Mecoprop

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 1989 to 17 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC of November 18, 1987 adapting to technical progress for the ninth time Council Directive 67/548/EEC, pp. 47 - 50.

Version / remarks:

1988

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1984

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF, Testing Guidelines for Toxicology Studies pp. 45 - 48.

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

- Route of administration: Since the test material is to be used as a herbicide and uptake of crop bearing residues by man cannot be ruled out, oral administration (via the diet) was selected.

Test material

Test material information

Constituent 1

Reference substance name:

Mecoprop

EC Number:

230-386-8

EC Name:

Mecoprop

Cas Number:

7085-19-0

Molecular formula:

C10H11ClO3

IUPAC Name:

2-(4-chloro-2-methylphenoxy)propanoic acid

Test material form:

solid

Details on test material:

- Stability: Stability on storage was ensured for the period of the study.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Chbb = THOM (SPF)

Details on species / strain selection:

This strain was selected since extensive experience is available on Wistar rats and the rat is the preferred animal species for reproduction studies according to the different test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Not specified
- Age at study initiation: F0 generation parental animals: 27 (± 1) days old when they arrived from the breeding facilities; 35 (± 1) days old at the beginning of treatment.
- Weight at study initiation: F0 generation parental male animals 126.2 g (117 - 134g), female animals 114.7 g (106 - 122 g).
During the acclimatisation period, animals with an extreme body weight were eliminated and sacrificed.
- Housing: During the study period, the rats were housed individually in type DKIII stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel., FRG (floor area of about 800 cm^ 2 ), with the following exceptions: During mating periods, the males designated for mating were kept individually in Makrolon cages, type M Ill (floor area of about 800 cm ^2 ); for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages. The M III cages were also suppl.ied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet: Ad libitum, except those animals selected for urinalyses had no access to food in the cages used to take urine samples.
- Water: Ad libitum, except those animals selected for urinalyses had no access to water in the cages used to take urine samples.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air conditioning guaranteed a range of temperature of 20 - 24 °C. There were no deviations from these limits.
- Humidity (%): Central air conditioning guaranteed a range of relative humidity of 30 - 70 %. There were no deviations from these limits.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours) in general.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The test material preparations were prepared at intervals of not more than 35 days.
- Mixing appropriate amounts with: The test material preparations were prepared by weighing in the test material and mixing this thoroughly with a small amount of the food. This mixture was then mixed in a BOSCH household mixer. An appropriate amount of food was then added to obtain the desired concentration, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.

Details on mating procedure:

Matings of F0 and F1 generation parental animals
- M/F ratio per cage: At least 70 days after the beginning of treatment, males and females were mated at a ratio of 1:1 (generally male No. 1 with female No. 101, male No. 2 with female No. 102 etc.).
Throughout the mating period, each female animal was mated with a male animal that had been determined before.
- Length of cohabitation: Overnight. Normally, the female animal was placed toward 16.00 hours in the cage with its male partner and was again separated from the male animal between 7.00 and 9.00 hours of the following morning. Deviations from the specified times were possible on weekends and public holidays.
- Proof of pregnancy: A vaginal smear of the female animals was prepared after each mating and examined for sperm. If sperms were detected, the partners were no longer mated with each other. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
- Further matings after two unsuccessful attempts: Yes. Animals was mated overnight for a maximum of 3 weeks.

Reevaluation of fertility
If an animal had not produced any offspring after the 2 matings (F1a and F1b litters) or after the mating for the F2 generation, those animals treated with the test material were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with a mating partner with proven fertility of the controls.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical investigations to determine the active ingredient content, the stability and homogeneity of the test material were carried out before the beginning of the study, during the study period and after the end of the study. Furthermore, the test material was investigated for any additional constituents and trace contaminants.
For the determinations of the homogeneity representative samples were taken from the top, the middle and the bottom of the containers.
Analyses of the stability of the test material in the diet over 33 days was carried out before the start of the study for another rat feeding study.
The homogeneity of the test material in the maintenance diet was analytically investigated before the start of the study in the aforementioned rat feeding study and in addition for this reproduction study at the beginning of the study period.
In order to check the correctness of the concentration, samples of each one of the doses were sent for analysis to the analytical laboratory at the beginning of the study and then at approximately three-monthly intervals.
The content of the test material in the test material/food mixes was determined by HPLC-method.

