Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Dec 2010 to 29 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Acid Yellow RN 2903
IUPAC Name:
Acid Yellow RN 2903
Details on test material:
Identification: Acid Yellow RN 2903
Description: Yellow to orange powder
Batch Number: Vers. Kilo 6, 26.05.2009
Purity: 65.7 %
Purity Correction: Yes
Correction Factor for the Purity: 1.522
Stability of Test Item: Stable under storage conditions
Stability of Test Item Dilution: Stable in water for 4 days
Expiry Date: 01-Aug-2015
Storage Conditions: At room temperature (20 ± 5 °C), away from direct sunlight.
Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals:
Group 1: 10 males and 10 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 10 males and 10 females
Total Number of Animals Used: 30 males and 30 females
Total Number of Animals Ordered: 32 males and 32 females (including reserve animals)
Age (at Delivery): Ca. 7 weeks
Body Weight Range (at Acclimatization): Males: 186 to 214 g , Females: 144 to 166 g
Identification:
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Randomly allocated to groups by body weight.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ACCOMODATION:
In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch 72.

DIET:
Pelleted standard Harlan Teklad 2914C (batch no. 39/10) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants. Results are included in the report.

WATER:
Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of respective samples are included in the report.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
bidistilled
Details on oral exposure:
PREPARATION OF DOSE FORMULATIONS:
The dose formulations were corrected to 100% according to its active ingredient purity of 65.7%.

The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study D04538), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

Acid Yellow RN 2903 was weighed into a glass beaker on a tared Mettler balance. A part of the vehicle was added and the mixture was homogenized with an Ultra Turrax. Thereafter, the remaining vehicle was added to give the appropriate final concentration of the test item in the dilution. The mixtures were then stirred on a magnetic stirrer and stored in aliquots for each treatment day at room temperature (20  5 °C).

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

TREATMENT:
Frequency of Administration: Daily
Dose Levels (active ingredient):
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day
Dose Volume: 10 mL/kg body weight for aqueous vehicles
Dose Concentrations (active ingredient):
Group 1: 0 mg/mL/day
Group 2: 10 mg/mL/day
Group 3: 30 mg/mL/day
Group 4: 100 mg/mL/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories Study D04538 (non-GLP).
Duration of Pre-Randomization Period: 1 day
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: 28 days
Duration of Recovery Period: 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a method provided by the Sponsor. The test item was used as analytical standard.

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at room temperature and stored there at -20 ± 5 °C until analysis.

Concentration, homogeneity and stability (8 d) of dose formulations were determined in samples taken after experimental start and during week 3 of treatment.
The following acceptance criteria were applied to analytical results: sample contents had to be within a range of ±20% of nominal content. Formulations were considered homogeneous if the maximum deviation from mean calculated from top, middle and bottom samples was not more than 15%. The results obtained from storage stability samples had not to deviate more than 10% from time-zero reference (content or mean of homogeneity samples).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
dose levels of 100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
MAIN STUDY:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 100 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg / day
RECOVERY:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg / day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories Study D04538 (non-GLP).

- Post-exposure recovery period in satellite groups:
yes; 0 mg/kg/day and 1000 mg/kg/day
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Viability / Mortality: Twice daily
Clinical Signs:
- Acclimatization Period: Once daily
- Treatment Period: Twice daily on days 1 to 3; once daily thereafter
- Recovery Period: Once daily
Detailed Behavioral Observations:
- Acclimatization Period: Once weekly
- Treatment Period: Once weekly (during weeks 1-3; in week 4 included in FOB)
- Recovery Period: Not performed during recovery
Food Consumption:
- Acclimatization Period: Weekly
- Treatment Period: Weekly
- Recovery Period: Weekly
Body Weights:
- Pre-Randomization: Once
- Acclimatization Period: Once weekly
- Treatment Period: Once weekly
- Recovery Period: Once weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period (Day 28) / at the end of recovery.
- Anaesthetic used for blood collection: Isofluran
- Animals fasted: yes
- How many animals: Five males and five females per group
Routine haematological parameters were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: Five males and five females per group
Routine biochemical parameters were examined.

Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

URINALYSIS: Yes
- Time schedule for collection of urine: see above
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Routine parameters were examined

Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.


