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Effects on fertility

Description of key information

A two-generation reproduction toxicity study in Wistar rats with continuous glutaraldehyde administration in the drinking water was conducted in accordance with the OECD TG 416; the study conducts followed GLP (BASF, 2001). The study showed that glutaraldehyde does not affect the reproductive performance and fertility. In a second two-generation toxicity study published by Neeper-Bradley et al. (2000) similar results were observed.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-06-04 to 2001-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
(1998)
Qualifier:
according to guideline
Guideline:
other: EC Commission Directive 87/302/EEC of November 18, 1987; Part B: Methods for the determination of toxicity: Two-generation reproduction toxicity test; Official Journal of the European Communities, No. L 133, pp. 47-50 (1988)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Physical state: homogeneous colorless-clear liquid
- Analytical purity: 50.5%
- Lot/batch No.: 50-4402
- Stability under test conditions: stability assured for at least 2 years if stored under the cited storage conditions (according to the certiflcate of Analysis, Feb. 22, 2001); stability was additionally verified by reanalysis after the experimental phase of several other studies with the test compound.
- Storage condition of test material: refrigerator (4 to 10°C), under nitrogen
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: The animals of the F0 generation were about 34 +/- 1 day old at test starting
- Weight at study initiation: mean body weight for the F0 males was 100.5 (89.3 - 116.6) g; mean body weight for the F0 females was 89 (77.6 – 99.6) g
- Fasting period before study: no
- Housing: during the study period, the rats were housed individually in type DK III stainless steel wire mesh cages (floor area ca. 800 cm2), with the following exceptions: from day 18 of gestation until day 14 after birth, the pregnant animals and their Iitters were also housed in Makrolon type M III cages; pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/ mouse/hamster, meal (Provimi Kliba SA, Kaiseraugst, Switzerland, offered ad libitum throughout the study
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): accommodations were fully air- conditioned
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was added to an appropriate amount of drinking water and was thoroughly mixed with a stirrer.
The drinking water solutions were prepared twice a week.
Details on mating procedure:
Each male was paired with one female of the same group; pairing happened overnight over a period of maximum 2 weeks. For this purpose, the female was placed into the cage of the male from ca. 4.00 p.m. to 7.00 – 9.00 a.m. of the following day. Mating was carried out with the same partner in each case. After each mating, a vaginal smear was taken from the female and checked for presence of sperm. If sperm was present, the female was considered as fertilized and the day was designated as day 0. The following day was then day 1 post-coitum (p.c.).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to check the correctness of the concentrations, samples of each one of the doses were drawn for concentration control analyses at the start of the administration period, thereafter in intervals of about 3-months during the study, and at study termination. The samples were subjected to gas chromatography (GC) analysis.
Duration of treatment / exposure:
Parental animals (F0 generation): about 20 weeks, including the premating exposure period of about 10 - 12 weeks.
F1 parental generation: about 17 weeks.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
F0 GENERATION:
After the acclimatization period, the F0 generation parental animals continuously received the test substance at the appropriate concentrations in the drinking water until they were sacrificed. At least 76 days after the beginning of treatment, males and females from the same dose group were paired at a ratio of 1:1. The females were allowed to litter and rear their pups (FI generation pups) until day 4 or 21 post-parturition. After weaning of FI pups the F0 generation parental animals were sacrificed.

F1 GENERATION:
After weaning, 27 males and 27 females of the F1 pups of each test group were taken as the basis of the F1 generation parental animals.
All selected animals were exposed continuously to the test substance at the same dose level as their parents from their growth into adulthood until or up to about 16 hours before they were sacriflced.
At least 76 days after assignment of the F1 generation parental animals, the males and females were paired at a ratio of 1:1. The partners were randomly assigned to one another, and pairings of siblings were avoided. The females were allowed to litter and rear their pups (F2 generation pups) until day 4 or 21 post-parturition. One week after the F2 generation pups had been weaned, the FI generation parental
animals were sacrificed.
Dose / conc.:
100 ppm (nominal)
Remarks:
in water; 12 mg/kg bw/day of test material; mean values referring to the test material intake in parental males and females of the F0 and the F1 generation during premating.
Dose / conc.:
500 ppm (nominal)
Remarks:
in water; 58 mg/kg bw/day of test material; mean values referring to the test material intake in parental males and females of the F0 and the F1 generation during premating.
Dose / conc.:
2 000 ppm (nominal)
Remarks:
in water; 199 mg/kg bw/day of test material; mean values referring to the test material intake in parental males and females of the F0 and the F1 generation during premating.
No. of animals per sex per dose:
27 animals/sex/group
Control animals:
yes, concurrent no treatment
Parental animals: Observations and examinations:
MORTALITY AND CLINICAL SYMPTOMS
The animals were checked daily for mortality (dead and moribund animals) and clinical symptoms of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after of parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 1-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups will start consumption of solid food; therefore there will be no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.

WATER CONSUMPTION
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 4-5, 7-8, 14-15 post parturition (pp).
- F0 and F1 dams from day 15 pp onwards: water consumption was not determined for the F0 and F1 dams from day 15 pp onwards, since during this period the pups will start consumption of water; therefore there will be no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.

TEST SUBSTANCE INTAKE
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the adequate formula, taking into account, the daily water consumption, the dose level and the body weight.
Oestrous cyclicity (parental animals):
The oestrus cycle was evaluated daily for length and normality for all F0 and F1 parental females, over a period of at least 3 weeks prior mating and through mating period until mating was definitively evidenced. A last evaluation was done at necropsy.
Sperm parameters (parental animals):
Following necropsy and organ weighing, the right testis and cauda epididymis were taken from the F0 and F1 males of all test groups for the evaluation of following parameters:
- Sperm motility:evaluated by microscopy all groups, expressed as %
- Sperm morphology: examined in the control and the 2000 ppm group by microscopy following vital staining (eosin), expressed as %
- Sperm head count (cauda epididymis): examined in the control and the 2000 ppm group by microscopy, expressed as sperm heads/10E+6/g cauda epididymis
- Sperm head count (testis): examined as above, expressed as sperm heads/10E+6/g testis
Litter observations:
The pups (F1 and F2 litters) were examined on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: day 1 - day 4, day 5 - day 7, day 8 - day 14 and day 15 - day 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation.
The number of surviving pups was determined for day 0, day 4, day 7, day 14 and day 21 and served for the calculation of the viability index and the lactation index, according to adequate formula.
Day 4 after birth preceded standardization of the litters whereas day 21 after birth followed standardization of the litters.
The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to adequate formula.
The pups were weighed on day 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities.
Within necropsy of sacrificed pups (F1 and F2 generations), the brain, spleen and thymus were weighed. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.
All female pups selected for the parental F1 generation (27/group) were evaluated daily for vaginal opening indicative of sexual maturation, starting from day 27 after birth. The selected male pups also were evaluated for sexual maturation by examination for preputial separation starting from day 40 after birth.
Postmortem examinations (parental animals):
PARENTAL F0 AND F1 ANIMALS
The parental F0 and F1 animals were sacrificed for the purpose of necropsy. Animals that died also were subjected to necropsy as soon as possible after death.
Following organs were weighed: whole body, liver, kidneys, adrenals, testes, epididymes (total and caudal), prostate, seminal vesicles (with coagulating glands and their fluid), ovaries, uterus (with cervix uteri and oviducts), spleen, brain and pituitary.
Following tissues/organs were sampled and fixed (4% formaldehyde or Bouin´s solution: Vagina, cervix uteri, uterus, oviducts, ovaries, left testicle, left epididymis, seminal vesicles, coagulating glands, prostate, pituitary, liver, kidneys, spleen, brain, adrenals and all gross lesions.
Following tissues/organs were subjected to light microscopical examination: all gross lesions and all tissues/organ samples from the animals of the control and the 2000 ppm group. In the 100 and 500 ppm groups, all tissue/organ samples of animals suspected of impaired fertility were examined. Particular attention was given to correlate gross lesions with microscopical findings.
The ovaries of each animal were subjected to a differential ovarian follicle count (DOFC).
Postmortem examinations (offspring):
F1 AND F2 PUPS
The brain, spleen and thymus of the sacrificed pups were weighed. The determination of the relative organ weight was based on the pup body weight on day 21 after birth.
The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology.
Skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings.
Stillborn pups as well as pups that died during weaning also were subjected to necropsy.
Statistics:
The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered:
Dunnett test
Fisher´s exact test
Wilcoxon test
Kruskall-Wallis test.
Reproductive indices:
REPRODUCTIVE INDICES FOR THE MALES:
Male mating index (%) = N (males with confirmed mating) x 100 / N (males placed with females)
Male fertility index (%) = N (males with proved fertility) x 100 / N (males placed with females)

Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “with proved fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

REPRODUCTIVE INDICES FOR THE FEMALES:
Female mating index (%) = N (females mated) x 100 / N (females placed with males)
Female fertility index (%) = N (pregnant females) x 100 / N (mated females)
Gestation index (%) =N (females with live pups on day of birth) x 100 / N (pregnant females)
Postimplantation loss (%) = N (implantations) – N (pups delivered)x100 / N (implantations)

Females were defined as mated when vaginal sperm was evidenced, or when they gave birth to a litter, or had fetuses in the uterus.
Females were defined as pregnant when they gave birth to a litter or had pups of fetuses in th
Offspring viability indices:
F1 AND F2 PUPS
Live birth index (%) = N (liveborn pups at birth) x 100 / N (total pups born)
Viability index (%) = N (live pups on day 4 after birth) x 100 / N (total live pups on day of birth)
Lactation index (%) = N (live pups on day 21 after birth) x 100 / N (live pups on day 4 after birth)
Clinical signs:
no effects observed
Description (incidence and severity):
F0 PARENTAL MALES
Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed.

F0 PARENTAL FEMALES
Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed.

F0 PARENTAL FEMALES DURING GESTATION
No treatment-related clinical symptoms were seen.

F0 PARENTAL FEMALES DURING LACTATION
No treatment-related clinical symptoms were seen.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F0 PARENTAL MALES
No mortality was observed.

F0 PARENTAL FEMALES
No treatment-related mortality was observed. In fact one control female was found dead on the first day of mating; necropsy revealed an incidental occurring malignant lymphoma that had infiltrated several organs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F0 PARENTAL MALES
The mean body weights and body weight changes for the treated F0 males were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance.

F0 PARENTAL FEMALES
The mean body weights and body weight changes for the treated F0 females were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance (this was also true for the post weaning period).

F0 PARENTAL FEMALES DURING GESTATION
The mean body weights and body weight changes for the treated F0 females were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance.

F0 PARENTAL FEMALES DURING LACTATION
The mean body weight and body weight gain of the 2000 ppm F0 females during the lactation period was impaired compared to control. In fact, at the end of the lactation period (i.e. day 21 of lactation), the mean body weight of the 2000 ppm females was about 227 g versus ca. 262 g for control females, and therefore it was about 13% below control. The mean body weight change over the whole lactation period (from day 1 to day 21) was about 8 g for the 2000 ppm females versus 28 g for the control females, corresponding to 72% below control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0 PARENTAL MALES
Food consumption for the F0 males of all treated groups was quite similar to that observed in the control group. In fact, a statistically significant but transient decrease in food consumption was observed at 2000 ppm during the first week of treatment only. This decrease was of no biological relevance as the food uptake thereafter reached or even exceeded that of control animals.

F0 PARENTAL FEMALES
Food consumption for the F0 females of all treated groups was quite similar to that observed in the control group. In fact, a statistically significant but transient decrease in food consumption was observed at 500 and 2000 ppm during the first week of treatment only. This decrease was of no biological relevance as the food uptake thereafter reached or even exceeded that of control animals.

F0 PARENTAL FEMALES DURING GESTATION
During gestation, food consumption for the F0 females of all treated groups was similar to that observed in the control group.

