AAA reaction product

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-05-14 to 2004-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance to recognized testing guidelines with no deviations to study protocol.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Principles of method if other than guideline:

Na

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): DEA/ACID ANHYDRIDE REACTION PRODUCT

- Physical state: highly viscous amber liquid

- Composition of test material, percentage of components:
Monomers: <17-35% (9-16% diethanolamine; 2-8% tetrahydrophthalic acid; 5-11% trimellitic acid; <0.1% tetrahydrophthalic anhydride; <0.8% trimellitic anhydride)
Dimers/trimers: <24-45%
Polymers: <10-25%
Not removable water: 5-15%

- Lot/batch No.: 200501.UN2810

- Expiration date of the lot/batch: september 2003

- Stability under test conditions: stable under storage condition

- Storage condition of test material: in the refrigerator in the original container away from direct sunlight

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rat, HanBrl:WIST (SPF)
- Age at study initiation: 6 weeks
- Weight at study initiation: Males = 150.6 g (mean), female = 119.1 g (mean)
- Fasting period before study:
- Housing: groups of five in Makrolon type 4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): ad libitum diet (Pelleted rat standard Provimi Kliba 3433 analyzed for contaminants)
- Water (e.g. ad libitum): ad libitum tap water analyzed for bacteria, chemicals and contaminants
- Acclimation period: 7 days period. Only healthy animals were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): standard laboratory conditions with a target range for temperature of 22 ± 3 °C.
- Humidity (%): relative humidity between 30-70 %.
- Air changes (per hr): 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light / 12 hours dark and music during the light period.

IN-LIFE DATES: From: 20 May 2003 To: 24 June 2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG 300; Batch No 442989/1 54802033; Expiry date 27 Feb 2008.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

PREPARATION
- Formulations of AAA reaction product in the vehicle (PEG 300) were prepared weekly. AAA reaction product was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored in the refrigerator (2-8°C).
Homogeneity of the AAA reaction product formulations were maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Justification for use and choice of vehicle (if other than water): AAA reaction product was soluble in PEG 3000
- Concentration in vehicle: 2, 10 and 50 mg/mL (Nominal concentrations)
- Amount of vehicle (if gavage): in accordance to 2, 10 and 50 mg/mL (Nominal concentrations)
- Lot/batch no. (if required): 442989/1 54802033
- Purity: no information

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.
It is noted that although the dose formulations were made and used before the expiry date of the test item and analytical samples were taken and deep-frozen (ca. –20°C) before the expiry date of the test item, the final analytical method was not provided until after the expiry date had passed.

The results in terms of concentration, homogenity and stability was within the limits of the established chemical analysis.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 50, 250 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Male: 5 animals at 10 mg/kg bw/day; 5 animals at 50 mg/kg bw/day; 5 animals at 250 mg/kg bw/day
Female: 5 animals at 10 mg/kg bw/day; 5 animals at 50 mg/kg bw/day; 5 animals at 250 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a non-GLP 5-day dose-range-finding study (RCC Study Number 848627) in which AAA reaction product was administered by gavage to 2 rats per group and sex.
- Rationale for animal assignment (if not random): NA
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA

Positive control:

No, not included and required for this type of study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: NA

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: NA
- Dose groups that were examined: NA

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia was used
- Animals fasted: Yes, 18 hours before blood sampling
- How many animals: all animals
- Parameters examined: in accordance to the standard requirements in the OECD testing guidelne (407)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, 18 hours before blood sampling
- How many animals: all animals
- Parameters examined: in accordance to the standard requirements in the OECD testing guidelne (407)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: control and all dose groups
- Battery of functions tested: grip strength and motor activity (modifierd Irwin screen test)

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were sacrifised after 4 weeks, weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals and Epididymides. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.


HISTOPATHOLOGY: Yes, samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution. 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 from control and high-dose groups were examined.

Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain (three levels)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes with optic nerve (fixed in Davidson's solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland (exorbital)
Liver
Lungs (infused with formalin at necropsy)
Lymph nodes (mesenteric, mandibular)
Mammary gland area
Nasal cavity Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands (mandibular, sublingual)
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord (cervical, midthoracic, lumbar)
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland)
Tongue
Trachea
Urinary bladder (infused with formalin at necropsy)
Uterus
Vagina
Gross lesions

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
• Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett’s test if the ANOVA was significant at the 5% level and by Dunn’s test in the case of a significant Kruskal-Wallis test (p0.05).

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

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical observations: No test item-related findings of toxicological significance were noted during daily observations in the test item- treated rats. No test item-related findings of toxicological significance were noted during weekly behavioral observations in the test item-treated rats.

Laboratory findings: Mean hemoglobin levels and hematocrit was reduced in males and females treated with 250 mg/kg bw/day when compared with the controls. The differences for males remained within the lower limit of the 95% tolerance limits of the historical control data, but the values for the females exceeded the lower limit of the historical control data. The mean red blood cell count was reduced in males and females treated with 250 mg/kg bw/day. These differences were considered to be test item- related. All other changes were considered to be minor and unrelated to the treatment with the test item.

Effects in organs: Test item-related changes included increased kidney and liver weights in males and females treated with 250 mg/kg bw/day. There were no item-related necropsy or histopathological findings. In particular, there were no histopathological findings to correlate with the observed increase in weight of kidneys (males and females at 250 mg/kg bw/day) and liver (females at 250 mg/kg bw/day).

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

dissolved

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The 28-day oral toxicity of AAA-reaction product was evaluated in accordance to OECD 407 using dose levels of 10, 50 and 250 mg/kg bw/day. A NOAEL of 50 mg/kg bw/day was established based on changes in hematology parameters and slightly increased mean kidney and liver weights in males and females treated with 250 mg/kg bw/day.

Executive summary:

Oral administration of AAA-reaction product to Wistar rats at doses of 10, 50 and 250 mg/kg bw/day, for 28 days (OECD 407) resulted in no deaths, no clinical signs of toxicological relevance following daily observations, weekly behavioral observations (weeks 1-3) or functional observational battery (week 4), including grip strength and locomotor activity. No differences in mean food consumption or mean body weight were noted, and no changes in the clinical biochemistry parameters were seen. There were no necropsy or histopathological findings related to AAA-reaction product.

Toxicological findings related to AAA-reaction product were generally restricted to changes in hematology parameters (reduced mean hemoglobin and hematocrit at 250 mg/kg bw/day, as well as reduced mean red blood cell count), and slightly increased mean kidney and liver weights in males and females treated with 250 mg/kg bw/day. These changes were, however, not accompanied by microscopical changes.

Based on the results of this study, 50 mg/kg bw/day of AAA-reaction product was established as the no-observed-adverse-effect-level (NOAEL).