Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-11-06 until 2011-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
August 1998
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Glyoxal
EC Number:
203-474-9
EC Name:
Glyoxal
Cas Number:
107-22-2
Molecular formula:
C2H2O2
IUPAC Name:
oxalaldehyde
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B62

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2
- Stability under test conditions:The stability of the test substance under storage conditions over the test period was guaranteed by
the manufacturer, and the manufacturer holds this responsibility

Test animals

Species:
rat
Strain:
Wistar
Remarks:
strain Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to test guidelines.
This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 35 wks
- Weight at study initiation: (P) Males: 123.7-149.2 g; Females: 96 -122.2 g
- Housing: housed individually in Makrolon type M III cages (exeptions: During overnight matings, male and female mating partners were housed together in
Makrolon type M III cages; Pregnant animals and their litters were housed together until PND 21 (end of lactation)).
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal
- Water: ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the male in the cage of the female mating partner from about 16.00 h until 7.00-9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
During the first week of the premating period, F0 parental animals received drinking water with Glyoxal 40% concentrations based on the body weight of randomization and historical water consumption data given.
During the remaining premating period, the drinking water concentrations of Glyoxal 40% were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
During the mating period of the F0 parental animals, each group and sex received the concentrations of Glyoxal 40% used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception. During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test drinking water when they were separated the following morning. This test drinking water cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
After the mating period the drinking water concentrations of Glyoxal 40% for the F0 males were again adjusted weekly on the basis of body weight and water consumption data from the preceding week, until the end of their study. During gestation period, the Glyoxal 40% concentrations in the drinking water of the F0 females were the same as those used during the last week of the premating period.
During lactation period, the Glyoxal 40% concentrations in the drinking water of the F0 females were 50% of those used during the last week of the premating period. This drinking water adjustment, derived from historical body weight and water consumption data, maintained the dams at the desired doses of Glyoxal 40% during this period of increased water intake.
to be continued at frequency of treatment
Frequency of treatment:
Post weaning, drinking water Glyoxal 40% levels for F0 parental females awaiting necropsy were the same as used during the last week of the respective premating period.
Until all litters were weaned (when the last selected F1 pup reached age of PND 21), the drinking water for the weaned F1 pups selected as F1 parental animals was prepared with Glyoxal 40% concentrations on the basis of historical body weight and water consumption data for rats of similar age.
During the first week of the premating period of F1 parental animals, drinking water concentrations of Glyoxal 40% were formulated on the basis of actual body weight on day 0 and historical water consumption data. Subsequently, drinking water Glyoxal 40% levels for each F1 group and sex were adjusted as described for F0 parental animals.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: determined on the first day of the premating period and then once a week at the same time of the day

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: once a week in a period of 4 days; exceptions: during pregnancy, water consumption of the F0 and F1 females with evidence of sperm was determined for GD 0-1, 7-8, 14-15 and 20-21; during lactation, water consumption of the F0 and F1 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 14-15 and 21-22
- Generally, food consumption was determined once a week for male and female F0 and F1 parental animals, with the following exceptions: During pregnancy, food consumption of the F0 and F1 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20; During lactation, food consumption of the F0 and F1 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-14, and 14-21

Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 and F1 female with scheduled sacrifice.
Sperm parameters (parental animals):
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 and F1 males of all dose groups. The following parameters were determined: sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis). Sperm motility examinations and the preparation of the specimens for sperm morphology were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
GROSS NECROPSY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Liver, Kidneys, Adrenal glands, Testes, Epididymides
Cauda epididymis, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Spleen, Brain, Pituitary gland, Thyroid glands (with parathyroid glands)
The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% formaldehyde solution or in BOUIN’s solution:
Vagina, Cervix uteri, Uterus, Ovaries (fixed in BOUIN´s solution), Oviducts, Left testis** (fixed in BOUIN´s solution), Left epididymis** (fixed in BOUIN´s solution)
Seminal vesicles, Coagulation glands, Prostate, Pituitary gland, Adrenal glands, Stomach (forestomach and glandular stomach), all gross lesions, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands)


Postmortem examinations (offspring):
GROSS NECROPSY
All pups with scheduled sacrifice (i.e. pups culled on PND 4 or sacrificed on PND 21) were sacrificed by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.All stillborn pups and all pups that died before weaning were examined externally,
eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic
evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed.

