week 1 and reduced body weight gains were evident during gestation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males (750 mg/kg bw/day): reduction in dietary intake noted. Females (750 mg/kg bw/day): reduction in dietary intake evident during 2-week prepairing period and first 2 week of gestation. No effects during final week of gestation or during lactation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males (750 mg/kg bw/d): reduced during weeks 1 and 2. Females (750 mg/kg bw/d) reduced during week 1
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily measurement of water bottles revealed an increase in water intake for males treated with 750 mg/kg bw/day during the treatment period, and for females treated with 750 mg/kg bw/day during the pre-pairing, gestation and lactation phases of the study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males 750 mg/kg bw/d: reduction in haemoglobin, erythrocyte and haematocrit counts, with increases in mean cell haemoglobin and mean cell volume, when compared to controls. Males 300 mg/kg bw/d: increases in mean cell haemoglobin and mean cell volume.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A reduction in total protein and an increase in albumin/globulin ratio was detected for males treated with 750 mg/kg bw/day when compared to controls
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects were detected
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 750 and 300 mg/kg bw/day showed an increase in kidney weights, both absolute and relative to terminal body weights. There were no further organ weight changes considered to be related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant macroscopic abnormalities detected.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The read-across study supplied supports the feasibility of the approach: see report in the summary endpoint.

Applicant's summary and conclusion

Conclusions:
The oral administration of Direct Orange 39 (C.I. 40215) to rats by gavage, at dose levels of 750, 300 and 30 mg/kg bw/day, resulted in treatment-related changes at 750 and 300 mg/kg bw/day. Effects at 300 mg/kg bw/day were considered not to represent an adverse effect. Therefore a ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day.
Executive summary:

The oral administration of Direct Orange 39 (C.I. 40215) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation period for females) at dose levels of 750, 300 and 30 mg/kg bw/day resulted in treatment-related changes at 750 and 300 mg/kg bw/day.

There were three unscheduled deaths during the study. One death was due to an open wound and therefore unrelated to treatment with the test item. One animal was found dead and another was killed in extremis due to emaciation. Histopathological examinations showed the deaths of these two animals to be attributable to inappropriate dosing technique rather than a toxic effect of treatment. The instances of noisy respiration were also considered to be due to dosing technique rather than a toxic effect of treatment.

Males treated with 750 mg/kg bw/day showed a reduction in haemoglobin, erythrocytes and haematocrit counts, with corresponding increases in mean cell haemoglobin and mean cell volume. Increases in mean cell volume and mean cell haemoglobin were also

higher for males treated with 300 mg/kg bw/day when compared to controls. These findings are consistent with an anaemic response, however, there were no differences in reticulocyte counts and in the absence of extramedullary haemopoiesis observed following histopathological of the spleen and/or bone marrow, this finding was considered not to represent an adverse effect of treatment.

Blood chemical analysis revealed a reduction in total protein level and an increase in A/G ratio when compared to controls for males treated at the highest dose level. In the absence of any structural defects in systemic organs, it is likely that this change may be of limited toxicological significance and related to metabolic stimulation.

An increase in water intake was evident for animals of either sex treated with 750 mg/kg bw/day and organ weight assessments revealed an increase in kidney weights, both absolute and relative to terminal body weights, for animals of either sex treated with

750 and 300 mg/kg bw/day. In the absence of any histopathological renal changes, these increases were considered not to represent an adverse effect of treatment.

Furthermore, an increase in absolute and body weight-relative liver weights when compared to control values, was evident for animals of either sex treated with 750 mg/kg bw/day and for females treated with 300 mg/kg bw/day. The increases in liver weights were considered to represent and adaptive response to test item administration and in the absence of any histopathological correlates, these increases were considered not to represent an adverse health effect.

The most significant findings detected during the study were the lower overall body weight gains and a reduction in dietary intake detected for males treated with the highest dose level when compared to controls. Actual body weight losses were also evident for females treated with 750 mg/kg bw/day, together with a reduction in dietary intake, with reductions in body weight gains detected during gestation when compared to controls. Reduced dietary intake was also detected during the first two weeks of gestation when compared to controls. Slightly smaller litter sizes were evident at 750 mg/kg bw/day when compared to controls. Sex ratio or offspring viability was not affected. Offspring of slightly lower bodyweights were evident for females treated with 750 mg/kg bw/day when compared to controls. Slightly lower numbers of corpora lutea and implantation sites were noted for females treated with 750 mg/kg bw/day when compared to controls. Slightly higher pre- and post- implantation losses were also evident at 750 mg/kg bw/day

when compared to controls. Furthermore, slightly longer gestation lengths were evident at the highest dose level when compared to controls. These intergroup differences were most likely attributable to maternal stress. The reduced bodyweight gains and dietary intake observed at the highest dose level, and did not represent true reproductive toxicity. Due to the effect on litter size, the reductions in body weight gains and dietary intake at 750 mg/kg bw/day were considered to represent an adverse effect of treatment

and contributed to the minor fluctuations in litter values. The lack of specificity of reproductive effect was concluded to be more likely to be a consequence of an impaired health of adult females that contributed to slightly reduced reproductive performance.