F0 PARENTAL FEMALES DURING LACTATION
Food consumption for the 2000 ppm F0 females during lactation was found to be statistically significantly reduced between day 4 to day 7; the overall food consumption from day 1 to day 14 post parturition was about 7% below control.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
F0 PARENTAL MALES
Water consumption was statistically significantly reduced in the F0 males of the 2000 ppm group (about 22% below control during the premating period). For the F0 males of the 500 ppm group, water consumption was slightly reduced compared to control (about 6% below control during the premating period). Water consumption of the 100 ppm F0 males was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES
Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group (about 25% below control during the premating period). For the F0 females of the 500 ppm group, water consumption was slightly reduced compared to control (about 10% below control during the premating period). Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES DURING GESTATION
Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group during gestation (about 29% below control). Water consumption for the 500 ppm F0 females during gestation was slightly reduced compared to control (about 9% below control). Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES DURING LACTATION
Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group during lactation (about 21% below control). During the lactation phase, water consumption of the 500 ppm F0 females was similar to control. Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain.

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Most gross lesions described above could be confirmed/supplemented histopathologically. Moreover, histopathology revealed no adverse treatment-related effects. Considering the reproductive organs of the males and females, which were sacrificed at study termination, only incidental findings were reported, including e.g. dilated uterus horn (4 control females, one 100 ppm female, one 500 ppm female and seven 2000 ppm females), focal tubular atrophy in the left testicle (one male of the 2000 ppm group) and focal epithelial vacuolization in the left epididymis (2 control and one 2000 ppm males). Moreover, no histopathological effects were reported for the vagina, the cervix uteri and the oviducts of the females, and the seminal vesicles and coagulating glands of the males.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data were similar between treated and control groups
Reproductive performance:
no effects observed
Description (incidence and severity):
Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights, gross and histopathological findings of these organs were similar between treated and control groups. - Most of the F0 and F1 parental rats proved to be fertile; single cases of infertility were not treatment-related.

F0 PARENTAL MALES
Excepted for one male of the control group and one male of the100 ppm group, mating was confirmed for all males of the parental F0 generation in all test groups. Thus the mating index for all groups varied between 96 and 100%, independently of treatment or not. The fertility index of the males varied between 92 and 100% independently of treatment or not. For all considered sperm parameters, data were quite similar for the treated and the control groups, indicating that there was no treatment-related effect on these parameters.

F0 PARENTAL FEMALES
The mean cycle from oestrus to oestrus for the F0 female of the treated groups varied between 5.5 +/- 1.41 and 6.3 + 1.85 days, versus 5.3 +/- 1.43 days for the control F0 females. The cycles were generally regular and in a few cases of all groups including control, an unusually prolonged oestrus cycle duration (>= 9 days) was observed. This effect was considered to be spontaneous in nature.
The female mating index for the F0 females ranged between 96 and 100% for all groups including the control. The mean duration of the sperm detection period was about 2.3 and 3 days and showed to treatment-relationship. Because of the non-pregnancy of respectively one control female and one 500 ppm female, the fertility index was about 96% for each of the control and 500 ppm group. In each of the 100 ppm and the 2000 ppm group, all females became pregnant, resulting in a fertility index of 100%for each of these two groups. Moreover, the percentages reported above were within the range of historical control values.
The mean gestation period was quite similar in all groups and ranged from 21.8 to 22.1 days. As all pregnant females had live F1 pups in their litters, the gestation index for all groups was 100%. The mean number of implantation sites was quite similar for all groups, ranging from 259 to 302 per group. Postimplantation loss ranged from 3.5 to 5.4% and showed no treatment-relationship. This indicates that no embryo-/fetolethality resulted from the treatment. The mean number of F2 pups delivered per dam was quite similar for all groups, ranging from10.8 to 11.6. The number of stillborn pups was about 2 -3 per group and was comparable for all groups. The total number of liveborn pups per group ranged between 247 (control) and 290 (500 ppm group); the number of live pups per litter was about 10.1 to 11.2 and was therefore within the same range for all groups. The live birth index for all groups varied between 98 and 100%.

DIFFERENTIAL OVARIAN FOLLICLE COUNT IN F0 A PARENTAL FEMALES
These values indicate that there was no treatment-related adverse effect on the follicle incidence and distribution.
SYSTEMIC TOXICITY
F0 PARENTAL MALES
No mortality was observed. Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed. The mean body weights and body weight changes for the treated F0 males were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance. Food consumption for the F0 males of all treated groups was quite similar to that observed in the control group. In fact, a statistically significant but transient decrease in food consumption was observed at 2000 ppm during the first week of treatment only. This decrease was of no biological relevance as the food uptake thereafter reached or even exceeded that of control animals. Water consumption was statistically significantly reduced in the F0 males of the 2000 ppm group (about 22% below control during the premating period). For the F0 males of the 500 ppm group, water consumption was slightly reduced compared to control (about 6% below control during the premating period). Water consumption of the 100 ppm F0 males was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES
No treatment-related mortality was observed. In fact one control female was found dead on the first day of mating; necropsy revealed an incidental occurring malignant lymphoma that had infiltrated several organs. Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed. The mean body weights and body weight changes for the treated F0 females were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance (this was also true for the post weaning period). Food consumption for the F0 females of all treated groups was quite similar to that observed in the control group. In fact, a statistically significant but transient decrease in food consumption was observed at 500 and 2000 ppm during the first week of treatment only. This decrease was of no biological relevance as the food uptake thereafter reached or even exceeded that of control animals. Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group (about 25% below control during the premating period). For the F0 females of the 500 ppm group, water consumption was slightly reduced compared to control (about 10% below control during the premating period). Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES DURING GESTATION
No treatment-related clinical symptoms were seen. The mean body weights and body weight changes for the treated F0 females were within the control range, with occasional isolated in- or decreases, which were statistically significant but without any biological significance. During gestation, food consumption for the F0 females of all treated groups was similar to that observed in the control group. Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group during gestation (about 29% below control). Water consumption for the 500 ppm F0 females during gestation was slightly reduced compared to control (about 9% below control). Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain.

F0 PARENTAL FEMALES DURING LACTATION
No treatment-related clinical symptoms were seen. The mean body weight and body weight gain of the 2000 ppm F0 females during the lactation period was impaired compared to control. In fact, at the end of the lactation period (i.e.day 21 of lactation), the mean body weight of the 2000 ppm females was about 227 g versus ca. 262 g for control females, and therefore it was about 13% below control. The mean body weight change over the whole lactation period (from day 1 to day 21) was about 8 g for the 2000 ppm females versus 28 g for the control females, corresponding to 72% below control. Food consumption for the 2000 ppm F0 females during lactation was found to be statistically significantly reduced between day 4 to day 7; the overall food consumption from day 1 to day 14 post parturition was about 7% below control. Water consumption was statistically significantly reduced in the F0 females of the 2000 ppm group during lactation (about 21% below control).. During the lactation phase, water consumption of the 500 ppm F0 females was similar to control. Water consumption of the 100 ppm F0 females was similar to control and within the normal variation range for the used rat strain

F1 PARENTAL MALES
No unscheduled mortality was observed. Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed. The mean body weight of the F1 males of the 2000 ppm group was statistically significantly lowered throughout the study period (about 8% below the mean body weight of control males) and at the end of this period, body weight gain for the 2000 ppm F1 males was about 7% below control value. Food consumption for the F1 males of the 2000 ppm group was impaired compared to control (about 7% below control value); in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 males. Water consumption was statistically significantly reduced in the F1 males of the 2000 ppm group (about 28% below control during the premating period). Water consumption of the 500 ppm F1 males also was reduced to about 14% below control; for the F1 males of the 100 ppm, water consumption was quite similar to that of controls and was within the normal biological variation.

F1 PARENTAL FEMALES
No unscheduled mortality was observed. Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed. The mean body weight of the F1 females of the 2000 ppm group was about 4% below control value during the premating period. During the premating period, food consumption for the F1 females of the 2000 ppm group was impaired compared to control (about 8% below control value); in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females. During the premating period, water consumption was statistically significantly reduced in the F1 females of the 2000 ppm group to about 33% below control; the F1 females of the 500 ppm groups also showed reduced water consumption (about 14% below control). Water consumption of the 100 ppm F1 females was quite similar to that of controls and was within the normal biological variation.

F1 PARENTAL FEMALES DURING GESTATION
No treatment-related clinical symptoms were observed during gestation. The mean body weight of the F1 females of the 2000 ppm group was about 5% below control value during this period. Food consumption for the F1 females of the 2000 ppm group was impaired compared to control and was about 7% below control value; in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females. A clear reduction in water consumption during the gestation period (day 0 to 20 post coitum) was reported for both, the 2000 ppm- and the 500 ppm F1 females. In fact, the 2000 ppm females consumed about 35% less water than the control whereas the 500 ppm F1 females consumed about 18% less than control.

F1 PARENTAL FEMALES DURING LACTATION
No treatment-related clinical symptoms were observed during lactation. The mean body weight of the F1 females of the 2000 ppm group was about 12% below control value during the this period. The mean body weight gain for the 2000 ppm females during lactation was about 64% below that body weight gain of the corresponding control females when calculated for the period day 1 to day 21 post parturition. Food consumption for the F1 females of the 2000 ppm group was impaired during the lactation period from day 1 to day 14 post parturition and was about 10% below control value; in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females. During lactation, the water consumption of the 2000 ppm F1 females was statistically reduced to about 26% below control.

REPRODUCTION TOXICITY
F0 PARENTAL MALES
Excepted for one male of the control group and one male of the100 ppm group, mating was confirmed for all males of the parental F0 generation in all test groups. Thus the mating index for all groups varied between 96 and 100%, independently of treatment or not. The fertility index of the males varied between 92 and 100% independently of treatment or not. For all considered sperm parameters, data were quite similar for the treated and the control groups, indicating that there was no treatment-related effect on these parameters.

F0 PARENTAL FEMALES
The mean cycle from oestrus to oestrus for the F0 female of the treated groups varied between 5.5 +/- 1.41 and 6.3 + 1.85 days, versus 5.3 +/- 1.43 days for the control F0 females. The cycles were generally regular and in a few cases of all groups including control, an unusually prolonged oestrus cycle duration (>= 9 days) was observed. This effect was considered to be spontaneous in nature.
The female mating index for the F0 females ranged between 96 and 100% for all groups including the control. The mean duration of the sperm detection period was about 2.3 and 3 days and showed to treatment-relationship. Because of the non-pregnancy of respectively one control female and one 500 ppm female, the fertility index was about 96% for each of the control and 500 ppm group. In each of the 100 ppm and the 2000 ppm group, all females became pregnant, resulting in a fertility index of 100%for each of these two groups. Moreover, the percentages reported above were within the range of historical control values.
The mean gestation period was quite similar in all groups and ranged from 21.8 to 22.1 days. As all pregnant females had live F1 pups in their litters, the gestation index for all groups was 100%. The mean number of implantation sites was quite similar for all groups, ranging from 259 to 302 per group. Postimplantation loss ranged from 3.5 to 5.4% and showed no treatment-relationship. This indicates that no embryo-/fetolethality resulted from the treatment. The mean number of F2 pups delivered per dam was quite similar for all groups, ranging from10.8 to 11.6. The number of stillborn pups was about 2 -3 per group and was comparable for all groups. The total number of liveborn pups per group ranged between 247 (control) and 290 (500 ppm group); the number of live pups per litter was about 10.1 to 11.2 and was therefore within the same range for all groups. The live birth index for all groups varied between 98 and 100%.

F1 PARENTAL MALES
Excepted for one male of the 100 ppm group mating was confirmed for all males of the parental F1 generation in all test groups. Thus the mating index for all groups varied between 96 and 100%, independently of treatment or not. The fertility index of the males varied between 93 and 100% independently of treatment or not. For all considered sperm parameters, data were quite similar for the treated and the control groups, indicating that there was no treatment-related effect on these parameters.

