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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 Jan. 2004 to 1 Feb. 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Data generated according to international accepted valid testing method
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
The objective of this study was to provide information on the possible health hazards likely to arise from repeated dietary exposure to Glyoxylic Acid 50 % over a relatively short period of time (about 8 weeks of treatment).
It further comprise a reproduction/developmental toxicity screening test to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, development of the conceptus and parturition.
The study was performed in accordance with the requirements of the OECD 422 Guideline for testing of chemicals.
The design was based on a 4 week repeated dose toxicity study to assess the overall toxic potential and included a screen for reproductive and developmental effects. The test substance was Glyoxylic Acid 50 (a 50% w/w solution of Glyoxylic Acid in water) which was incorporated into the diet of CD rats.
GLP compliance:
yes (incl. certificate)
Remarks:
Huntingdon Life Sciences, Wooley Road, Alconbury, Huntingdon, Cambridgeshire, PE284HS, England
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Glyoxylic acid 50
- Physical state: liquid
- Analytical purity: 50.0 % , w/v aqueous solution
- Lot/batch No.: SCA210704-03
- Expiration date of the lot/batch: one year from manufacture
- Storage condition of test material: at ambient temperature or in a refrigerator, unless otherwise directed by the Sponsor

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Species: Rat
- Strain: Crl:CD (SD) IGS BR
- Age ordered: 7 weeks of age males and females not siblings
- Weight range ordered: to be within an 15 g range for each sex/batch
- Supplier: Charles River (UK)
- Acclimatisation: 3 weeks
- Weight at study initiation: bodyweights were in the range of 367 to 442 g for males and 212 to 253 g for females and animals were 70 days of age

Animal - housing, diet and water supply:
- Rodent facility: Full barrier - to minimise entry of external biological and chemical agents.
- Air supply: Filtered, not recirculated.
- Temperature: Maintened within the range of 19-23 °C.
- Relative humidity: Maintened within the range of 40-70 %
- Lighting: 12 hours light: 12 hours dark.
- Animal per cage: Five of the same sex, unless reduced by mortality or isolation.
- Cage material: Polypropylene or stainless steel.
- Diet name: standard rodent breeding diet (SDS VRF1 Certified Diet manufactured by Special Diets Services Ltd., Witham, Essex, England) ad libitum
- Water supply: Potable water from the public supply ad libitum

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on oral exposure:
The test substance (Glyoxylic Acid: 50 % aqueous solution) was administered continuously via the diet throughout the treatment period.
The SDS VRF 1 diet for the Control group was blended with the same amount of water as the 18000 ppm Glyoxylic acid 50 diet i.e. 9000 ppm water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the aspen chew blocks. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
The test substance (Glyoxylic Acid: 50 % aqueous solution) was administered continuously via the diet throughout the treatment period.
Doses / concentrations
Remarks:
Doses / Concentrations:
2000, 6000 and 18000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
The test consisted of one control and three treated groups of Crl:CDr (SD)IGS BR rats (10 males and 15 females per group) divided in two subgroups:Toxicity subgroup: 5 males and 5 females/group
Reproduction subgroup: 5 males and 10 females/group
Control animals:
other: Basal diet including water at the same level as for high ppm group.
Details on study design:
The dietary concentrations used in this study (0, 2000, 6000 and 18000 ppm) were selected in conjunction with the Sponsor, based on the results of a preliminary toxicity and palatability test by dietary administration to CD rats for 14 days (Huntingdon Life Sciences Report No.
CRI032/040104).

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. In addition, a more detailed physical examination, and assessment of behaviour in a standard arena, were performed for each animal each week to monitor general health and any possible signs of neurotoxicity.


BODY WEIGHT: Yes
- Time schedule for examinations:
Each male and toxicity subgroup female was weighed on the day that treatment commenced, weekly thereafter, and at necropsy. Reproductive subgroup females were weighed on the first day of treatment, weekly until pairing and on Days 0, 7, 14, 17 and 20 after mating, Days 1 and 4 of lactation, and at necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption for all males and females was recorded daily throughout the study prior to pairing. Food consumption was not recorded for toxicity subgroup males or reproductive subgroup animals during pairing when cohabitation altered baseline values. Food intake was then recorded daily for reproductive subgroup females during gestation and lactation, and resumed for males and toxicity subgroup females until termination.

Water consumption: Assessed visually each day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 5
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
Haematocrit (Hct)
Haemoglobin (Hb)
Red blood cell count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the same time and using the same animals as for peripheral haematology
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total Bilirubin (Bili)
Bile acids
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Magnesium (Mg)
Total protein (Total Prot)
Albumin (Alb)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: following the completion of 4 weeks of
treatment
- Dose groups that were examined: all toxicity subgroups
- Battery of functions tested: sensory activity / grip strength / motor activity

HISTOLOGY:
- Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four
to four micron thickness and stained with haematoxylin and eosin, except the testes which
were stained using a standard periodic acid/Schiff (PAS) method.