The stability of the test material in the maintenance diet over a 33-day period was confirmed. The homogeneous distribution of the test material in the rat food used was also confirmed. According to the results of the concentration control analyses the minimum content of the test material in the food was generally guaranteed.

Duration of treatment / exposure:

The duration varied between generations. The study was conducted for approximately 44 weeks.

Frequency of treatment:

The F0 generation parental animals continuously received the test material at the appropriate dose levels via the diet until or up to about 16 hours before they were sacrificed, with the exception of infertile animals and those animals which were taken for urinalyses.

Details on study schedule:

F0 generation:
The females were allowed to litter and rear their pups (F1a generation pups) until day 4 or 21 after parturition. At least 10 days after the last weaning of the F1a generation pups, the F0 generation parental animals were mated again at a ratio of 1:1. The male partners were selected by lot, thus ruling out the possibility that mating was carried out with the same partners which produced the F1a generation pups.
The females were allowed to litter and rear their pups (F1b generation pups) until day 4 or 21 after parturition.
After the F1b generation pups had been weaned, the fasted (about 16 hours) F0 generation parental animals were sacrificed (with the exception of animals which were taken for the reevaluation of their fertility).

F1 generation parental animals and their progeny:
After weaning, 25 males and 25 females (each litter was taken into account) of the F1a pups were chosen per group (by lot) as the basis of the F1 generation parental animals. If fewer than 25 litters were available for selection or if one sex was missing in a litter, more animals were taken from different litters from the relevant test group to give the full number.
These animals were exposed continuously to the test material from their growth into adulthood until or up to about 16 hours before they were sacrificed (with the exception of infertile animals and those animals which were taken for urinalyses). At least 98 days after formation of the F1 generation parental animals, the males and females were mated at a ratio of 1:1. The male partners were selected by lot avoiding any mating between siblings.
The females were allowed to litter and rear their pups (F2 generation pups) until day or 21 after parturition.
After the F2 generation pups had been weaned, the fasted (about 16 hours) F1 generation parental animals were sacrificed (with the exception of animals which were taken for the reevaluation of their fertility).

Infertile animals:
If an animal of the F0 (after both mating intervals) or F1 generation parental animals had not produced any offspring, these animals were again mated for not more than 3 weeks with one fertile male or female animal of the control group each.
Thereafter the male animals were sacrificed shortly after sperm was detected in the vaginal smear of the relevant female partner. The relevant females were sacrificed before littering (if an animal after these matings had proved to be fertile) or about 10 days after the last mating (if an animal after these matings likewise had proved to be infertile).
Those parental animals from test groups 01, 02 and 03 (F0 generation parental animals) and 11, 12 and 13 (F1 generation parental animals) whose fertility had to be reevaluated, were offered test material-free diet only during the specific nightly matings (when they were kept together with one fertile animal of the control group). In the remaining times (i.e., the period between the individual matings as well as after the last mating until sacrifice), these animals were offered food with the test material depending to which group they belonged.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

100 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

500 ppm (nominal)

No. of animals per sex per dose:

25 animals per sex per dose for the F0 generation parental animals and F1 generation parental animals.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of various previous studies.
In a sub-chronic study, the test material was administered to 90 Wistar rats (45 males, 45 females) via their diet for 3 months. For comparison, a group of untreated animals (15 per sex) was used as control. The doses were 0, 50, 150, 450 ppm.
Food consumption and body weight were determined once a week; the rats’ state of health was checked each day. At the beginning of the study and toward the end of the administration period all the animals were subjected to ophthalmological examinations.
Two clinico-chemical and haematological examinations as well as two urinalyses were carried out. All animals were subjected to a gross-pathological examination. Finally, an extensive histopathological examination was carried out. The following findings were obtained and assessed or discussed in relation to the test material:
450 ppm group:
Clinico-chemical findings: Increase in the creatinine values in the plasma of female rats.
Organ weights: Increase of absolute and relative kidney weights in the animals of both sexes.
150 ppm group:
Organ weights: Increase of absolute and relative kidney weights in the male animals; increase of relative kidney weights in the female animals.
50 ppm group: No changes to which a test material-induced relevance can be attributed.