F1 PARENTAL FEMALES
The mean cycle from oestrus to oestrus for the F1 females of all group (i.e. control and treated) was generally regular and varied between 4.4 and 4.8 days. The female mating index for the F1 females ranged between 96% (100 ppm group) and 100% (all remaining groups including control). In fact, one F1 female of the 100 ppm group showed neither sperm in the vaginal smear nor indications of successful implantation. The mean duration of the sperm detection period was about 2.2 and 2.9 days and showed to treatment-relationship; these values were within the normal biological range for the used rat strain. Because of the non-pregnancy of respectively one female of the 100 and one of the 2000 ppm group, the fertility index was about 96% (100 ppm and 2000 ppm) and 100% (control and 500 ppm group). The percentages reported above were within the range of historical control values. The mean gestation period was quite similar in all groups and ranged from 21.8 to 22.1 days. Four pregnant F1 females of the 2000 ppm group did not deliver (implantation sites were seen at necropsy), resulting in a gestation index of 85% for this group. For all other groups, the gestation index was 100%. The mean number of implantation sites was quite similar for all groups, ranging from 256 to 285 per group. The mean postimplantation loss was about 3.7% in control, 2.5% in the 100 ppm group, 15.3% in the 500 ppm group and 2.8% in the 2000 ppm group. Because of its isolated occurrence and the lack of dose-response relationship, the increase in resorption rate observed at 500 ppm was considered to be incidental. The number of stillborn pups was about 0 to 2 per group. The total number of liveborn pups per group ranged between 242 (2000 ppm) and 261 (100 ppm group); the number of live pups per litter was about 9.7 to 11 and was therefore within the same range for all groups. The live birth index for all groups varied between 97 and 100%.

NECROPSY OF F0 PARENTS
The mean absolute organ weights for the treated F0 animals were not significantly different from those of the control animals. The relative kidney and spleen weight of the 2000 ppm group were statistically significantly increased compared to control. In fact, the mean relative kidney weight of the 2000 ppm male and female F0 rats respectively was 5.2% and 6.5% above control value whereas the mean relative spleen weight was 11.3% above control for the F0 females only.
Animals of all groups which were sacrificed at the end of the experiment showed gross lesions, e.g. in the liver (focal constriction), the kidneys (cyst or pelvic dilatation), the testes (reduced size) or the ovaries (cyst). These lesions however occurred isolated and there was to indication of dose-effect relationship. Necropsy of a control female that died on day 76 of treatment: gross pathology revealed a series of lesions including among other presence of a mass in the spleen, enlarged lymph nodes, enlarged adrenal glands, acinar pattern in the liver and foci in the lungs. Histopathology revealed a malignant lymphoma.
Two control females, one female of the 100 ppm group and one female of the 500 ppm did not become pregnant. Excepted for an area of diffuse sparse hair reported for the 100 ppm female, gross pathology of these females revealed no abnormalities. Excepted for a reduction in size of the testes and epididymides of one control male, gross pathology of the corresponding males revealed no abnormalities.
Most gross lesions described above could be confirmed/supplemented histopathologically. Moreover, histopathology revealed no adverse treatment-related effects. Considering the reproductive organs of the males and females, which were sacrificed at study termination, only incidental findings were reported, including e.g. dilated uterus horn (4 control females, one 100 ppm female, one 500 ppm female and seven 2000 ppm females), focal tubular atrophy in the left testicle (one male of the 2000 ppm group) and focal epithelial vacuolization in the left epididymis (2 control and one 2000 ppm males). Moreover, no histopathological effects were reported for the vagina, the cervix uteri and the oviducts of the females, and the seminal vesicles and coagulating glands of the males.

NECROPSY OF F1 PARENTS
The mean terminal body weight of the 2000 ppm males was significantly reduced to about 8.3% below control value. Following organs showed changes in mean absolute weights compared to control: the testes of the 2000 ppm males (reduction of 6.8% below control), the prostate of the 2000 ppm males (reduction of 10.3% below control), the brain of the 100 ppm and the 2000 ppm males (respectively increase of 3.7% and 2.5% above control). The mean terminal body weight of the 2000 ppm females also was reduced to about 8.25 below control value. An increase in absolute weight was reported for the ovaries of the 100 ppm females only (10.4% above control); no further significant changes in absolute organ weights for the females were seen.
Considering the mean relative organ weights, following changes were reported for the males of the 2000 ppm group: increase in relative kidney weight (8.6% above control), increase in relative spleen weight (10.5% above control), increase in relative brain weight (11.9% above control). For the females of the 2000 ppm group, increases in relative kidney, brain and pituitary weights of respectively 8.9%, 10.6% and 20% above control were reported.
Gross treatment-related lesions were reported for the glandular stomach and consisted of small erosions and ulcers within the mucosa. These lesions occurred with a higher incidence in the females of the 2000 ppm group compared to control and to the remained treated groups (i.e., 17 cases versus respectively 6 cases in the control group, 4 cases in the 100 ppm group and 3 cases in the 500 ppm group). In males, no such increase in incidence of lesions in the glandular stomach was seen (one case in the control group, 0 cases in the 100 ppm group, 3 cases in the 500 ppm group and 4 cases in the 2000 ppm group); therefore and in contrast to the females, no association of these lesions to the treatment could be done for the males.
Further gross lesions were reported, which occurred isolated with no indication of dose-effect relationship.
Two females of the 100 ppm group and one female of the 2000 ppm did not become pregnant. Gross pathology of the two 100 ppm females revealed no abnormalities. In contrast, gross pathology of the 2000 ppm female revealed black erosion/ulcer in the mucosa of the glandular stomach. Gross pathology of the corresponding males revealed no abnormalities
Most gross lesions could be confirmed histopathologically. In some cases however, no such confirmation could be reached. This for example was true for the erosion/ulcer in the mucosa of the glandular stomach in following animals: one male and one female of the control group, one 100 ppm female, one 500 ppm female, one 2000 ppm male and two 2000 ppm females. For these animals, no reasonable histological confirmation of the lesions in the glandular stomach was obtained. Considering the reproductive organs, only incidental findings were reported for both males and females, including e.g. dilated uterus horn (9 control females, one 100 ppm female and 11 females of the 2000 ppm group) and focal/diffuse tubular atrophy in the left testicle (one control male, one 2000 ppm male). No histopathological effects were seen in the vagina, the cervix uteri and the oviducts of the females, and the seminal vesicles and coagulating glands of the males.
Treatment-related findings in the mucosa/submucosa of the glandular stomach: In the females, focal erosions in the mucosa were reported for 5 controls, two 100 ppm females, two 500 ppm females and 14 females of the 2000 ppm group. Slight to moderate inflammatory edema in the submucosa was reported for 3 control females, 3 females of the 100 ppm group, 2 females of the 500 ppm group and 10 females of the 2000 ppm group. Focal erosions were not always associated with inflammatory edema and vice versa. The number of females displaying either focal erosion or inflammatory edema or both together was: 5 control females, 3 females of the 100 ppm group, 2 females of the 500 ppm group and 15 females of the 2000 ppm group.

DIFFERENTIAL OVARIAN FOLLICLE COUNT IN F0 AND F1 PARENTAL FEMALES
These values indicate that there was no treatment-related adverse effect on the follicle incidence and distribution.
Dose descriptor:
NOAEL
Remarks:
reproductive performance or fertility
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose except for slight decreases in water consumption due to a palatability problem.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
Clinical signs:
no effects observed
Description (incidence and severity):
F1 PARENTAL MALES
Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed.

F1 PARENTAL FEMALES
Neither treatment-related clinical symptoms nor disturbances of the general behaviour were observed.

F1 PARENTAL FEMALES DURING GESTATION
No treatment-related clinical symptoms were observed during gestation.

F1 PARENTAL FEMALES DURING LACTATION
No treatment-related clinical symptoms were observed during lactation.
Mortality:
no mortality observed
Description (incidence):
F1 PARENTAL MALES
No unscheduled mortality was observed.

F1 PARENTAL FEMALES
No unscheduled mortality was observed.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 PARENTAL MALES
The mean body weight of the F1 males of the 2000 ppm group was statistically significantly lowered throughout the study period (about 8% below the mean body weight of control males) and at the end of this period, body weight gain for the 2000 ppm F1 males was about 7% below control value.

F1 PARENTAL FEMALES
The mean body weight of the F1 females of the 2000 ppm group was about 4% below control value during the premating period.

F1 PARENTAL FEMALES DURING GESTATION
The mean body weight of the F1 females of the 2000 ppm group was about 5% below control value during this period.

F1 PARENTAL FEMALES DURING LACTATION
The mean body weight of the F1 females of the 2000 ppm group was about 12% below control value during this period. The mean body weight gain for the 2000 ppm females during lactation was about 64% below that body weight gain of the corresponding control females when calculated for the period day 1 to day 21 post parturition.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 PARENTAL MALES
Food consumption for the F1 males of the 2000 ppm group was impaired compared to control (about 7% below control value); in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 males.

F1 PARENTAL FEMALES
During the premating period, food consumption for the F1 females of the 2000 ppm group was impaired compared to control (about 8% below control value); in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females.

F1 PARENTAL FEMALES DURING GESTATION
Food consumption for the F1 females of the 2000 ppm group was impaired compared to control and was about 7% below control value; in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females.

F1 PARENTAL FEMALES DURING LACTATION
Food consumption for the F1 females of the 2000 ppm group was impaired during the lactation period from day 1 to day 14 post parturition and was about 10% below control value; in contrast, food uptake in the 100 and 500 ppm was similar to of the control F1 females.


Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
F1 PARENTAL MALES
Water consumption was statistically significantly reduced in the F1 males of the 2000 ppm group (about 28% below control during the premating period). Water consumption of the 500 ppm F1 males also was reduced to about 14% below control; for the F1 males of the 100 ppm, water consumption was quite similar to that of controls and was within the normal biological variation.

F1 PARENTAL FEMALES
During the premating period, water consumption was statistically significantly reduced in the F1 females of the 2000 ppm group to about 33% below control; the F1 females of the 500 ppm groups also showed reduced water consumption (about 14% below control). Water consumption of the 100 ppm F1 females was quite similar to that of controls and was within the normal biological variation.

F1 PARENTAL FEMALES DURING GESTATION
A clear reduction in water consumption during the gestation period (day 0 to 20 post coitum) was reported for both, the 2000 ppm- and the 500 ppm F1 females. In fact, the 2000 ppm females consumed about 35% less water than the control whereas the 500 ppm F1 females consumed about 18% less than control.