SEXUAL MATURATION (F1)
All female F1 pups selected to become the F1 parental generation females (25/group) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined. All male F1 pups selected to become the F1 parental generation males (25/group) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined
Statistics:
DUNNETT test (two-sided): Food consumption, water consumption, body weight and body weight change (parental animals and pups); estrous cycle length; mating days; duration of gestation; number of delivered pups per litter; developmental landmarks (days up to preputial separation or opening of the vagina);
FISHER's exact test: Number of live and dead pups and different indices (e.g. mating index, fertility index and gestation index) and number of litters with necropsy findings in pups; developmental landmarks (preputial separation or opening of the vagina);
KRUSKAL-WALLIS and WILCOXON test (two-sided): Absolute and relative pup organ weights;
WILCOXON test (one-sided): Proportion of pups with necropsy findings per litter.
Reproductive indices:
For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters.
Offspring viability indices:
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters. The implantations were counted and the postimplantation loss (in %) was calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weights in the males were observed for the most part of the study (weeks 2 – 16, maximum -17%) and in the females for the most part of lactation ( PND 4 – 21, maximum -6%). Decreased body weight gain in the males was observed for the most part of the study (maximum -61%, average -25%) and in the females during gestation (maximum -9%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly below control during the entire treatment period (maximum -18%, average -13%). Food consumption of the male F0 rats in the mid and low dose groups was comparable to the concurrent control throughout the entire study.
Food consumption of the high-dose F0 females was comparable to the concurrent control during premating and lactation, only during early gestation (GD 0 – 7) a slight but statistically significant decrease was noted (-7%). Food consumption of the mid and low-dose F0 females was comparable to the control animals during premating, gestation and lactation. The statistically significantly increased food consumption of the mid-dose F0 females during premating weeks 2 - 5 was considered as spontaneous in nature and not treatment-related.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of the high-dose F0 males (400 mg/kg bw/d) was statistically significantly below control during the entire study period (maximum -43%, average during premating -33%). Water consumption of the mid dose males (100 mg/kg bw/d) was significantly decreased during several segments of the study (weeks 4 - 6 and weeks 7 - 16). Water consumption of the male F0 low-dose rats (25 mg/kg bw/d) was basically comparable
to the concurrent control throughout the entire study. The statistically significantly decreased water consumption in the low-dose males during study weeks 12 - 13 was considered as a single, spontaneous event.
Water consumption of the high-dose F0 females (400 mg/kg bw/d) was statistically significantly below control during the entire premating (maximum -37%), gestation (maximum -41%) and during several segments of lactation (PND 1 - 2, 7 - 8 and 14 – 15; maximum -17%). Water consumption of the mid and low-dose F0 females (100 and 25 mg/kg bw/d) was comparable to the control animals during premating, gestation and lactation.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased red blood cell (RBC) counts, haemoglobin and hematocrit values in males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in rats of both sexes was observed.
In addition, decreased cholesterol and inorganic phosphate levels in rats of both sexes and decreased creatinine, total protein, globulin levels in males were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Erosions or ulcers: The lesion was characterized by acute coagulative necrosis of the superficial epithelium with deposition of fine granular dark brown pigment. A focal/ multifocal slight (grade 2) inflammatory (granulocytic) cell infiltration of the lamina propria was detected in one female of
dose group 100 mg/kg/bw/d, in 4 females of dose group 400 mg/kg/bw/d and in one female of the control (in this test animal associated with proliferation of fibrous tissue indicating a subacute inflammation). The gross finding erosion/ulcer of the glandular stomach
was correlated with the features of a histomorphological erosion/ ulcus in 2 females of dose group 100 mg/kg/bw/d and in 3 females of dose group 400 mg/kg/bw/d.
All the other histopathological findings noted occurred either incidentally as single cases or were equally distributed over the dose groups and the control and are interpreted being spontaneous in nature and without any relation to treatment. Most of the gross lesions could be correlated with a sound histomorphological finding.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.1 days in control, 3.9 days in the low-dose group and 4.0 days in the mid- and high-dose groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Reproductive performance:
no effects observed
Description (incidence and severity):
The female mating index calculated after the mating period for F1 litter was 100% in the control and low- and mid-dose groups and 96% in the high-dose group. For all but one F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. The exception was high-dose male No. 93 paired with high-dose female No. 193. Thus, the male mating index was 100% in the control, low- and mid-dose groups and 96% in the high-dose group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Fertility and reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: General, systemic toxicity.
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: changes in clinical chemistry

