Glutaral

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

(2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

(1998)

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Glutaral

EC Number:

203-856-5

EC Name:

Glutaral

Cas Number:

111-30-8

Molecular formula:

C5H8O2

IUPAC Name:

glutaraldehyde

Test material form:

liquid

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

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.

Doses / concentrations 3

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

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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.

3

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

1

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

2

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.

3

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

Results: F1 generation

General toxicity (F1)

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.

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

Results: F2 generation

General toxicity (F2)

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.

Effect levels (F2)

1

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

2

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

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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