F1 PARENTAL FEMALES DURING LACTATION
During lactation, the water consumption of the 2000 ppm F1 females was statistically reduced to about 26% below control.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean terminal body weight of the 2000 ppm males was significantly reduced to about 8.3% below control value. Following organs showed changes in mean absolute weights compared to control: the testes of the 2000 ppm males (reduction of 6.8% below control), the prostate of the 2000 ppm males (reduction of 10.3% below control), the brain of the 100 ppm and the 2000 ppm males (respectively increase of 3.7% and 2.5% above control). The mean terminal body weight of the 2000 ppm females also was reduced to about 8.25 below control value. An increase in absolute weight was reported for the ovaries of the 100 ppm females only (10.4% above control); no further significant changes in absolute organ weights for the females were seen.
Considering the mean relative organ weights, following changes were reported for the males of the 2000 ppm group: increase in relative kidney weight (8.6% above control), increase in relative spleen weight (10.5% above control), increase in relative brain weight (11.9% above control). For the females of the 2000 ppm group, increases in relative kidney, brain and pituitary weights of respectively 8.9%, 10.6% and 20% above control were reported.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross treatment-related lesions were reported for the glandular stomach and consisted of small erosions and ulcers within the mucosa. These lesions occurred with a higher incidence in the females of the 2000 ppm group compared to control and to the remained treated groups (i.e., 17 cases versus respectively 6 cases in the control group, 4 cases in the 100 ppm group and 3 cases in the 500 ppm group). In males, no such increase in incidence of lesions in the glandular stomach was seen (one case in the control group, 0 cases in the 100 ppm group, 3 cases in the 500 ppm group and 4 cases in the 2000 ppm group); therefore and in contrast to the females, no association of these lesions to the treatment could be done for the males.
Further gross lesions were reported, which occurred isolated with no indication of dose-effect relationship.
Two females of the 100 ppm group and one female of the 2000 ppm did not become pregnant. Gross pathology of the two 100 ppm females revealed no abnormalities. In contrast, gross pathology of the 2000 ppm female revealed black erosion/ulcer in the mucosa of the glandular stomach. Gross pathology of the corresponding males revealed no abnormalities
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Most gross lesions could be confirmed histopathologically. In some cases however, no such confirmation could be reached. This for example was true for the erosion/ulcer in the mucosa of the glandular stomach in following animals: one male and one female of the control group, one 100 ppm female, one 500 ppm female, one 2000 ppm male and two 2000 ppm females. For these animals, no reasonable histological confirmation of the lesions in the glandular stomach was obtained. Considering the reproductive organs, only incidental findings were reported for both males and females, including e.g. dilated uterus horn (9 control females, one 100 ppm female and 11 females of the 2000 ppm group) and focal/diffuse tubular atrophy in the left testicle (one control male, one 2000 ppm male). No histopathological effects were seen in the vagina, the cervix uteri and the oviducts of the females, and the seminal vesicles and coagulating glands of the males.
Treatment-related findings in the mucosa/submucosa of the glandular stomach: In the females, focal erosions in the mucosa were reported for 5 controls, two 100 ppm females, two 500 ppm females and 14 females of the 2000 ppm group. Slight to moderate inflammatory edema in the submucosa was reported for 3 control females, 3 females of the 100 ppm group, 2 females of the 500 ppm group and 10 females of the 2000 ppm group. Focal erosions were not always associated with inflammatory edema and vice versa. The number of females displaying either focal erosion or inflammatory edema or both together was: 5 control females, 3 females of the 100 ppm group, 2 females of the 500 ppm group and 15 females of the 2000 ppm group.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean cycle from oestrus to oestrus for the F1 females of all group (i.e. control and treated) was generally regular and varied between 4.4 and 4.8 days.
Reproductive performance:
no effects observed
Description (incidence and severity):
F1 PARENTAL MALES
Excepted for one male of the 100 ppm group mating was confirmed for all males of the parental F1 generation in all test groups. Thus the mating index for all groups varied between 96 and 100%, independently of treatment or not. The fertility index of the males varied between 93 and 100% independently of treatment or not. For all considered sperm parameters, data were quite similar for the treated and the control groups, indicating that there was no treatment-related effect on these parameters.

F1 PARENTAL FEMALES
The mean cycle from oestrus to oestrus for the F1 females of all group (i.e. control and treated) was generally regular and varied between 4.4 and 4.8 days. The female mating index for the F1 females ranged between 96% (100 ppm group) and 100% (all remaining groups including control). In fact, one F1 female of the 100 ppm group showed neither sperm in the vaginal smear nor indications of successful implantation. The mean duration of the sperm detection period was about 2.2 and 2.9 days and showed to treatment-relationship; these values were within the normal biological range for the used rat strain. Because of the non-pregnancy of respectively one female of the 100 and one of the 2000 ppm group, the fertility index was about 96% (100 ppm and 2000 ppm) and 100% (control and 500 ppm group). The percentages reported above were within the range of historical control values. The mean gestation period was quite similar in all groups and ranged from 21.8 to 22.1 days. Four pregnant F1 females of the 2000 ppm group did not deliver (implantation sites were seen at necropsy), resulting in a gestation index of 85% for this group. For all other groups, the gestation index was 100%. The mean number of implantation sites was quite similar for all groups, ranging from 256 to 285 per group. The mean postimplantation loss was about 3.7% in control, 2.5% in the 100 ppm group, 15.3% in the 500 ppm group and 2.8% in the 2000 ppm group. Because of its isolated occurrence and the lack of dose-response relationship, the increase in resorption rate observed at 500 ppm was considered to be incidental. The number of stillborn pups was about 0 to 2 per group. The total number of liveborn pups per group ranged between 242 (2000 ppm) and 261 (100 ppm group); the number of live pups per litter was about 9.7 to 11 and was therefore within the same range for all groups. The live birth index for all groups varied between 97 and 100%.

DIFFERENTIAL OVARIAN FOLLICLE COUNT IN F0 A PARENTAL FEMALES
These values indicate that there was no treatment-related adverse effect on the follicle incidence and distribution.
Dose descriptor:
NOAEL
Remarks:
reproductive performance or fertility
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose except for slight decreases in water consumption due to a palatability problem.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
Clinical signs:
no effects observed
Description (incidence and severity):
The F1 pups showed no treatment-related clinical symptoms of toxicity
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup viability, day 0 to day 4 after birth:
The mean number of delivered F1 pups per dam as well as the rate of liveborn and stillborn pups was not affected by the treatment. During the first 4 days following birth, 11 cases of pup mortality were reported for the 2000 ppm group, versus 2 cases in the control group. Five cases were reported for the 100 ppm group and 2 cases for the 500 ppm group. The viability index for the 2000 ppm group was therefore about 95% versus 98% for the control group. In fact, the reduced pup viability observed at 2000 ppm was mainly related to one dam only, which lost 9 of 14 pups because of bad nursing. Moreover, the viability index of 95% reported for the 2000 ppm group still was within the range of historical control data.

Pup viability, day 4 to day 21after birth:
The pup mortality in each group for this period was indicated by the lactation index. The lactation index ranged from of 97% (control) to 100% (500 ppm group) and no treatment-related differences were evident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of the F1 pups (males + females) of the 2000 ppm group on day 21 were statistically significantly reduced compared to control. In fact, the mean body weight of the 2000 ppm F1 pups was about 15% below control. In contrast, the differences in mean body weight seen between control and each of the 100- and the 500 ppm group were negligible as they were in the range of biological variation.
Sexual maturation:
no effects observed
Description (incidence and severity):
Vaginal opening in the selected F1 female pups occurred within day 30 and 41 post parturition. Preputial separation in the selected F1 male pups occurred between day 42 and 50 post parturition. Differences between treated and control group were not statistical significant and further were within the range of biological variation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in absolute organ weights were reported for the thymus and the spleen of the F1 pups from the treated groups when compared to those of the control group. In fact, the mean thymus weight in the pups of the 100, the 500 and the 2000 ppm group respectively was about 10%, 12% and 19% below control value. The mean spleen weight was 16% and 31% below control value respectively for the pups of the 500 and the 2000 ppm group. Considering the relative pup organ weight, statistically significant differences were reported for the brain and the spleen of the 500 and the 2000 ppm groups when compared to control. In fact, the mean relative brain weight was increased about 7% over control value for the pups of the 500 ppm group, and about 19% over control value for those of the 2000 ppm group. The mean relative spleen weight for the pups of the 500 ppm and the 2000 ppm group respectively was about 11% and 17% below control value.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopical examination of stillborn pups, pups that died intercurrently, culled or surplus pups of the F1 litter revealed no treatment-related abnormalities. In fact, for all groups (i.e. including control) spontaneous findings were seen, which were without any dose-relationship and/or were known to occur at similar or higher incidences within historical control. One pup of the 2000 ppm group was reported to show a malpositioned tail and was subjected to further skeletal examination; this anomaly was related to several misshapen sacral vertebrae.
Other effects:
no effects observed
Description (incidence and severity):
On day 0, the sex distribution (%males, %females) was as follows:
52.2% males and 47.8% females for control
49.6% males and 50.4% females for the 100 ppm group
47.9% males and 52.1% females for the 500 ppm group
47.0% males and 53.0% females for the 2000 ppm group
The sex distribution and sex ratio of the live F1 pups of the day of birth therefore were quite similar for all groups.
On day 21, the sex distribution (%males, %females) was as follows:
51.6% males and 48.4% females for control
51.8% males and 48.2% females for the 100 ppm group
48.0% males and 52.0% females for the 500 ppm group
47.8% males and 52.2% females for the 2000 ppm group
The sex distribution and sex ratio of the live F1 pups of day 21after birth also were quite similar for all groups.
DEVELOPMENTAL TOXICITY
F1 OFFSPRING

Pup viability, day 0 to day 4 after birth:
The mean number of delivered F1 pups per dam as well as the rate of liveborn and stillborn pups was not affected by the treatment. During the first 4 days following birth, 11 cases of pup mortality were reported for the 2000 ppm group, versus 2 cases in the control group. Five cases were reported for the 100 ppm group and 2 cases for the 500 ppm group. The viability index for the 2000 ppm group was therefore about 95% versus 98% for the control group. In fact, the reduced pup viability observed at 2000 ppm was mainly related to one dam only, which lost 9 of 14 pups because of bad nursing. Moreover, the viability index of 95% reported for the 2000 ppm group still was within the range of historical control data.

Pup viability, day 4 to day 21after birth:
The pup mortality in each group for this period was indicated by the lactation index. The lactation index ranged from of 97% (control) to 100% (500 ppm group) and no treatment-related differences were evident.

Sex ratio:
On day 0, the sex distribution (%males, %females) was as follows:
52.2% males and 47.8% females for control
49.6% males and 50.4% females for the 100 ppm group
47.9% males and 52.1% females for the 500 ppm group
47.0% males and 53.0% females for the 2000 ppm group
The sex distribution and sex ratio of the live F1 pups of the day of birth therefore were quite similar for all groups.
On day 21, the sex distribution (%males, %females) was as follows:
51.6% males and 48.4% females for control
51.8% males and 48.2% females for the 100 ppm group
48.0% males and 52.0% females for the 500 ppm group
47.8% males and 52.2% females for the 2000 ppm group
The sex distribution and sex ratio of the live F1 pups of day 21after birth also were quite similar for all groups.

Body weight:
The mean body weight of the F1 pups (males + females) of the 2000 ppm group on day 21 were statistically significantly reduced compared to control. In fact, the mean body weight of the 2000 ppm F1 pups was about 15% below control. In contrast, the differences in mean body weight seen between control and each of the 100- and the 500 ppm group were negligible as they were in the range of biological variation.

Clinical symptoms of toxicity:
The F1 pups showed no treatment-related clinical symptoms of toxicity

Necropsy:
Statistically significant decreases in absolute organ weights were reported for the thymus and the spleen of the F1 pups from the treated groups when compared to those of the control group. In fact, the mean thymus weight in the pups of the 100, the 500 and the 2000 ppm group respectively was about 10%, 12% and 19% below control value. The mean spleen weight was 16% and 31% below control value respectively for the pups of the 500 and the 2000 ppm group. Considering the relative pup organ weight, statistically significant differences were reported for the brain and the spleen of the 500 and the 2000 ppm groups when compared to control. In fact, the mean relative brain weight was increased about 7% over control value for the pups of the 500 ppm group, and about 19% over control value for those of the 2000 ppm group. The mean relative spleen weight for the pups of the 500 ppm and the 2000 ppm group respectively was about 11% and 17% below control value.
The macroscopical examination of stillborn pups, pups that died intercurrently, culled or surplus pups of the F1 litter revealed no treatment-related abnormalities. In fact, for all groups (i.e. including control) spontaneous findings were seen, which were without any dose-relationship and/or were known to occur at similar or higher incidences within historical control. One pup of the 2000 ppm group was reported to show a malpositioned tail and was subjected to further skeletal examination; this anomaly was related to several misshapen sacral vertebrae.
Vaginal opening in the selected F1 female pups occurred within day 30 and 41 post parturition. Preputial separation in the selected F1 male pups occurred between day 42 and 50 post parturition. Differences between treated and control group were not statistical significant and further were within the range of biological variation.

F2 OFFSPRING

Pup viability, day 0 to day 4 after birth:
The mean number of delivered F2 pups per dam as well as the rate of liveborn and stillborn pups was not affected by the treatment. During the first 4 days following birth, 9 cases of pup mortality were reported for the 2000 ppm group, versus 6 cases in the control group. 8 cases were reported for the 100 ppm group and 2 cases for the 500 ppm group. The viability index of the F2 pups for the 2000 ppm group was therefore about 96% versus 97% for the control group. Moreover, the viability index of 96% reported for the 2000 ppm group still was within the range of historical control data.