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One F1 male animal of the control group 10 (No. 209) was found dead in study week 14. One F1 male animal of the low-dose group was sacrificed moribund because of poor general state and nutritional state in study week 7. One F1 female animal of the low-dose group was found dead on GD 22 (study week 14) after it was unable to deliver pups. None of the findings were considered to be treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High-dose parental F1 males (400 mg/kg bw/d) had statistically significantly lower body weights from test week 2 until the end of treatment (final weight -15%). Their body weight gain was statistically significantly decreased when calculated as average for the entire study (-17%), showing maximal decreases during weeks 9 - 10 (-36%) and 13 - 14 (-72%). Mean body weights and body weight gain of the F1 males in the mid- and low-dose groups
(100 and 25 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study. Body weights of the high-dose F1 females were statistically significantly below control in premating week 0 (about 7%) and from premating week 2 until the end of premating (maximum -8%), during the whole gestation period (maximum -9%) and during the entire lactation period (maximum -10%). Their body weight gain was statistically significantly
decreased when calculated as average for the entire premating period (-8%), showing a maximal decrease during weeks 1 - 2 (-15%). Mean body weight gain of the high-dose F1 females was still below control during gestation (-11%) without gaining statistical significance, while it was comparable to the concurrent control during lactation period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F1 males was statistically significantly below control during the entire treatment period (maximum -17%, average -11%). The mid-dose F1 males consumed slightly less food only at the beginning of premating (during weeks 0 – 1; -7%).
Food consumption of the male F1 rats in the low-dose group was comparable to the concurrent control throughout the entire study.
Food consumption of the high-dose F1 females was statistically significantly below control during several segments of premating (up to -7%) and during several segments of gestation (up to -8%). In these females no influence on food consumption was noted during lactation.
In the mid-dose F1 females food consumption was slightly below control during gestion gaining statistical significance during GD 14 - 20 ( -7%). In these females no influence on food consumption was noted during premating and lactation.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of the high-dose F1 males (400 mg/kg bw/d) was statistically significantly below control during the entire study period (maximum -45%, average during premating -31%). Water consumption of the mid dose males (100 mg/kg bw/d) was significantly decreased during several segments of the study (weeks 0 – 2, 8 - 9 and 10 - 15). Water consumption of the male F1 low-dose rats (25 mg/kg bw/d) was comparable to the concurrent control throughout the entire study.
Water consumption of the high-dose F1 females (400 mg/kg bw/d) was statistically significantly below control during the entire premating (maximum -38%), gestation (maximum -47%) and during the first segment of lactation (PND 1 - 8; maximum -24%). Water consumption of the mid-dose F1 females was statistically significantly below control during the major part of the premating period (maximum -15%), during the whole gestation period (maximum -23%) and during PND 21 - 22 (about -17%).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In all dosed F1 males, the haemoglobin concentration was higher compared to controls. This was the only measured red blood cell parameter changed in these rats. The calculated mean corpuscular haemoglobin concentration (MCHC) in males of the 400 mg/kg bw/d dose group was also statistically significantly increased, because of the higher haemoglobin concentration. Because of this isolated altered haemoglobin parameter, this effect was
regarded as treatment-related but not adverse. In males of the 100 and 400 mg/kg bw/d dose groups the relative reticulocyte counts were decreased compared to controls. Reticulocyte counts are used to specify the kind of anemia. In the mentioned rats no anemia was present, but contrarily the haemoglobin values were higher compared to controls. Therefore, the statistically significantly decreased reticulocyte counts were regarded as maybe treatment-related, but not adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In rats of both sexes of the 100 and 400 mg/kg bw/d dose groups, the alanine aminotransferase (ALT) activity was lower compared to controls. In female and male rats of the 400 mg/kg bw/d dose group and additionally in males of the 100 mg/kg bw/d group the alkaline phosphatise (ALP) activity was decreased.In rats of both sexes of the 400 mg/kg bw/d dose group, the inorganic phosphate and the globulin values were decreased. Additionally, in males of the 400 mg/kg bw/d dose group the urea concentration was increased and in males of this dose group and the 100 mg/kg bw/d dose group the creatinine levels were decreased. In females of the 400 mg/kg bw/d dose group the total protein and calcium levels were lower compared to controls. In the F1 males of the 100 mg/kg bw/d dose group the albumin levels were higher compared to controls, but this increase was not dose-dependent, and the values were within the historical control range of 4.5 months old rats (albumin: 36. 12 – 39.76 g/L). Therefore, this alteration was regarded as not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The absolute brain, liver, spleen and thyroid glands weight decrease of the high dose males and the absolute spleen weight decrease of high dose females is regarded to be related to the terminal body weight decrease.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Erosions/ulcera of the mucosa of the glandular stomach were diagnosed in 1 control male, 1 female test animal of dose group 25 mg/kg/bw/d, 1 female test animals of dose group 100 mg/kg/bw/d, and in 4 female test animals of dose group 400 mg/kg/bw/d; Focal discoloration was observed in one female of the low dose and hyperemia in one female of the high dose group as well
- Unilateral abscess was noted in the region of the left cauda epididymis of one male of dose group 100 mg/kg/bw/d
- Exophthalmia of the right eye was recorded for one male of dose 100 mg/kg/bw/d
- Unilateral cyst was seen in one male of the low dose group, one male of the high dose group showed slight enlarged kidneys, two females of dose group 25 mg/kg/bw/dwere detected with unilateral retraction
- Yellow focus was recorded in the liver for one male and female of the control each and for one male of the high dose group
- Discoloration of the lungs was found in one male of the low dose group
- A grey-red mass of 30 mm diameter adjacent to the thymus with adhesion to the sternum was recorded for one male of the low dose group
- Unilateral cysts in the ovaries were diagnosed in two female rats of the control and in one female each of the low, mid and high dose group
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Glandular stomach: Erosions were detected in one female of dose 25 mg/kg/bw/d and 100 mg/kg/bw/d each and in 4 females of dose 400 mg/kg/bw/d. These erosions were accompanied by slight submucosal edema. One male of the control, one male and female of the high dose group (400 mg/kg/bw/d) each showed slight (multi)focal submucosal inflammatory cell infiltrations without visible erosions of the epithelium. All the other histopathological findings noted occurred either incidentally as single cases or were equally distributed over the dose groups and the control and are interpreted being
spontaneous in nature.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating for the F2 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 4.1 days in the low-dose group and 4.2 days in the mid- and high-dose groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F1 parental males, which were placed with females to generate F2 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. The female mating index calculated after the mating period for F2 litter was 100% in all test groups.
The fertility index varied between 92% (test group 13), 96% (test group 11 and 12) and 100% (control). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data.