NEUROBEHAVIOURAL EXAMINATION: Yes (see tables)
FOB = Functional observational battery (including grip strength and locomotor activity):during week 4 of treatment
- Battery of functions tested: sensory activity / grip strength / motor activity

Behavioural Assessments were undertaken for all animals at weekly intervals throughout the study. Motor Activity, Forelimb/Hindlimb Grip Strength and Sensory Reactivity were undertaken for all animals during the final week of treatment.

Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see tables)
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Samples of the tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution.

ORGAN WEIGHTS: Yes (see tables)
The organs from all animals were weighed before fixation on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

HISTOPATHOLOGY: Yes (see tables)
All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. As test item-related morphologic changes were detected in the spleen and test item pigments were found in the lung of high-dose animals, the same organs from male and female animals of the mid- and low-dose groups were examined.

Attempts were made to correlate gross observations with microscopic findings.

A peer review was performed by Dr. K. Weber. The findings of the study pathologist and the peer-reviewing pathologist compared favorably. The peer review report is retained in the raw data.

All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of lung and spleen.
Other examinations:
Thyroid hormones (T3, T4 and TSH) were not evaluated as no changes in the absolute and/or relative organ weights of the pituitary and/or thyroid gland (in conjunction with changes in liver weights) were noted at necropsy.
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increase in reticulocyte count noted in males at 300 and 1000 mg/kg/day and increased methemoglobin concentrations in males at 100, 300 and 1000 mg/kg/day after four weeks of treatment. Methemoglobin decreased to values comparab
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant changes in clinical biochemistry parameters indicating effects on liver and / or kidney function were noted. These changes included increased urea and creatinine levels in males at 300 and 1000 mg/kg/day. As not further findings
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopically, increased extramedullary hematopoiesis was noted in males at 300 and 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Details on results:
Analysis of Dose Formulations:
Absence of Acid Yellow RN 2903 in the vehicle was demonstrated. The application formulations during the study were found to comprise the test item in the range of 92.6% to 102.7% and were, therefore, within the range of acceptance criteria (20%) of nominal content. Homogeneity of the formulations was also approved as the maximum deviation from the corresponding mean value was not more than 4.7% (accepted range up to 15%). The stability of dose formulations for a period of 8 days at room temperature was confirmed with a maximum variation of 2.2% based on values within 10% of mean recovery.
The results indicate the accurate use of the test item Acid Yellow RN 2903 and the vehicle (bidistilled water) during this study.

Viability / Mortality
All animals survived the scheduled treatment and recovery periods.

Clinical Signs
No clinical signs were noted in all animals during treatment and recovery.


Detailed Behavioral Observations (Weekly)
No abnormalities were recorded in the detailed behavioral observations in all animals during the treatment period.

Functional Observational Battery
No test item-related findings were evident at the functional observational battery performed during the fourth week of treatment.
Grip Strength : There were no differences in the mean fore and hind limb grip strength in all treated animals compared to control.
Locomotor Activity: No test item-related differences in locomotor activity were noted between the groups.
Increased locomotor activity in females at 100 mg/kg/day from 10 to 20 minutes were considered to be incidental as no dose respons relationship was noted and only one measurement interval was affected.

Food Consumption
No test item-related effects on absolute or relative food consumption were noted.
Slightly increased food consumption in females at 300 mg/kg/day during the treatment phase was considered to be incidental as there was no dose response-relationship.

Body Weights
No test item-related differences in body weight or body weight gain were noted between the groups during treatment and recovery.
Increased body weight gain in females treated with 300 mg/kg/day was considerd to be in correlation with increased food consumption and therefore, also to be incidental.

Clinical Laboratory Investigations
Hematology:
The following statistically significant differences from controls were noted at the end of treatment and considered to be test item-related:
• Increased relative and absolute reticulocyte count in males at 300 and 1000 mg/kg/day (p<0.05 to p<0.01)
• Increased methemoglobin levels in males at 100, 300 and 1000 mg/kg/day (p<0.05 at 100 and 300 mg/kg/day, p<0.01 at 1000 mg/k g/day)
All changes reversed during the treatment-free recovery period.
Other slight differences, even when attaining statistical significance, were considered not to be test item-related as there was not dose response-relationship and the values were within the range of historical data.