Pup viability, day 4 to day 21after birth:
The pup mortality in each group for this period was indicated by the lactation index. The lactation index was 99% in control, 98% in the 100 ppm group, 100% in the 500 ppm group and 97% in the 2000 ppm group; no treatment-related differences were evident.

Sex ratio:
On day 0, the sex distribution (%males, %females) was as follows:
55.2% males and 44.8% females for control
45% males and 55% females for the 100 ppm group
50.4% males and 49.6% females for the 500 ppm group
46.6% males and 53.4% females for the 2000 ppm group
The sex distribution and sex ratio of the live F2 pups of the day of birth therefore were quite similar for all groups.
On day 21, the sex distribution (%males, %females) was as follows:
52.7% males and 47.3% females for control
49.2% males and 50.8% females for the 100 ppm group
48.9% males and 51.1% females for the 500 ppm group
49.2% males and 50.8% females for the 2000 ppm group
The sex distribution and sex ratio of the live F2 pups of day 21after birth also were quite similar for all groups.

Body weight:
The mean body weight of the F2 pups (males + females) of the 2000 ppm group was statistically significantly reduced compared to control from day 14 post parturition upwards. In fact, the mean body weight of the 2000 ppm F2 pups was about 11% below control on day 21. The mean body weight gain at 2000 ppm was statistically impaired from day 7 to day 14 post parturition. In fact, from day 4 to day 21, the mean body weight gain of the 2000 ppm F2 pups was about 14% below control. The changes in body weight observed for the 2000 ppm F2 pups were considered to be treatment-related. The body weight of the F2 pups in the 100 and the 500 ppm groups were inconspicuous.

Clinical symptoms of toxicity:
The F2 pups showed no treatment-related clinical symptoms of toxicity

Necropsy:
A statistically significant decrease in absolute organ weight was reported for the spleen of the 2000 ppm F2 pups when compared to those of the control group. In fact, the mean spleen weight for the 2000 ppm F2 pups was about 15% below control value.
Considering the relative pup organ weight, a statistically significant difference was reported for the brain only, which showed an increase in relative weight for the 2000 ppm F2 pups compared to control. In fact, the mean relative brain weight was increased about 14% over control value. The changes in absolute and relative organ weights reported above were related to the significant delays in mean body weight gains, which were reported for the 2000 ppm F2 pups.
The macroscopical examination of stillborn pups, pups that died intercurrently, culled or surplus pups of the F1 litter revealed no treatment-related abnormalities. In fact, for all groups (i.e. including control) spontaneous findings were seen, which were without any dose-relationship and/or were known to occur at similar or higher incidences within historical control. One pup of the 500 ppm group was reported to show an anophthalmia and was subjected to further examinations according to Wilson ´s method; the bilateral anophthalmia was confirmed.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Clinical signs:
no effects observed
Description (incidence and severity):
The F2 pups showed no treatment-related clinical symptoms of toxicity
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup viability, day 0 to day 4 after birth:
The mean number of delivered F2 pups per dam as well as the rate of liveborn and stillborn pups was not affected by the treatment. During the first 4 days following birth, 9 cases of pup mortality were reported for the 2000 ppm group, versus 6 cases in the control group. 8 cases were reported for the 100 ppm group and 2 cases for the 500 ppm group. The viability index of the F2 pups for the 2000 ppm group was therefore about 96% versus 97% for the control group. Moreover, the viability index of 96% reported for the 2000 ppm group still was within the range of historical control data.

Pup viability, day 4 to day 21after birth:
The pup mortality in each group for this period was indicated by the lactation index. The lactation index was 99% in control, 98% in the 100 ppm group, 100% in the 500 ppm group and 97% in the 2000 ppm group; no treatment-related differences were evident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of the F2 pups (males + females) of the 2000 ppm group was statistically significantly reduced compared to control from day 14 post parturition upwards. In fact, the mean body weight of the 2000 ppm F2 pups was about 11% below control on day 21. The mean body weight gain at 2000 ppm was statistically impaired from day 7 to day 14 post parturition. In fact, from day 4 to day 21, the mean body weight gain of the 2000 ppm F2 pups was about 14% below control. The changes in body weight observed for the 2000 ppm F2 pups were considered to be treatment-related. The body weight of the F2 pups in the 100 and the 500 ppm groups were inconspicuous.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in absolute organ weight was reported for the spleen of the 2000 ppm F2 pups when compared to those of the control group. In fact, the mean spleen weight for the 2000 ppm F2 pups was about 15% below control value.
Considering the relative pup organ weight, a statistically significant difference was reported for the brain only, which showed an increase in relative weight for the 2000 ppm F2 pups compared to control. In fact, the mean relative brain weight was increased about 14% over control value. The changes in absolute and relative organ weights reported above were related to the significant delays in mean body weight gains, which were reported for the 2000 ppm F2 pups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopical examination of stillborn pups, pups that died intercurrently, culled or surplus pups of the F1 litter revealed no treatment-related abnormalities. In fact, for all groups (i.e. including control) spontaneous findings were seen, which were without any dose-relationship and/or were known to occur at similar or higher incidences within historical control. One pup of the 500 ppm group was reported to show an anophthalmia and was subjected to further examinations according to Wilson ´s method; the bilateral anophthalmia was confirmed.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio:
On day 0, the sex distribution (%males, %females) was as follows:
55.2% males and 44.8% females for control
45% males and 55% females for the 100 ppm group
50.4% males and 49.6% females for the 500 ppm group
46.6% males and 53.4% females for the 2000 ppm group
The sex distribution and sex ratio of the live F2 pups of the day of birth therefore were quite similar for all groups.
On day 21, the sex distribution (%males, %females) was as follows:
52.7% males and 47.3% females for control
49.2% males and 50.8% females for the 100 ppm group
48.9% males and 51.1% females for the 500 ppm group
49.2% males and 50.8% females for the 2000 ppm group
The sex distribution and sex ratio of the live F2 pups of day 21after birth also were quite similar for all groups.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Dose descriptor:
LOAEL
Remarks:
(developmental toxicity)
Generation:
F2
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Reproductive effects observed:
no
Test substance intake:
---------------------
__________________________________________________________
Animals Mean intake of test material(mg/kg bw/day)
100 ppm 500 ppm 2000 ppm
F0 males 9.4 44.8 152
F0 females 12.1 57.4 191.2
F0 females,GP 12 53.9 167.3
F0 females,LP 17.1 84.5 286.6
F1 males 12.8 60.4 213.1
F1 females 15.0 69.7 239.1
F1 females,GP 13.4 56.4 188.8
F1 females,LP 18.6 92.9 304.7
__________________________________________________________
GP, gestation period
LP, lactation perio
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The effects of glutaraldehyde (GA 50%) on the reproduction of Wistar rats were investigated in a two-generation study, performed in accordance with GLP and OECD Guideline 416, where the rats received the test substance in drinking water (BASF, 2001). The nominal test concentrations of the test substance were 100, 500 and 2000 ppm. Symptoms of systemic toxicity were seen in parental animals (F0 & F1, both sexes) at the highest tested concentration of 2000 ppm, and mainly consisted of decreased food and water consumption and impairments in body weight changes; furthermore, F1 parental females showed treatment-related lesions in the glandular stomach. A developmental toxicity affecting the F1 and F2 pups was seen at 2000 ppm, i.e. at a dose level, which clearly was toxic to the parental animals. In contrast even at the highest test concentration of 2000 ppm, the reproductive performance and fertility remained inconspicuous. The NOAEL for parental toxicity was determined to be 500 ppm, which corresponded to about 45 and 60 mg/kg bw/day of test substance (GA 50%) for the males and females, respectively. The NOAEL for reproductive performance and fertility was determined to be 2000 ppm, i.e., about 150 and 190 mg/kg bw/day of test substance for the males and females, respectively. The NOAEL for developmental toxicity was determined to be 500 ppm.

In a second two-generation toxicity study published by Neeper-Bradley et al. (2000) similar results were observed. In this study, performed similar to OECD Guideline 416, rats received glutaraldehyde at concentrations of 50, 250, and 1000 ppm in drinking water. Systemic toxicity was observed at 250 and 1000 ppm. Parental body weights and body weight gains were significantly reduced at 1000 ppm at some periods, particularly during prebreed. Food consumption was significantly reduced at 1000 ppm for F0 and F1 parents during the prebreed and gestation periods, and at 250 ppm for F0 males during prebreed and gestation and F1 females during gestation and lactation. Water consumption was reduced throughout the prebreed period for the F0 and F1 parents of the 250 and the 1000 ppm groups. No indication of impairment of fertility was observed up to and including the highest tested concentration. For F1 and F2 offspring of the 1000 ppm group, body weight was reduced from day 21 to day 28 of lactation. The NOAELs were determined to be 50, 1000, and 250 ppm for systemic toxicity, effects on fertility, and developmental toxicity, respectively. Overall it can be concluded that glutaraldehyde did not affect the reproductive performance and fertility at a dose level that was found to cause systemic toxicity in the parental animals (i.e. 1000 ppm). Symptoms of developmental toxicity in F1 und F2 pups only were seen at a dose level, which clearly were toxic to parental animals.

Effects on developmental toxicity

Description of key information

Referring to developmental toxicity and teratogenicity, a series of studies including range-finders is available, which had been conducted with mice, rabbits and rats. All studies with rats and rabbits were conducted according to the OECD TG 414, and followed GLP. Neither an embryo/fetotoxic nor a teratogenic potential could be evidenced for glutaraldehyde.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-03-20 until 1990-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1981)
Qualifier:
according to guideline
Guideline:
other: "Commission Directive 87/302/EEC of 18 November 1987 adapting to technical progress for the ninth time Council Directive 67/548/EEC, pp. 24 - 26" (1988)
Qualifier:
according to guideline
Guideline:
other: "EPA/FIFRA Pesticide Assessment Guidelines", Subdivision F, § 83-3, pp. 126 -130, NTIS (Nov. 1984)
Qualifier:
according to guideline
Guideline:
other: "EPA/TSCA New and Revised Health Effects Test Guidelines [Developmental Toxicity Study] ", NTIS (Oct. 1984).
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
_ Name of test material (as cited in study report): Glutaraldehyde
- Physical state: colourless-fluid
- Analytical purity: 50.3% in water
- Lot/batch No.: no data
- Stability under test conditions: stability of the test substance (content of active ingredient > 51%) proven by reanalysis at study ending.
- Storage condition of test material: refrigerator, under N2, in the dark
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach/Riss, Germany
- Age at study initiation: about 68 to 77 days old at study start (corresponding to day 0, detection of sperms in the vaginal smear)
- Weight at study initiation: ca. 230 g
- Housing: individual housing in type D III stainless steel wire mesh cages (floor area about 800 cm2)
- Diet (e.g. ad libitum): ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water was available ad libitum during days 0 to 6 p.c. and 16 to 20 p.c., while doubly distilled water with or without addition of glutaraldehyde was available ad libitum during the treatment period (days 6 to 16 p.c.)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): the animals were kept in fully air-conditioned rooms, not further specified
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The test solutions were prepared in doubly distilled water. The test material was weighed in a beaker depending an the dose group and topped up with doubly distilled water, which also was weighed). Thereafter, the solutions were placed on a magnetic stirrer for about 15 minutes. The solutions were filled into Makrolon@ drinking water bottles using a semi-automatic device.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test material in doubly distilled water were carried out before the beginning of the study.
Furthermore, samples of the test solutions were sent to the analytical laboratories of BASF AG twice during the study period for verification of the concentrations.
The test solutions were analyzed by colorimetrically after reaction with thiobarbituric acid and FeCl3.
Details on mating procedure:
Three to four untreated females were mated with one untreated fertile male rat of the same breed. Mating took place from ca. 16.00 hours to ca. 7.30 hours on the following day; the mating period was therefore about 15 to 16 hours.
Duration of treatment / exposure:
day 6 to day 16 of gestation
Frequency of treatment:
continuously in drinking water
Duration of test:
21 days
Dose / conc.:
50 ppm (nominal)
Remarks:
in water; 5.2 mg/kg bw/day actual ingested
Dose / conc.:
250 ppm (nominal)
Remarks:
in water; 25.7 mg/kg bw/day actual ingested
Dose / conc.:
750 ppm (nominal)
Remarks:
in water; 68 mg/kg bw/day actual ingested
No. of animals per sex per dose:
25 females/group
Control animals:
yes, concurrent no treatment
Details on study design:
CHOICE OF TEST DOSES
Dose selection was based on 2 range-finding studies
In the first range-fincling study (BASF AG, 1991, Report No: 13R0599/89035), the test substance was administered to pregnant rats (10/dose) with the drinking water from day 6 to day 16 post coitum at doses of 0, 100 and 500 ppm, corresponding to 0, 10 and 50 mg/kg body weight/day. Some findings assessed as possibly being substance-related were reported for the 500 ppm level, including a diminished food consumption (day 6 to 8 p.c.), a reduced water intake (day 6 to 13 p.c.), and foci in the glandular stomach (2 cases only). The dose level of 100 ppm was defined as the NOAEL for both, the dams and the fetuses.
In the second range-finding study (BASF AG, 1991, Report No: 10R0599/89048), the test substance was administered by gavage to pregnant rats (10/dose) from day 6 to day 15 post coitum) at doses of 0, 10 and 50 mg/kg bw/day. A series of findings assessed as possibly being substance-related was reported for the 50 mg/kg bw/day dose level, including reduced food consumption, reduced mean body weights, diminished body weight gains, reduced corrected body weight gain, reduced nutritional state, labored breathing and piloerection seen in some cases, one case of vaginal hemorrhage on days 15 and 16 p.c. with absence of viable fetuses, lowering of total protein and globulin concentration, and increased relative kidney weights. Moreover, all animals showed thickening of margo plicatus, and in 3 cases, the animals additionally showed lesions of the glandular stomach. A slight increase in post implantation loss due to a marginally increased number of mainly late resorptions also was reported. In the 10 mg/kg bw/day group, one case of margo plicatus was reported.