In a combined study three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months. Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex, respectively. A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The food consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week. At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically. Blood samples for haematological and clinico-chemical examinations were taken 5 times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups). Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period. The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks. All animals used were assessed by gross pathology. This was followed by a comprehensive histopathological examination. The following findings were obtained and assessed or discussed to be test material-related:
400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.

On the basis of the results of these studies, which indicated renal impairment being more pronounced in males than in females in dosages ranging from 450 ppm down to 100 ppm, the following doses were chosen for the reproduction study with the test material:
20 ppm: As the expected no adverse effect level.
100 ppm: As a concentration with possibly minimal toxic effects (e.g. increased kidney weights).
500 ppm: As the dose in which toxic effects were expected (e.g. kidney impairment) in the parental animals, but which should not induce mortality in these animals.

- Rationale for animal assignment: The assignment of the animals to the different test groups was carried out in accordance with a randomisation program, separately for males and females according to their weight, one day before the beginning of the administration period (day -1).
- Fasting period before blood sampling for clinical biochemistry: No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily a check was made for any dead or moribund animals. If animals were in a moribund state, they were sacrificed and dissected like animals that died.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All parental animals were checked each day for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis
The nesting, littering, and lactation behaviour of the dams were generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.
The littering behaviour of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the period between about 15.00 hours of the one, and about 15.00 hours of the following day (= 24 hours). Deviations from this procedure were possible on Saturdays, Sundays and on public holidays, when the weighings of the dams took place as early as about 7.00 hours.
If an animal was not able to litter or was in a moribund state, these animals were sacrificed and examined in the laboratory of pathology.

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day in each case (in the morning); if possible, the weighings were carried out until the end of the study. The body weight change of the animals was calculated from these results. As exceptions, the following particularities applied to the female parental animals:
a) During each mating period (1st and 2nd matings of the F0 and mating of the F1 generation parental animals) the females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum.
b) Females showing no positive evidence of sperm within one mating period were not weighed during this mating interval.
c) Females with litters were weighed on the day of parturition (day 0 p.p.) and on days 4, 7, 14 and 21 postpartum.
d) Females without litters were not weighed during the lactation period of the dams used in parallel.
e) After weaning of the last F1a pups the female F0 generation parental animals were weighed weekly until the beginning of the 2nd mating interval (in parallel to the male F0 generation parental animals).
Furthermore, male and female animals whose fertility had to be reevaluated and those controls which were chosen as partners for these reevaluations were not weighed during the additional matings and the interval until they were sacrificed

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- During the first 10 (F0 generation parental animals) and 14 (F1 generation parental animals) test weeks food consumption was determined once a week (each time for a period of 7 days) for males and females. After the 10th (F0 generation parental animals) and 14th (F1 generation parental animals) test week food consumption of the females during pregnancy (animals with evidence of sperm) was determined for days 0 - 7, 7 - 14 and 14 - 20 p.c..
- During the lactation period (animals with litter) food consumption was determined for days 0 - 4, 4 - 7 and 7 - 14 p.p..
- Food consumption was not determined between days 14 and 21 after parturition, since during this time pups will begin to consume considerable amounts of the rat food offered, and therefore there was no point in such a measurement.
- Food consumption of the males was not determined any longer after the 10th (F0 generation parental animals) or 14th (F1 generation parental animals) test week until sacrifice. Furthermore, there was no determination of food consumption in the females during the mating period, animals without positive evidence of sperm (during the gestation period of the dams used in parallel) and animals without litter (during the lactation period of the dams used in parallel).
- The food consumption of those animals whose fertility had to be reevaluated and those controls which were chosen as partners for these reevaluations was not determined either during the additional matings or the interval until they were sacrificed
- The intake of test material was calculated from the amount of food consumed and expressed in mg/kg body weight per day.
- The calculation of the group values/day was carried out according to the following formula:

(FC x D) / BWx = intake of test substance in mg/kg body weight/day

Where:
FC: Mean daily food consumption in grams (on day x + y; y = 3, 4, 6 or 7 depending on the study period).
D: Dose in ppm.
BWx: Mean body weight on a specific day in g x (on day x).