Details on results (P1)

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.2 / 11.9 / 12.7 and 12.0 implants/dam in test groups 0 -
3). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups, and the mean number of F2 pups delivered per dam remained unaffected (10.9 / 11.2 / 11.4 and 11.1 pups/dam in test groups 0 - 3). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% (control and test group 11) and 100% (test groups 12 and 13). Moreover, the number of stillborn pups was comparable between the groups.
Thus, Glyoxal 40% did not adversely affect reproduction and delivery of the F1 generation parental females.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Fertility and reproductive performance.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Sex:
not specified
Basis for effect level:
other: General, systemic toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: changes in clinical chemistry

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 99% (test groups 00 and 02 - 03) and 97% (test group 01).The lactation index indicating pup mortality on PND 4 - 21 varied between 98% (test group 00), 99% (test groups 01 and 02) and 94% (test group 03).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F1 male and female pups (400 mg/kg bw/d) were statistically significantly below control during PND 7 - 21 (up to 11% on PND 21). Body weight gain was decreased in the high-dose F1 pups from PND 4 - 21 up to 12% below control [PND 4 - 7 males about 16%, females about 19% and both sexes combined about 16%]. No test compound-related influence on F1 pup body weights and pup body weight gain were
noted in the mid- and low-dose group (100 and 25 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The decreased absolute thymus weights and the increased relative brain weights (high dose group) are considered to be secondary to the lower body weight of the respective animals at weaning. As there was no dose response the increased absolute and relative spleen weights in the mid-dose F1 pups were considered neither adverse nor toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, aglossia, microphthalmia, hemorrhagic thymus, diaphragmatic hernia, empty stomach, hydronephrosis, dilated renal pelvis, hydroureter and mandibular micrognathia. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Developmental toxicity.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: slightly decreased pre-weaning pup body weights/pup weight gain observed at the LOEL of 400 mg/kg bw/d

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
For one female pup a thread-like tail was recorded during lactation (PND 0 - 21). This observation was considered not to be treatment-related. There were no other clinical observations in any of the test groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 95%** [**:p≤0.01] (test group 12), 98% (test groups 11) and 99% (control and test group 13). The lactation index indicating pup mortality on PND 4 - 21 varied between 99% (control and test group 13) and 100% (test groups 11 and 12). Thus, the test substance did not influence pre-weaning pup survival in any of the treated groups (25, 100 and 400 mg/kg bw/d). The statistically significant lower number of pups surviving days 0 to 4 in test group 12 (100 mg/kg bw/d) was considered as spontaneous in
nature, since the affected pups were all from one litter (dam No. 361) and there was no dose response.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F2 male and female pups (400 mg/kg bw/d) were statistically significant below control on PND 7 (-9%) only. Body weight gain was decreased in these pups during PND 4 - 7 (-13% for both sexes together). No other preweaning weight changes were noted in these animals.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The increased absolute and relative brain weights (high dose group) and the decreased relative thymus weights in the mid-dose F2 pups were considered neither adverse nor toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F2 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach, dilated renal pelvis and thread-like tail. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
400 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Developmental toxicity.
Dose descriptor:
NOEL
Generation:
F2
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: slightly decreased pre-weaning pup body weights/pup weight gain observed at the LOEL of 400 mg/kg bw/d.

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Concentration control analyses

Overall, the measured values were in the expected range of the target concentrations (90-110%), demonstrating the correctness of the drinking water preparations.

Diet analyses

With regard to the analytical findings of chemical and microbiological contaminants and the

duration of application, the diet was found to be suitable.

Drinking water analyses

On the basis of the analytical findings, the drinking water was found to be suitable.

Bedding and enrichment analyses

On the basis of the analytical findings, bedding and cage enrichment were found to be suitable.

Applicant's summary and conclusion

Categories Display