Taking into consideration the results above, it became obvious that the administration of glutaraldehyde with the drinking water was the more appropriate route of administration, because the dams showed only minimal signs of maternal toxicity in concentrations which produced frank toxicity in the dams when given by gavage; however, a high dose of 500 ppm (i.e., ca. 50 mg/kg bw/day) seemed to be too low to fulfill the recommendations af the test guidelines, since the highest dose level to be selected should induce some overt maternal toxicity such as slight weight loss. Thus the dose levels for the present study were fixed as follows: 0, 50, 250, 750 ppm.
Maternal examinations:
MORTALITY AND CLINICAL SYMPTOMS
the animals were checked daily, at least once for clinical symptoms and twice for mortality on working days. On Saturdays, Sundays or public holidays, the animals were only checked once a day.

BODY WEIGHT
Body weight was determined on day on day 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c., and body weight change was calculated on the basis of the values obtained.

FOOD CONSUMPTION
Except for day 0 p.c., food consumption was determined on the same days as body weight, and only pregnant dams were considered for calculation.

WATER CONSUMPTION
Except for day 0 p.c., water consumption was determined on the same days as body weight and additionally on day 16 p.c., and only pregnant dams were considered for calculation.

TEST SUBSTANCE INTAKE
The intake of test substance (IT, in mg/kg bw/day) was calculated according to following formula:
ITx = WC x D /BWx

D = dose in ppm
WC = mean daily water consumption on day x + y, y = 1, 2 or 3; in g
BWx = body weight on day x; in g
Only pregnant dams were considered for calculation

POST-MORTEM EXAMINATIONS
On day 20 p.c. all dams were sacrificed for the purpose of necropsy. The sacrificed dams were subjected to gross pathology and the uterus and ovaries were removed.
Only pregnant dams, which were sacrificed at the end of the study period, were considered for the evaluations of following parameters: gravid uterine weight, mean net maternal body weight gain, reproduction data.
Following sacrifice, the corrected body weight gain (i.e. the net maternal body weight change) was calculated from the terminal body weight by subtraction of (1) the uterus weight and (2) the body weight measured on day 6 p.c.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination; the examinations included:
- Gravid uterus weight;
- Number of corpora lutea;
- Number of distribution of implantations sites classified as live fetuses and dead implantations, with dead implantations comprising early resorptions, late resorptions and dead fetuses.

The conception rate (CR) as well as the pre- and post- implantation losses (Pre-I¸ Post-I) were calculated according to following formula:
- CR = Number of pregnant animals x 100 / Number of fertilized animals
- Pre-I = (Number of corpora lutea - Number of implantations) x 100 / Number of implantations
- Post-I = (Number of implantations - Number of live fetuses) x 100 / Number of implantations
Fetal examinations:
GENERAL EXAMINATION
The fetuses were extracted from the uterus and were examined for following parameters:
Fetal weight, sex (measurement of the anogenital distance, later confirmed by internal examination), external abnormalities, viability, placentae, umbilical cords, fetal membranes, and fetal liquids. Individual placental weights were recorded.
One half of the fetuses per dam were placed in ethyl alcohol whereas the other half was fixed in Bouin´s solution for further evaluation.

SKELET
The skeletons of the fetuses fixed in ethyl alcohol were stained according to the method of Dawson (Stain Technol. 1: 123, 1926) for examination under a stereomicroscope.

SOFT TISSUES
The fetuses fixed in Bouin´s solution were examined for effects in the organs according to the method of Barrow and Taylor (J. Morph. 127: 291-306, 1969).
Statistics:
Food and water consumption, body weight and body weight change, corrected maternal body weight gain, gravid uterine weight, weight of the fetuses, weight of the placentae, corpora lutea, implantations, pre- and post implantation losses, resorptions and live fetuses, were assessed by means of Dunnett´s Test.
Conception rate, maternal mortality and all fetal findings were assessed by means of Fisher´s Exact Test.
Historical control data:
Historical control data were added to the study report.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related signs of toxicity were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The maternal body weights and body weight changes were quite similar for the treated and the control group. The values were within the range of biological variation and differences between the groups were without biological relevance.

Net maternal body weight change from day 6 p.c.: No significant differences between treated and control groups were seen. The values for all groups ranged between 43.9 +/- 8.45 g ( 50 ppm group) and 48.5 +/- 9.32 g (750 ppm group).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The maternal food consumption was quite similar for the treated and the control group. The values were within the range of biological variation and differences between the groups showed no treatment-relationship.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was inconspicuous in the 50 ppm group whereas a slight decrease in water consumption (up to 12%) was reported for the 250 ppm group from day 10 to day 15 p.c. In the 750 ppm group, water consumption was clearly reduced to about 19% below control during the period ranging from day 6 to day 16 p.c.

The approximate test substance intake (mg/kg bw/day) was 0, 5.2, 25.7 and 68.0 mg/kg bw/day for 0, 50, 250 and 750 ppm, respectively.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterine weight was quite similar for all treated and the control groups; the values for all groups ranged between 74.9 +/- 25.13 g (control) and 82.2 +/- 16.14 g (250 ppm group).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy revealed no treatment-related abnormalities.
Description (incidence and severity):
Females excluded because of non pregnancy:
Following females were partly or totally excluded from data evaluation/calculation because of non pregnancy: 5 females of the control group, 3 females of the 50 ppm group, 2 females of the 250 ppm group, and 2 females of the 750 ppm group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre- and post implantation losses were inconspicuous.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Number of resorptions was inconspicuous.
Dead fetuses:
no effects observed
Description (incidence and severity):
Number of live fetuses was inconspicuous.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A conception rate of 92% respectively for the 250 and 750 ppm groups was reported, versus 80% for control. Conception rate was inconspicuous.
Conception rate, number of corpora lutea, number of implantation sites were inconspicuous.
Dose descriptor:
NOEL
Remarks:
maternal toxicity
Effect level:
50 ppm
Basis for effect level:
other: No adverse effetcs observed at this dose.
Dose descriptor:
LOEL
Remarks:
maternal toxicity
Effect level:
250 ppm
Basis for effect level:
water consumption and compound intake
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on sex ratio, placental weight, fetal weight were reported. No treatment-related external malformations were seen. In fact, a single case of malformation (aglossotomia) was reported for a fetus of the 750 ppm group; this type of malformation is known to occur in the historical control at a low frequency and was therefore considered to be a spontaneous finding. One case of fused placenta was reported for one fetus of the 50 ppm group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations of the skull, the sternum (e.g. dislocated ossification centers), the vertebral column and/or the ribs were reported for 9 fetuses of the control group, 8 fetuses of the 50 ppm group, 2 fetuses of the 250 ppm group and 9 fetuses of the 750 ppm group. The only statistically significant difference was the lower number of fetuses affected of the 250 ppm group. Variations concerning the ribs (e.g. shortened 13th ribs or accessory 14th ribs), the sternum and the vertebral column). Signs of retardations (e.g. incomplete or missing ossification of vertebral bodies) were seen in all groups including control. A statistically significantly increased number of 50 ppm fetuses with incomplete ossification of the sternebrae, as well as an increased litter incidence of fetuses with incomplete ossification of the sternebrae were reported, which indeed were considered to be of spontaneous nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Organ examination revealed no treatment-related effects. In fact, one case of organ malformation (situs inversus) was reported for one control fetus. Variations were seen, including dilated renal pelvis and hydroureter, which showed no dose-response relationship and were within the normal range of biological variation for the used strain.
Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
750 ppm
Sex:
male/female
Basis for effect level:
other: No indication for an embryo-/fetotoxic potential up to the highest tested dose.
Remarks on result:
other: 68 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
68 mg/kg body weight/day
Effect level:
750 ppm
Sex:
male/female
Basis for effect level:
other: No indication for a teratogenic potential at the highest tested dose.
Remarks on result:
other: 68 mg/kg bw/day
Abnormalities:
effects observed, non-treatment-related
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-03-05 until 1990-04-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study followed OECD guideline and was conducted in accordance with GLP. However following deficiencies were observed: (1) The correctness of the prepared concentrations showed some discrepancies. In fact, within the first analyses on samples taken at the study start, the actual values were significantly increased compared to the nominal values (up to 20%). In contrast, the analyses at the end of the study period revealed values about 20% below the nominal values (intermediate and high dose), whereas additional analyses conducted on frozen samples revealed concentrations, which were about 10% above the nominal values (intermediate and high dose). These discrepancies however do not affect the validity of the study. (2) Only 4 foetuses were available in the 45 mg/kg bw/day group for the evaluation of the teratogenic potential of the test substance. Because of this low number of foetuses, no clear assessment of teratogenicity was possible.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
other: "Commission Directive 87/302/EEC of 18 November 1987 adapting to technical progress for the ninth time Council Directive 67/548/EEC, pp. 24 - 26" (1988)
Qualifier:
according to guideline
Guideline:
other: "EPA/FIFRA Pesticide Assessment Guidelines", Subdivision F, § 83-3, pp. 126 -130, NTIS (Nov. 1984)
Qualifier:
according to guideline
Guideline:
other: "EPA/TSCA New and Revised Health Effects Test Guidelines [Developmental Toxicity Study] , NTIS (Oct. 1984).
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
_ Name of test material (as cited in study report): Glutaraldehyde
- Physical state: colourless-fluid
- Analytical purity: 50.3% in water
- Lot/batch No.: 89-599
- Stability under test conditions: stability of the test substance (content of active ingredient > 51%) proven by reanalysis at study ending.
- Storage condition of test material: refrigerator, underN2, in the dark
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach/Riss, Germany
- Age at study initiation: about 26 to 27 weeks old
- Weight at study initiation: about 2.596 kg /animal
- Housing: individual housing in type K 300/8 stainless steel wire mesh cages (floor area about 2860 cm2)
- Diet (e.g. ad libitum): pelleted Kliba maintenance diet type 23-341-4 for rabbits supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): the animals were kept in fully air-conditioned rooms, not further specified
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The test solutions were prepared twice a week after the stability of aqueous solutions of test material for a period of at least 4 days had been proven.
For the preparation of the solutions an appropriate amount of test material was weighed, subsequently topped up with doubly distilled water and intensively stirred.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test material in doubly distilled water were carried out before the beginning of the study in the analytical laboratories of BASF AG.
Furthermore, samples of each aqueous preparation of the test material were sent to the analytical laboratories of BASF AG twice during the study period for verification of the test concentrations. Due to unexpected pronounced differences between the nominal and the actual analytical values, additional analyses of the test material solutions were carried out on frozen samples after the end of the study.
The test solutions were analyzed colorimetrically after reaction with thiobarbituric acid and FeCl3.
Details on mating procedure:
The female rabbits were fertilized by means of artificial insemination, using pooled ejaculate from male Himalayan rabbits of the same breed. The day of insemination was designated as day 0 (study start) and the following day as day 1 post-insemination (p.i.).
Duration of treatment / exposure:
From day 7 to day 19 p.i. (corresponding to the period of organogenesis)
Frequency of treatment:
7 days/week
Duration of test:
23 days (13 days of treatment and 10 days post-exposure)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
glutaraldehyd actual ingested
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
glutaraldehyd actual ingested
Dose / conc.:
45 mg/kg bw/day (actual dose received)
Remarks:
glutaraldehyd actual ingested
No. of animals per sex per dose:
15 females/group
Control animals:
yes, concurrent no treatment
Details on study design:
CHOICE OF TEST DOSES
Dose selection was based on 2 range-finding studies:
In the first study (BASF AG,1991, Report No: 23R0599/89036) the test substance was given to the rabbits in the drinking water from day 7 to day 20 post-insemination (p.i.) at following concentrations: 0, 100 and 500 ppm. Each test group comprised 6 pregnant rabbits and the theoretical intake of test substance was calculated to be 0, 5 and 25 mg/kg bw/d, respectively. The lowest tested dose of 100 ppm (mean actual substance intake of ca. 7.1 mg/kg bw/day) was associated with a clearly reduced food consumption and a slightly reduced water intake during the treatment period. At 500 ppm (corresponding to a mean actual substance intake of ca. 23.4 mg/kg bw/day) also resulted in clearly reduced food consumption and drastically reduced water intake during the treatment period; furthermore, an increased post implantation loss was reported. Neither embryo/foetotoxic nor teratogenic treatment-related effects were seen.
In the second study (BASF AG, 1991, Report No: 20R0599/89038), the test substance was given to the rabbit by gavage from day 7 to day 19 p.i. Following doses were tested: 0, 5 and 25 mg/kg bw/d. Each test group comprised 6 pregnant rabbits. No clear substance-related effects were seen at 5 mg/kg bw/day; in contrast, at 25 mg/kg bw/day food consumption was clearly reduced during the first days of treatment and lowered calcium, glucose, protein and albumin concentrations also were reported. Two cases of microfocal gastritis in the fundus/pylorus region were regarded as questionable effect since one dam of the control group showed similar but more severe findings. Neither embryo/foetotoxic nor teratogenic treatment-related effects were seen.
On the basis of the findings mentioned above, it was decided that gavage would result in a higher and more uniform substance intake.Since only marginal toxicity was seen at 25 mg/kg bw/d, the test doses for the main study were chosen to be as follows: 0, 5, 15 and 45 mg/kg bw/day.
Maternal examinations:
MORTALITY AND CLINICAL SYMPTOMS
The animals were checked daily, at least once for clinical symptoms and twice for mortality on working days. On Saturdays, Sundays or public holidays, the animals were only checked once a day for mortality. In case of presence of clinical signs of toxicity, the animals were checked several times a day.