CLINICAL CHEMISTRY AND URINALYSES
Blood was taken from the retroorbital venous plexus in the morning from non-fasted, not anesthetised animals. The blood sampling procedures and the subsequent analysis of the serum samples were carried out in a randomised sequence. The list of randomisation instructions was compiled with a computer using a random number generator. For urinalyses the individual animals were transferred to metabolism cages and urine was collected at about 4 °C overnight. The urine samples were evaluated in a randomised sequence.
Male and female F0 parent urine collection was on nominal days 190, 191 and 197.
Male and female F0 parent blood collection was on nominal days 204/205.
Male and female F1 parent urine collection was on nominal days 152, 153 and 154.
Male and female F1 parent blood collection was on nominal days 155 and 158.
The assays of serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results.
The results of the clinico-chemical examinations are expressed in units of the International System (SI). The following examinations were carried out in 12 animals per test group and sex of the F0 and the Fl parental generation.

- Clinico-chemical examinations: An automatic analyser (Hitachi 737; by Boehringer, Mannheim, FRG) was used to examine the clinico-chemical parameters.
The following parameters were determined:
Enzymes: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase.
Blood chemistry: Sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium.

- Urinalyses: With the exception of the sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test RL, by Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model by Boehringer, Mannheim, FRG).
The specific gravity was determined using a urine refractometer.
The sediment was evaluated microscopically.
The following examinations were carried out: Volume, appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, standardisation of litters F1a, F1b and F2 generation pups.
- The individual litters were standardised in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible in single litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardisation of litters was not performed in litters with < 8 pups.
- Excess pups: With the exception of the F1a generation pups, which were chosen as the basis of the F1 generation parental animals, all pups were sacrificed (by means of CO2) after standardisation or weaning.

PARAMETERS EXAMINED
- Pup number and status at delivery: All pups derived from the F0 parents (F1a and F1b litters) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.
- Pup viability/mortality: A daily check was made for any dead or moribund pups. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of the lactation period were determined. However, pups which were culled or died accidentally were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4 (before and after standardisation of litters), 7, 14 and 21.
- Sex ratio: On the day of birth (day 0) and on the first few days after birth the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle: Normally, the anogenital distance is considerably greater in male than in female pups.
During the remaining rearing period the sex of the pups was assessed by external appearance and the sex was finally confirmed at necropsy.
- Pup body weight data: The pups were weighed on the day after birth (day 1 p.p.) and on days 4 (before standardisation), 7, 14 and 21 after birth. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. before standardisation of the litters.
- Pup clinical observations: The pups were examined each day for clinical symptoms (including gross-morphological findings).
- Development stages: Different development stages of the pups were monitored. A physiological development was assumed if the following parameters had been fulfilled on the days given in brackets:
Pinna unfolding (on day 4 after birth before standardisation)
Opening of the auditory canal (on day 13 after birth)
Opening of the eyes (on day 15 after birth)
The examinations to establish whether the various development stages had been reached were carried out in the morning in each case.

GROSS EXAMINATION OF DEAD PUPS:
With the exception of the F1a generation pups, which were chosen as the basis of the F1 generation parental animals, all pups were sacrificed (by means of CO2) after standardisation or weaning. These pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and organ) examination. Thereafter, stillborn pups and those that died during the rearing period as well as pups which were culled on day 4 p.p. or "surplus" (i.e. those pups with scheduled sacrifice after weaning) pups which showed any remarkable findings during rearing or abnormalities in the macroscopic assessment were treated with different methods.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Behavioral tests
Up to weaning, different behavioral tests with the pups were performed in order to record any variations from normal. All surviving pups were tested for the gripping reflex, the hearing test (acoustic startle) and the pupillary reflex test.
- Gripping reflex: On day 13 after birth (± 1 day), the pups were checked for this reflex. For this purpose, the front extremities of the animals were placed on a bar about 3 mm in diameter. The animals had to cling to this bar and pull themselves up. If this aim was achieved, the test was regarded as positive (yes response), if it was not, the test was regarded as negative (no response).
- Hearing test (acoustic startle): On day 21 after birth (± 1 day), the animals were placed in a soundproof box (internal dimensions 49.5 x 49.5 x 38.5 cm). After a short acclimatisation period, the animals were exposed (twice at most) to a sound (0.1 seconds, 5 000 Hz, about 90 dB). A startle reflex (movement of the ears or a jerk) was considered to be a response to this stimulus.
- Pupillary reflex (pupil constriction): On day 20 after birth (± 1 day), the animals pupils were allowed to dilate by introducing the pups to a low light environment. The pupillary constriction reflex was assessed by shining a pencil torch on the eye and observing the reaction of the pupil. Miosis was assessed as the presence of the reflex.

