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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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)
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
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:
Crj: CD(SD)
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 exposure:
The test substance (GA: 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.
Exposure: Continuously from 14 days before pairing, during 14-day pairing period, gestation, littering and lactation period until day 4.
Details on mating procedure:
- all 10 males in each group (toxicity and reproductive subgroups) were paired with the 10 reproductive subgroup females after all animals had received 2 weeks of treatment.
- M/F ratio per cage: one to one
- Length of cohabitation: up to 2 weeks was allowed although all animals mated and were separated within 1 week
- Proof of pregnancy: each morning, following pairing, the trays beneath the cages were checked for ejected copulation plugs and a wet vaginal smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation
- Pairing was on a one-to-one basis within treatment groups; up to 2 week was allowed although all animals mated and were separated within 1 week
- From day 20 after mating reproductive subgroup animals were checked 3 times daily for evidence of parturition.
- The female were permitted to deliver their young naturally and rear their own offspring until Day 4 of lactation.
- Numbers of live and dead offspring were recorded during the parturition process.
- All offspring were examined at approximately 24 hours after birth (Day 1) and the number of offspring born (live and dead) was recorded
- Litters were observed daily for evidence of abnormal appearance or behaviour. Daily records were maintained for mortality and consequent changes in litter size.
- Reproductive subgroup males were killed during Week 6 of treatment.
- Reproductive subgroup females and their offspring were killed on Day 4 of lactation/age (during Weeks 6 or 7 of treatment for the females).
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
The test substance (GA: 50 % aqueous solution) was administered continuously via the diet throughout the treatment period.
Details on study schedule:
- Selection of parents from F1 generation when pups were 4 days of age.
- Age at mating of the mated animals in the study: 12 weeks
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

Parental animals: Observations and examinations:
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

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: animals were killed during Week 6 of treatment,
- Maternal animals: animals and their offspring were killed on Day 4 of lactation/age (during Weeks 6 or 7 of treatment for the females)

HISTOPATHOLOGY / ORGAN WEIGHTS:
Any abnormality in the appearance or size of any organ or tissue was recorded and the required tissue samples preserved in appropriate fixative.
Any photographs of unusual findings were taken at the discretion of the necropsy supervisor. The retained tissues were checked before disposal of the carcass. Testes and epididymides were fixed in Bouin’s solution prior to transfer to 70 % industrial methylated spirit. Samples (or the whole) of the other tissues listed below from all animals were preserved in 10 % neutral buffered formalin: Adult males : Seminal vesicles with coagulating gland, prostate.
Dams: Ovaries, uterus with cervix and oviducts, vagina, pituitary, mammary tissue - tissues from the dams were not subjected to histological processing and microscopic examination.
Postmortem examinations (offspring):
SACRIFICE
- offspring were killed by intraperitoneal injection of sodium pentobarbitone
- 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. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. For dams, the number of uterine implantation sites was recorded.
- Offspring found dead or killed for reasons of animal welfare were subjected to a macroscopic
examination, which included an assessment of the stomach for milk content. Offspring
examined at termination on Day 4 of age were subjected to a macroscopic examination.
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
Reproductive indices:
The following were calculated for males and reproductive subgroup females separately:
Percentage mating = Animals with evidence of mating x 100/Animals paired
Conception rate = Animals achieving a pregnancy x 100/Animals with evidence of mating
Fertility index = Animals achieving a pregnancy x 100/Animals paired
Gestation index = Number of live litters born x 100 /Number pregnant
Offspring viability indices:
Post-implantation survival index = Total number of offspring born/ Total number of implantation sites
x100
Live birth index = Total number of offspring on Day 1 of age/ Total number of offspring born
x100
Viability index = Number of live offspring on Day 4 of age/ Number of live offspring on Day 1 of age
x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.
There was no effect on food consumption.

Bodyweight gains of males in the 18000 ppm and 6000 ppm groups (Groups 3 and 2 respectively) in both the toxicity and reproductive subgroups were lower than in the respective controls; the reduction in gains showed a dose response and at 18000 ppm was very marked during the second week of treatment on both phases, with the difference attaining statistical significance in the reproductive subgroup. The consistency of the reduced weight gains and dose response in both study subgroups indicates that the effect was due to treatment.

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 (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
18 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effect (maximum dose tested)
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
18 000 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effect (maximum dose tested)
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
6 000 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reduced body weight gain at 18000 mg/kg bw/d.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

There was a slightly higher incidence of macropathology findings among offspring examined at termination on day 4 of age in groups treated with Glyoxylic acid 50 compared with controls. However, only a small number of offspring were involved and as there was no consistency in the nature of the findings between the groups and no indication of an adverse effect on the survival or growth of the offspring, this minor difference was considered to be of no toxicological importance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
18 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effect (maximum dose tested)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Treatment at all dietary concentrations was well tolerated and there were no mortalities.
Bodyweight gain and food intake of reproductive subgroup females during gestation and early lactation was unaffected by treatment.
Achieved dosages for females at the start of gestation (Days 0-7) were 97, 296 and 910 mg/kg/day for females in the 2000, 6000 and 18000 ppm groups.
Achieved dosages during Days 1-4 of lactation were about 49-56 % higher (126, 383 and 1257 mg/kg/day respectively) than those at the end of gestation, reflecting increased physiological demand on the dams by the litters.
Achieved dosages largely reflected the three-fold difference in dietary concentrations of Glyoxylic acid 50.
There was no effect of treatment on the sensory reactivity, grip strength or motor activity of the animals monitored during Week 5.
Mating performance and fertility were unaffected by treatment.
Parental treatment had no effect on the survival, growth or development of the offspring 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.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it was concluded that the NOAEL for reproductive/developmental toxicity within the scope of this screening test was 18000 ppm.

The study was considered to have met the objectives of the OECD Guideline 422.

The reproduction/developmental toxicity screening test found no evidence of impaired performance at these dietary concentrations.
Executive summary:

In a combined repeated dose toxicity study with a reproduction toxicity screening test a subgroup of 10 female and 5 male

Crj:CD (SD) rats per dose were exposed to dietary concentrations of 0, 2000, 6000 or 18000 ppm Glyoxylic acid for about 8 weeks to screen for reproductive and developmental effects.

The NOAEL for reproductive/developmental toxicity within the scope of this screening test was 18000 ppm ( (i.e. for females 910 and 1257 mg/kg bw/d, respectively at the start of gestation and during days 1 -4 of lactation).

The reproduction/developmental toxicity screening test found no evidence of impaired performance at these dietary concentrations.

Mating performance and fertility were unaffected by treatment. Bodyweight gain and food intake of reproductive subgroup females during gestation and early lactation was unaffected. The duration of gestation was unaffected aswell. Parental treatment had no effect on the survival, growth or development of the offspring up to day 4 of age.

The study was considered to have met the objectives of the OECD Guideline 422.