Clinical Biochemistry:
The following, generally statistically significant differences from controls were noted and considered to be test item-related:
• Increased urea and creatinine levels in males at 300 and 1000 mg/kg/day (p<0.05 to p<0.01) after four weeks of treatment; no statistically significant differences after recovery
• Decreased phospholipid levels in males at 1000 mg/kg/day (p<0.05); however, similarly low levels also in males at 300 mg/kg/day but without statistical significance; after recovery, higher levels in control and high dose males were noted, but no statistical significance between controls and high dose
• Increased aspartate aminotransferase (ASAT) levels in males at 1000 mg/kg/day (p<0.05); after recovery, levels in high dose males were similar to levels after four weeks of treatment, but as control levels were also higher, the difference achieved no statistical significance
• Decreased total bilirubin levels in females at 100, 300 and 1000 mg/kg/day (all p<0.05); after recovery, levels in high dose females were similarly low, but as control levels were also lower, the difference achieved no statistical significance
• Decreased alkaline phosphatase (ALP) levels in females at 1000 mg/kg/day after four weeks of treatment (p<0.01) and after two weeks of recovery (p<0.05)
• Increased albumin levels in females at 100, 300 and 1000 mg/kg/day, mostly attaining statistical significance in correlation with significantly decreased globulin levels in females at 1000 mg/kg/day after four weeks of treatment. Furthermore, albumin/globulin ratio increased in females at 100, 300 and 1000 mg/kg/day; after recovery, no statistical difference was noted
Other slight differences, even when attaining statistical significance, were considered not to be test item-related as there was not dose response-relationship and the values were within the range of historical data.

Urinalysis:
Concerning urinalysis parameters, no differences between the groups were noted.

Pathology
Organ Weights:
There were no test item-related changes in the organ weights after treatment and recovery.
Differences in absolute and relative liver weights in females, even when attaining statistical significance, were considered not to be related to treatment with the test item, as there was no dose response relationship and no correlation with macroscopic or microscopic findings.
In females treated with 1000 mg/kg/day, significantly increased uterus weights were noted. In one female of this group, a nodule at the cervix was observed macroscopically resulting in increased organ weight. Therefore, this finding is considered to be incidental.
Significantly increased kidney weights were noted in females at 300 mg/kg/day and increased kidney/body weight ratio was noted in females at 1000 mg/kg/day. As there were no correlating microscopic findings, no changes in clinical biochemistry in the females and no urinary changes, this was considered not to be test item-related.

Macroscopic Findings:
No test item-related macroscopic findings were noted in male and female animals after four weeks of treatment or recovery.
Macroscopic findings recorded in individual males and females of all groups were considered to be within the range of normal background lesions in animals of this strain, sex and age and under conditions of the study.

Microscopic Findings:
Spleen (see tables)
In the main study animals, increased hematopoiesis was observed in males treated with 300 and 1000 mg/kg/day (5/5 = 100% of the males affected). After 2 weeks of recovery, a higher incidence of hematopoiesis was recovered in control males (3/5), whereas the incidence and severity in high dose males and females showed the trend to recover. After recovery, increased hemosiderin deposit was observed in males at 1000 mg/kg/day (4/5) in comparison with lower incidence in control males (1/5).

Lung
Alveolar macrophages which contained yellow pigments were observed in one female at 1000 mg/kg/day after four weeks of treatment and in one male treated with 1000 mg/kg/day in the recovery study. This finding was observed mainly in peri-bronchial/bronchiolar area without any further reaction such as inflammatory cell infiltration, necrosis or degeneration. Similar or related changes or any other changes considered to be treatment-related were not observed in the lung of the other animals. Therefore, this finding is considered to be caused by accidental regurgitation / aspiration of the test item after oral application. It is considered to be of technical nature but not to be a sign of systemic toxicity caused by the test item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Incidence and Mean Severity Grade of Main Findings inSpleen

Finding

Allocation A

Group 1

Allocation A

Group 2

Allocation A

Group 3

Allocation A

Group 4

Incidence /
Mean Severity

5M

5F

5M

5F

5M

5F

5M

5F

Hematopoiesis

1/1.0

2/1.0

2/1.0

3/1.0

5/1.6

3/1.3

5/1.8

4/1.5

Increased Hemosiderin

-

-

-

-

-

-

-

-

Finding

Allocation B

Group 1

 

 

 

 