BODY WEIGHT
Body weight was determined on day 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i., and body weight change was calculated on the basis of the values obtained.

FOOD CONSUMPTION
Food consumption was determined daily during the entire study period.

WATER CONSUMPTION
Not specified

POST-MORTEM EXAMINATIONS
On day 29 p.i. all surviving females were sacrificed for the purpose of necropsy and were examined macroscopically. The fetuses were dissected from the uterus and subjected to further examinations. Dams showing signs of abortion were sacrificed and examined as above. Following sacrifice, the net maternal body weight change (i.e. the corrected body weight gain) was calculated from the terminal body weight by subtraction of (1) the uterus weight and (2) the body weight measured on day 7 p.i.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination; the examinations included:
- Gravid uterus weight;
- Number of corpora lutea;
- Number of distribution of implantations sites classified as live fetuses and dead implantations, with dead implantations comprising early resorptions, late resorptions and dead fetuses.

The conception rate (CR) as well as the pre- and post- implantation losses (Pre-I¸ Post-I) were calculated according to following formula:
- CR = Number of pregnant animals x 100 / Number of fertilized animals
- Pre-I = (Number of corpora lutea - Number of implantations) x 100 / Number of implantations
- Post-I = (Number of implantations - Number of live fetuses) x 100 / Number of implantations


Fetal examinations:
GENERAL EXAMINATION
The fetuses were extracted from the uterus and were examined for following parameters:
Fetal weight, external abnormalities, viability, placentae, umbilical cords, fetal membranes, and fetal liquids. Individual placental weights were recorded.

SKELET
The skeletons of the fetuses fixed in ethyl alcohol were stained according to the method of Dawson (Stain Technol. 1: 123, 1926).

SOFT TISSUES
For the purpose of soft tissue examination, the fetuses were sacrificed and their abdomen and thorax were opened for in situ examination of the organs prior removal. Heart and kidney were sectioned for internal examination. The sex of the fetuses was determined by internal examination of the gonads. In case of abnormalities (e.g. hydrocephalus, microphthalmia or cleft palate), the head was separated from the remaining body and was fixed in Bouin´s solution for further procession and assessment according to Wilson´s method (Wilson and Warkany, Teratology: principles and techniques. The university of Chicago Press, Chicago and London, 1965).
Statistics:
Food and water consumption, body weight and body weight change, corrected maternal body weight gain, gravid uterine weight, weight of the fetuses, weight of the placentae, corpora lutea, implantations, pre- and post implantation losses, resorptions and live fetuses, were assessed by means of Dunnett´s Test.
Conception rate, maternal mortality and all fetal findings were assessed by means of Fisher´s Exact Test.
Historical control data:
Historical control data were added to the study report.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality was seen in the 45 mg/kg bw /day group, where 3 dams died on day 9 p.i., and 2 further on day 11 p.i. Almost all surviving dams of the 45 mg/kg bw/day group showed no defecation during one or more days of the treatment and the post-treatment period. This effect was related to the decrease in food consumption reported for the same days/periods of days. Moreover, 9 surviving dams of the 45 mg/kg bw/day also suffered from soft feces and/or diarrhea (6 cases). Blood was seen between day 14 and 18 p.i. in the bedding of 3 dams of this group. The remaining test groups were inconspicuous.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of the 45 mg/kg bw/day females was statistically significantly reduced from day 11 to day 29 p.i. (about 22% below control value at study termination). A severe loss in body weight in this group was observed during the treatment period (from day 7 to day 19 p.i.) and thereafter, from day 19 to day 21 p.i. From day 21 to day 29 p.i., the females of the 45 mg/kg bw/day group gained weight again. The impairments in body weight clearly were related to the reduced food consumption of the animals and were therefore considered to be treatment-related. Body weight and body weight change in the remaining test groups (5 and 15 mg/kg bw/day) were inconspicuous compared to control.

Net maternal body weight change from day 7 p.i.:
Whereas both, the 5 and the 15 mg/kg bw/day group were inconspicuous, the net maternal body weight change in the 45 mg/kg bw/day group significantly was reduced compared to control (i.e., -324.4 +/- 163.71 g versus -101.6 +/- 61.60 g in control; p<0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in the 45 mg/kg bw/day group was severely reduced during the treatment period (day 7 to day 19 p.i.) and from day 19 to day 21 post treatment. Thereafter food consumption turned back to or even exceeded control values. The impairment in food consumption observed at 45 mg/kg bw/day was considered to be treatment-related. Whereas food consumption in the 15 mg/kg bw/day group was inconspicuous, a slight impairment was seen at 5 mg/kg bw/day, which however rather was incidental and without any biological significance than treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Gravid uterine weight (GUW):
The mean gravid uterus weight in the 45 mg/kg bw/day group was found to be statistically significantly reduced compared to control, reaching about 7% of the control value (i.e., 24.4 +/- 51.69 g versus 337.9 +/- 81.49 g in control; p<0.01). This effect was considered to be treatment-related and furthermore was in line with the increased number of resorptions and postimplantation loss seen in the 45 mg/kg bw/day group. For the remaining groups (control, 5, 15 mg/kg bw/day), the gravid uterine weight was quite similar and/or within the range of biological variation
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy of the animals that died intercurrently revealed signs of irritation within the gastro-intestinal tract, including diffuse reddening of the fundus, thickening of the wall of the fundus and of the pylorus due to edema, and ulceration and/or distention of the cecum or the colon. No such signs were seen in the 45 mg/kg bw/day survivors that were sacrificed at the end of the study.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
In the 45 mg/kg bw/day group, the post-implantation loss also was clearly increased (94.3%).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
In the 45 mg/kg bw/day group, 9 of 15 animals had no viable fetuses at all but only early resorptions. A severely increased resorption rate was reported.
Description (incidence and severity):
In the 45 mg/kg bw/day group, only one female gave 4 alive fetuses on the scheduled date.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A conception rate of about 93% was reported for respectively the 5 and the 15 mg/kg bw/day group, whereas conception rate reached 100% in control and in the 45 mg/kg bw/day group. Moreover, all considered parameters (conception rate, number of corpora lutea, number of implantation sites were inconspicuous for the 5 and the 15 mg/kg bw/day groups; in fact, differences here were incidental and within the normal range of deviations known for the used test animal.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Females excluded because of non pregnancy:
Following females were partly or totally excluded from data evaluation/calculation because of non pregnancy: 1 female of the 5 mg/kg bw/day group, 1 female of the 15 mg/kg bw/day group, 5 females of the 45 mg/kg bw/day group (these animals died intercurrently).
Dose descriptor:
LOAEL
Remarks:
maternal toxicity
Effect level:
45 mg/kg bw/day (actual dose received)
Basis for effect level:
body weight and weight gain
clinical signs
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
mortality
pre and post implantation loss
other: Statistically significant reduction in mean gravid uterus weight (ca.7% of control value)
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Basis for effect level:
other: No treatment-related adverse effects observed at this dose.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal weight in the 5 and the 15 mg/kg bw/day groups were quite similar to control and/or within the range of biological variation. For the 45 mg/kg bw/day group, due to the limited number of live fetuses, no clear assessment of the parameter was possible. In fact, for this group, the fetal weight also was the lowest (mean of 26.9 g versus 41.7 g for control, 40.2 g for the 5 mg/kg bw/day group, and 41.3 g for the 15 mg/kg bw/day group) and furthermore was outside the historical control range (29.9 – 53.2 g, based on 188 control litters).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Fetal sex-distribution in the 5 and the 15 mg/kg bw/day groups was quite similar to control and/or within the range of biological variation. For the 45 mg/kg bw/day group, due to the limited number of live fetuses, no clear assessment of this parameter was possible.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The examination of the fetuses revealed neither malformations nor variations or unclassified abnomalities in the treated groups; in contrast, a single case of cheiloschisis (malformation) and a single case of pseudoankylosis (variation) were reported for the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations of skull, vertebral column and sternum were reported for the control group (4 cases) and the 15 mg/kg bw /day group (one case); in contrast, no such skeletal malformations were seen in the 5 and the 45 mg/kg bw/day groups. Variations such as e.g. accessory or shortened ribs were seen in single fetuses of all groups including the control; signs of retardations (e.g. incomplete or missing ossification of skull bones) also occurred in all groups. All the findings occurred without dose-relationship and/or were without any biologically relevance, and/or within the range of historical control data known for the rabbit strain used.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One case of hydrocephaly was reported for the control group. Three cases of agenesia of the gallbladder (2 fetuses of the 5 mg/kg bw/day group, one fetus of the 15 mg/kg bw/day group) further were reported. A case of truncus arteriosus communis also was seen in the 15 mg/kg bw/day group. Cases of separated origin of carotids, which was a very common finding for the strain used, as well as cases of hearts with traces of interventricular foramen/septum membranaceum were reported for all groups including the control. In addition, 3 cases of focal liver necrosis and one case of autolysis (dead fetus) were reported as unclassified observations for the 5 mg/kg bw/day group. None of all these findings was treatment-related. For details, see table below.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Placental weight in the 5 and the 15 mg/kg bw/day groups were quite similar to control and/or within the range of biological variation. For the 45 mg/kg bw/day group, due to the limited number of live fetuses, no clear assessment of this parameter was possible. In fact, for this group, the placental weight was the lowest compared to control (mean of 4.1 g versus 4.9 g for control, and 4.6 g for respectively the 5 and the 15 mg/kg bw/day group).
Dose descriptor:
LOAEL
Remarks:
embryotoxicity
Effect level:
45 mg/kg bw/day (actual dose received)
Basis for effect level:
fetal/pup body weight changes
other: Increased embryo- / fetolethality (only one female gave 4 alive fetuses, resorptions and post-implantation losses were significantly increased compared to the remaining groups). Reduction in mean placental weights.
Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Basis for effect level:
other: No treatment-related effects adverse effects observed at this dose.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
45 mg/kg bw/day (actual dose received)
Basis for effect level:
other: No indication for teratogenicity was seen up to 45 mg/kg bw/day; however, the number of available fetuses (only 4) in the 45 mg/kg bw/day group was too low for clear evaluation of teratogenicity.
Remarks on result:
not determinable
Abnormalities:
not specified
Developmental effects observed:
not specified