Those tissues subject to histological processing included the following regions:

Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar
and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section

For bilateral organs, sections of both organs were prepared. A single section was prepared
from each of the remaining tissues required for microscopic pathology.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
All statistical analyses were carried out separately for males and females.
All analyses were carried out using the individual animal as the basic experimental unit. The following data types were analysed at each timepoint separately: Bodyweight gains over appropriate study periods Blood chemistry and haematology parameters Organ weights, both absolute and relative to terminal bodyweight, Motor activity data, Grip strength data. For categorical data, the proportion of animals was analysed using Fisher’s Exact test (Fisher 1973) for each treated group versus the control. For continuous data, Bartlett’s test (Bartlett 1937) was first applied to test the homogeneity of
variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary. For bodyweight gains and organ weights, whenever Bartlett’s test was found to be
statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett’s test was used. The following sequence of statistical tests was used for blood chemistry and haematology, motor activity and grip strength data: If 75% of the data (across all groups) were the same value, for example c, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in proportions (Mantel 1963) and also pairwise Fisher's Exact tests (Fisher 1973) for each dose group against the control both for i) values =c, and for ii) values <=c versus values >c, as applicable.
Significant differences between Control and treated groups were expressed at the 5 % (p<0.05) or 1% (p<0.01) level. The following statistical cyphers were used throughout the report:
a - p < 0.05 ; b - p < 0.01 - using categorical or parametric tests

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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 specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
no effects observed
Details on results:
Treatment at all dietary concentrations was well tolerated and there were no mortalities.
There were no clinical signs or arena observations considered to be related to treatment.
Bodyweight gains of males in the 18000 ppm and 6000 ppm groups were noticeably lower than in control and the differences showed a dose response on both study subgroups.
No such difference was recorded among females. There was no effect on food consumption.

Achieved dosage of pure Glyoxylic Acid during the first week of treatment in the 2000 ppm, 6000 ppm and 18000 ppm Glyoxylic Acid 50 group were approximately 70 mg/kg/day, 200 mg/kg/day and 600 mg/kg/day respectively in males and 80, 240 and 730 mg/kg/day in females.
The findings of the haematology investigations during Week 5 were largely unremarkable; the only possible treatment-related change evident was a slightly shorter activated partial thromboplastin time for males at 6000 ppm or 18000 ppm; the differences did not attain statistical significance.

There was slightly greater variability in blood chemistry parameters, and the following were changes for which an effect of treatment could not be discounted: a statistically significantly elevated level of cholesterol among males at 18000 ppm; a dose related reduction in the level of alkaline phosphatase among females, with differences attaining significance at 6000 ppm and 18000 ppm; slightly but not significantly lower levels of alanine amino-transferase and aspartate amino-transferase among females in the 6000 ppm and 18000 ppm groups.

There were no effects of treatment on macroscopic pathology findings or absolute or bodyweight relative organ weights.

There were no microscopic pathology changes in the tissues examined that were considered to be related to treatment.



Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: dose corresponds to 200 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: dose corresponds to 730 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The study author concluded that the NOAEL for general toxicity in adult male rats was 2000 ppm (70 mg/kg bw/d) Glyoxylic Acid 50%, based on results showing changes in bodyweight gain at 6000 and 18000 ppm, which were discussed as an adverse effect. However, this effect on bodyweight gain at 6000 ppm has to be regarded as a non adverse effect, because of the following reasons:

i) Bodyweight development of male and female rats was not impaired in all dose groups.

ii) Bodyweight gain was not affected in females.

iii) Statistically significant reduction in the cumulative bodyweight gain was observed only from week 1-3 in the toxicity subgroup and from week 1-2 and week 1-4 in the reproductive subgroup at 18000 ppm. No significant changes were observed during all other treatment intervals or for the whole study period.

iv) Weekly bodyweight gains were not statistically significantly changed at all dose levels.

In conclusion, a NOAEL of 6000 ppm (200 mg/kg/d) can be derived due to the statistically significantly reduced cumulative bodyweight gain among males at 18000 ppm.

Applicant's summary and conclusion

Executive summary:

In a subacute toxicity study according to OECD TG 422, Glyoxylic acid 50% was administered to 5 Crj:CD (SD) rats per sex per dose by feed at dose levels of 0, 2000, 6000 or 18000 ppm for a period of 5 weeks (Myers, 2005). Bodyweight gains of males in the 18000 ppm and 6000 ppm groups were lower than in control. The change at 6000 ppm is regarded as a non adverse effect, because statistical significance was only observed temporarily at 18000 ppm, no such effect was recorded among females, bodyweight development was not impaired and weekly bodyweight gains were not statistically significantly changed. There was no effect on food consumption. The only possible treatment-related change evident concerning haematology was a slightly shorter activated partial thromboplastin time for males at 6000 ppm or 18000 ppm; the differences did not attain statistical significance. There were some changes in blood chemistry parameters for which an effect of treatment could not be discounted: a statistically significantly elevated level of cholesterol among males at 18000 ppm; a dose related reduction in the level of alkaline phosphatase among females, with differences attaining significance at 6000 ppm and 18000 ppm; slightly but not significantly lower levels of alanine amino-transferase and aspartate amino-transferase among females in the 6000 ppm and 18000 ppm groups. There were no effects of treatment on macroscopic pathology findings or absolute or bodyweight relative organ weights. There were no microscopic pathology changes in the tissues examined that were considered to be related to treatment.

Based on the effects observed in this study a NOAEL of 6000 ppm (200 mg/kg/d) for males due to the statistically significantly reduced cumulative bodyweight gain among males at 18000 ppm and 18000 (730 mg/kg bw/d) for females can be derived.