Allocation B

Group 4

Incidence /
Mean Severity

5M

5F

 

 

 

 

5M

5F

Hematopoiesis

3/1.7

-

 

 

 

 

2/1.5

1/1.0

Increased Hemosiderin

1/1.0

-

 

 

 

 

4/1.0

1/1.0

 

Applicant's summary and conclusion

Conclusions:
Discussion and Conclusion
Oral administration (gavage) of Acid Yellow RN 2903 to Wistar rats at doses of 100, 300 and 1000 mg/kg/day for a period of 28 days was generally well tolerated. Treatment had no effect on survival and there were no changes in daily or weekly observations or in parameters of the functional observational battery (performed during week 4) including grip strength and locomotor activity. Furthermore, no effects on body weights or food consumption were noted.
 
The following test item-related findings were observed:
 
Changes in hematology parameters comprised a slight increase in methemoglobin concentration in males at 100, 300 and 1000 mg/kg/day, only, and an increase in reticulocyte count in males at 300 and 1000 mg/kg/day after four weeks of treatment. Both findings were no longer present at the end of the treatment-free recovery period. Histopathologically, increased hematopoiesis in the spleen was noted in males at 300 and 1000 mg/kg. This is considered a compensatory change due to the effect on hematology parameters. After the recovery period, the hematopoiesis in the spleen was similar in controls and males previously dosed at 1000 mg/kg.
Compared to historical data, increased methemoglobin levels were also noted in control males and females. As a consequence, the use of historical data for this finding was not appropriate, in this study. As the change was mild and in the absence of related findings like Heinz bodies, altered hemoglobin concentration or altered general function (e.g. clinical signs like cyanosis, respiratory changes, decreased activity), it was considered to be of no biological concern, which was further supported by the reversibility during the treatment-free recovery period.

Changes in clinical biochemistry parameters possibly indicating effects on liver and / or kidney function included increased urea and creatinine levels in males at 300 and 1000 mg/kg/day. As no further findings indicating effects on kidney function were noted, this is considered not to be adverse. Furthermore,decreased phospholipid levels in males at 300 and 1000 mg/kg/day, increased aspartate aminotransferase (ASAT) levels in males at 1000 mg/kg/day, decreased bilirubin levels in females at 100, 300 and 1000 mg/kg/day, decreased alkaline phosphatase (ALP) levels in females at 1000 mg/kg/day, increased albumin levels in females at 100, 300 and 1000 mg/kg/day, decreased globulin levels in females at 1000 mg/kg/day, increased albumin/globulin ratio in females at 100, 300 and 1000 mg/kg/day were noted. The changes were generally slight and largely reversible during the recovery period. The only finding after recovery was a slightly decreased ALP, which was considered to be of no toxicological relevance. There were no morphological correlates pointing at the kidney or the liver as target organs.

Histologically, increased extramedullary hematopoiesis was noted in males at 300 and 1000 mg/kg/day after treatment, whereas after the recovery period, an increased incidence of hemosiderin deposits in the spleen of males at 1000 mg/kg was noted. The increased hematopoiesis was considered to be an adaptive response to increased methemoglobin levels (see above) in males. Increased hemosiderin is generally considered not to impair cell or organ function [see also reference (4)] and, in this study, thought to be a secondary effect due to the increased levels of methemoglobin [see also reference (5)]. Aditionally, methemoglobin reversed throughout the recovery period.
 
Based on above mentioned findings and considerations, a no observed effect level (NOEL) for the test item could not be established in male and female animals.
 
The no observed adverse effect level (NOAEL) was established at 1000 mg/kg/day in males and females, although slightly increased methemoglobin levels were noted in males, only, of all dose groups. This change was considered not to be of adverse nature, as the degree was minor and the performance of the whole organism was not impaired. In addition, there was evidence of adaptive responses and the increased methemoglobin levels reversed after cessation of treatment.
For females, changes in clinical biochemistry parameters were noted at all dose levels. However, these changes were largely reversible and were not considered to be adverse.
 