Reproduction and fetal parameters

Dosage levels (mg/kg bw/day)

0

5

15

45

Mated females (N)

15

15

15

15

Pregnant females (%)

100%

93%

93%

100%

Abortions (N)

0

0

0

0

Premature births (N)

0

0

0

0

Dams with viable fetuses (N)

100%

93%

93%

7%

Dams with resorptions (%)

0%

0%

0%

60%

Female mortality, N (%)

0 (0%)

0 (0%)

0 (0%)

5*(33%)

Pregnant at Ceasarian section (%)

100%

93%

93%

10*(67%)

Mean corpora lutea (total corpora lutea)

8.5 +/- 1.2 (128)

8.7 +/- 1.5 (122)

8.1 +/- 1.1 (113)

7.6 +/- 1.7 (76)

Mean implantation sites (total implantation sites)

6.9 +/- 1.6 (103)

7.6 +/- 1.8 (107)

6.6 +/- 2.1 (92)

7.2 +/- 1.2 (72)

Mean pre-implantation loss (%)

19.2% +/- 17.7

12.1% +/- 13.7

19.0% +/-21.8

4.0% +/- 9.1

Mean post-implantation loss (%)

13.6% +/- 14.6

7.4 +/- 13.9

12.0 +/- 19.1

94.3%**+/- 18.1

Mean total resorptions (%)

13.6% +/- 14.6

Early: 7.6% +/- 10.9

Late: 6.0% +/- 9.5

6.2 +/- 10.1

Early: 4.2% +/- 7.2 Late: 2.0% +/- 5.2

12.0 +/- 19.1

Early: 4.3% +/- 9.7

Late: 7.7% +/- 11.9

94.3%**+/- 18.1

Early: 91.4%**+/- 27.1

Late: 2.9% +/- 9.0

Dead fetuses (N)

0

1

0

0

Live fetuses

Total (N)

89

100

83

4

Mean (N)

5.9 +/- 1.7

7.1+/- 2.1

5.9+/- 2.4

4.0+/- 0.0

Mean (%)

86.4%+/- 14.6

92.6%+/- 13.9

88.0%+/- 19.1

57.1+/- 0.0

Females (%)

60.7%

48%

56.6%

100%

Males (%)

39.3%

52%

43.4%

0.0%

*, p<0.05; **, p<0.01

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Glutaraldehyde was investigated for prenatal toxicity in several studies in rabbits, rats and mice.

Rats

Two dose-range finding studies performed similar to OECD 414 and a prenatal toxicity study performed in accordance with OECD 414 were available. All three studies followed GLP.

In the first range-finding study, Wistar rats were exposed to 0, 100, and 500 ppm test substance (0, 11, and 51 mg/kg bw) in drinking water (BASF, 1991) for 11 days. The NOAEL for maternal toxicity was determined to be 100 ppm based on reduction in food and water consumption and on the presence of foci in the glandular stomach of 2 animals in the high dose group. No embryo-/fetotoxic treatment-related effects were seen. However, because of the early sacrifice of the dams, only a limited examination of the fetuses was possible.

In the second range-finding study, Wistar rats were exposed to 0, 10, and 50 mg/kg bw test substance via oral gavage (BASF, 1991) for 11 days. The NOAEL for maternal toxicity was determined to be 10 mg/kg bw based on reduced nutritional state, labored breathing, piloerection, one case of vaginal hemorrhage, reduced food consumption, reduced body weight and body weight gain, decreases in total protein and globulins, thickening of the margo plicatus in the forestomach of all animals, cases of hemorrhagic mucosal lesions in the glandular stomach, and increased number of late resorptions and post implantation loss. Marginal signs of embryo-/fetotoxicity were seen in the 50 mg/kg bw/day group only (increased incidence of ca.10% in late resorptions and post implantation loss compared to control) whereas there was no indication of any teratogenic effect at both dose levels. However, because of the early sacrifice of the dams, only a limited examination of the fetuses was possible.

On the basis of the results of the two range finding studies, administration via drinking water and dose levels of 0, 50, 250 and 750 ppm of test substance were selected for the conduct of the main prenatal developmental toxicity study (BASF, 1991). The NOEL for maternal toxicity was determined to be 50 ppm based on the reduction in water consumption of ca. 19% and 12% reported at 250 and 750 ppm, respectively. The reduction in water consumption was considered to be due to the bad tasting or smell of the drinking test solution, and the effect was seen as a physiological effect rather than a toxicological one. No indication for developmental toxicity potential of glutaraldehyde in rat at doses up to 750 ppm, corresponding to ca. 68 mg/kg body weight/day was observed.

In addition, Ema et al. (1992) performed a prenatal developmental toxicity study (non guideline, non GLP). Wistar rats were exposed via oral gavage to 0, 25, 50, 100 mg/kg bw test substance from gestation day 6 to 15. At a dose of 100 mg/kg bw test substance significantly higher lethality (5 out of 26 animals died), lower body weight gain, and reduced food consumption was observed in the dams. In the 50 mg/kg bw dose group 2 out of 21 dams died. No teratogenic potential could be evidenced for glutaraldehyde in rat fetuses, even at a dose which induced severe maternal toxicity. Fetotoxicity as demonstrated by a significant decrease in the weight of live fetuses was observed at the highest tested dose level of 100 mg/kg bw/day, which clearly was toxic to the dams. Thus, decrease in fetal weight seems to rather be a secondary effect of the females during gestation than being a direct effect of the treatment.

Rabbits

Two dose-range finding studies performed similar to OECD 414 and a prenatal toxicity study performed in accordance with OECD 414 were available. All three studies followed GLP.

In the first range-finding study, Himalayan rabbits were exposed to 0, 100, and 500 ppm test substance (0, 7.1, and 23.4 mg/kg bw) in drinking water (BASF, 1991) from gestation day 7 to 20. No NOAEL for maternal toxicity could be determined as even in the low dose group, a reduction in food and water consumption was observed. In addition, post-implantation loss was observed at 500 ppm. No embryo-/fetotoxic treatment-related effects were seen. However, because of the early sacrifice of the dams, only a limited examination of the fetuses was possible.

In the second range-finding study, Himalayan rabbits were exposed to 0, 5, and 25 mg/kg bw test substance via oral gavage (BASF, 1991) from gestation day 7 to 21. No NOAEL for maternal toxicity could be determined as even in the low dose group, a reduction in food and water consumption was observed. In addition, maternal toxicity in rabbit at the highest tested dose of 25 mg/kg bw/day included reduced food consumption and decreased total protein, albumin, calcium and glucose contents in blood. No embryo-/fetotoxic treatment-related effects were seen. However, because of the early sacrifice of the dams, only a limited examination of the fetuses was possible.

On the basis of the results of the two ran ge finding studies, administration via gavage and dose levels of 0, 5, 15 and 45 mg/kg bw of test substance were selected for the conduct of the main prenatal developmental toxicity study (BASF, 1991). The NOAEL for maternal toxicity was determined to be 15 mg/kg bw based on impaired food consumption, body weight loss, spontaneous deaths, adverse clinical symptoms, irritations of the gastrointestinal tract and reduced uterus weights. In the high dose group increased embryo- / fetolethality (only one female gave 4 alive fetuses, resorptions and post-implantation losses were significantly increased compared to the remaining groups) and a reduction in mean placental and fetal weights was observed. No indication for teratogenicity was seen up to 45 mg/kg bw/day; however, the number of available fetuses (only 4) in the 45 mg/kg bw/day group was too low for clear evaluation of teratogenicity. Thus fetotoxicity was only observed at the highest tested dose level of 45 mg/kg bw/day, which clearly was toxic to the dams and although teratogenicity could not be assessed, no teratogenicity was observed at dose levels which were not toxic to the dams.

Mice

In a teratogeniciy study from Marks et al (1980), pregnant outbred albino mice were given Sonacide (potentiated acid glutaraldehyde) by gavage on days 6-15 of gestation to 0.8, 1.0, 1.2, 2.0, 2.5 and 5.0 mL/kg day (equivalent to 16, 20, 24, 40, 50, 100 mg/kg day glutaraldehyde). The mice were killed on day 18, the general health and reproductive status of the dam were evaluated, and the fetuses examined and processed in order to characterize external, visceral, and skeletal malformations. The test substance (Sonacide) was highly toxic to the mice at doses 2.0 mL/kg day (40 mg/kg glutaraldehyde) and above. Six out of 35 animals died at this dose. There was a statistically significant dose dependent reduction in average weight gain during pregnancy. The maternal LOAEL was determined to be 16 mg/kg bw. Only at doses that were highly toxic to the pregnant dams a significant increase in the number of stunted fetuses as well as a significant increase in the average percent malformed fetuses was observed (at 100 mg/kg bw glutaraldehyde). Therefore, based on the results obtained in this study the NOAEL for teratogenicity was determined to be 50 mg/kg bw.

Conclusion

In pregnant rabbits, rats, and mice neither an embryo/fetotoxic nor a teratogenic potential could be evidenced at dose levels which were not toxic to the dams. For rabbits, the maternal NOAEL and the NOAEL for feto/embryotoxicity, respectively, were 15 mg/kg bw/day of the test substance (GA 50%). For rat, the maternal NOAEL and the NOAEL for feto/embryotoxicity and teratogenicity, respectively, were about 68 mg/kg bw/day of the test substance (GA 50%). For mice, the maternal LOAEL and teratogenicity NOAEL were 16 and 50 mg/kg bw active ingredient, respectively.

Human data:

The Dutch Expert Committee on Occupational Standards (2005) reviewed data obtained from Finnish hospital nurses and staff on the risk of spontaneous abortions and foetal malformations. According to the review, no increased risk of spontaneous abortions and foetal malformations was found in Finnish hospital nurses and staff,using glutaraldehyde as a sterilising agent.

Justification for classification or non-classification

Glutaraldehyde does not affect the reproductive performance and fertility, and neither possesses an embryo/fetotoxic nor a teratogenic potential. Therefore, no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008).

Additional information