In conclusion, the general function of the organism and the general well-being was not impaired by treatment and all findings were considered to have no biological or clinical relevance[see also references (6 and7)].
References:
4. J.R. Glaister: Principles of Toxicologic Pathology, Taylor & Francis, London and Philadelphia (1986)
5. A.W. Suttie: Histopathology of the Spleen, Toxicologic Pathology vol. 34, pp. 466 – 503, 2006.
6. ECETOC Technical Report 85 “Recognition of, and Differenciation between, Adverse and Non-adverse Effects in Toxicology Studies. European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels, Belgium, 2002.
7. R. W. Lewis at al.: “Recognition of Adverse and Nonadverse effects in Toxicity Studies” TOXICOLOGIC PATHOLOGY, vol 30, no 1, pp 66-74, 2002.
Executive summary:

General

In this subacute toxicity study, Acid Yellow RN 2903 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only.

 

The groups comprised 5 animals per sex and group which were sacrificed after 28 days of treatment. Additional 5 rats per sex were used in the control group and at 1000 mg/kg/day. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

 

Clinical signs, detailed behavioral observations, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery including locomotor activity and grip strength was performed during week 4 of treatment.

At the end of the treatment and recovery period, respectively, blood samples were withdrawn for hematology and plasma biochemistry analyses. Urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, spleen and lung were additionally examined to establish a no-effect level.

  

Mortality / Viability

All animals survived the scheduled treatment and/or recovery periods.

 

Clinical Signs

No clinical signs were noted in all animals during treatment and recovery.

  

Functional Observational Battery

No findings were evident at the functional observational battery performed during the fourth week of treatment.

Grip Strength: There were no differences in the mean fore and hind limb grip strength in all test item-treated animals compared to control.

Locomotor Activity: No differences in locomotor activity were noted between the groups.

  

Food Consumption

No test item-related effects on absolute or relative food consumption were noted.

 

Body Weights

No test item-related differences in body weight or body weight gain were noted between the groups during the treatment and recovery period.

  

Clinical Laboratory Investigations

The following changes in hematology and clinical biochemistry parameters were noted:

Hematology:

Significant changes in hematology parameters including increased reticulocyte count were noted in males at 300 and 1000 mg/kg/day and increased methemoglobin concentrations in males at 100, 300 and 1000 mg/kg/day after four weeks of treatment. After the 2-week recovery, the methemoglobin decreased to values comparable to the respective control males.

 

Clinical Biochemistry: 

Statistically significant changes in clinical biochemistry parameters indicating effects on liver and / or kidney function were noted. These changes included increased urea and creatinine levels in males at 300 and 1000 mg/kg/day. As not further findings indicating effects on kidney function were noted, this was considered not to be adverse. Furthermore,decreased phospholipid levels in males at 1000 mg/kg/day, increased aspartate aminotransferase (ASAT) levels in males at 1000 mg/kg/day with increasing tendency already at 100 and 300 mg/kg/day, decreased bilirubin levels in females at 100, 300 and 1000 mg/kg/day, decreased alkaline phosphatase (ALP) levels in females at 1000 mg/kg/day, increased albumin levels in females at 100, 300 and 1000 mg/kg/day, decreased globulin levels in females at 1000 mg/kg/day, increased albumin/globulin ratio in females at 100, 300 and 1000 mg/kg/day were noted. The changes were generally slight and largely reversible during the recovery period. The only finding after recovery was a slightly decreased ALP, which was considered to be of no toxicological relevance. There were no morphological correlates pointing at the kidney or the liver as target organs. 

 

Urinalysis:

Concerning urinalysis parameters, no differences between the groups were noted.

 

Organ Weights

No test item-related changes in absolute or relative organ weights were noted. 

 

Macroscopic / Microscopic Findings

No test item-related macroscopic findings were noted in male and female animals after four weeks of treatment or recovery.

Microscopically, increased extramedullary hematopoiesis was noted in males at 300 and 1000 mg/kg/day.

 

Conclusion

The no observed adverse effect level (NOAEL) was established at 1000 mg/kg/day in males and females, although slightly increased methemoglobin levels were noted in males, only, of all dose groups. This change was considered not to be of adverse nature, as the degree was minor and the performance of the whole organism was not impaired. In addition, there was evidence of adaptive responses and the increased methemoglobin levels reversed after cessation of treatment. For females, changes in clinical biochemistry parameters were noted at all dose levels. However, these changes were largely reversible and were not considered to be adverse.

 

In conclusion, the general function of the organism and the general well-being was not impaired by treatment and all findings were considered to have no biological or clinical relevance [see also references ].