Postmortem examinations (parental animals):

SACRIFICE
The animals were sacrificed by decapitation under CO2 anesthesia. Then the animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
Reevaluation of fertility: After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological assessments.
- Reevaluation of fertility: The uteri (including the contents) of the relevant female animals were assessed by the reproduction toxicology laboratory. If macroscopic implantations were seen, their number, position and condition were recorded. Uterus contents were then removed as completely as possible. The uteri of apparently non-pregnant animals, or empty uterus horns were placed in 10 % ammonium sulfide solutions for about 5 minutes in order to be able to recognise early resorptions or implantations. Then the uteri were rinsed carefully under running water. After these examinations of the uteri were completed at the reproduction toxicology laboratory, the uteri of the corresponding animals were transferred to the pathology lab for further fixation and evaluation.

The weight of the anesthetised animals and the weight of the liver, kidneys, and testes from all animals sacrificed at scheduled dates were determined.
Subsequently, the following organs or tissues of the F0 and F1 generation parental animals were fixed in 4 % formaldehyde solution:

Control and 500 ppm groups: Vagina, cervix, uterus, ovaries, testes, epididymides, seminal vesicles, coagulating gland, prostate gland, pituitary gland, liver, kidneys, all gross lesions.
For the 20 ppm and 100 ppm groups only the liver, kidney and gross lesions were examined.
Of the animals that were necropsied prematurely, the above mentioned organs were processed and investigated histopathologically.

Postmortem examinations (offspring):

SACRIFICE
Culled (i.e. all pups which were sacrificed on day 4 p.p. as a result of standardisation) and surplus pups (i.e. all pups reared until day 21 p.p. except those F1a generation pups selected as the basis of the F1 parental generation) were sacrificed on day 21 after birth or subsequent days by means of CO2.

GROSS NECROPSY
- Pups were examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson’s method and/or further processing of head according to Wilson's method).
- All stillborn pups and all pups that died during rearing were examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method).
- The stained skeletons were evaluated under a stereomicroscope or a magnifying glass.
- All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

- Clinical examinations: Dunnett’s Test was used for statistical evaluation of all parametric data such as food consumption (parental animals), body weights and body weight change (parental animals and pups). Fisher's Exact Test was used for statistical evaluation of all quantitative data such as developmental stages, gripping and pupillary reflex, hearing test, number of live and dead pups, and the different indices (e.g. male and female mating index, male and female fertility index, gestation index, live birth index, viability index, lactation index). Differences between control and treated groups were considered significant at p ≤ 0.05 or p ≤ 0.01.
- Clinical chemistry: Means and standard deviations have been calculated for each test group and tabulated together with the individual values. In order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups were compared with those for the control group using the analysis of variance (ANOVA and Dunnett’s test).
- Urinalyses: The assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi^2 test in appropriate two-by two contingency tables.

Reproductive indices:

Males
The mating partners, the number of mating days until vaginal sperm could be detected in the female mating partner, and the pregnancy status of the female partner were noted for F0 and F1 males. For the males, mating and fertility indices were calculated for F1a, F1b and F2 litters according to the following formulae:

Male mating index (%) = (number of males with confirmed mating† / number of males placed with females) x 100
† A female with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Male fertility index (%) = (number of males proving their fertility† / number of males placed with females) x 100
† A female giving birth to a litter or with pups/foetuses in utero

The mating partners, the number of mating days until vaginal sperm could be detected, and pregnancy status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1a, F1b and F2 litters according to the following formulae:

Females:
Female mating index (%) = (number of females mated† / number of females placed with males) x 100
† The number of females with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Female fertility index (%) = (number of females pregnant† / number of females mated‡) x 100
† The number of females that gave birth to a litter or with pups/foetuses in utero.
‡ The number of females with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant†) x 100
† The number of females that gave birth to a litter or with pups/foetuses in utero.
Furthermore, the total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1a, F1b and F2 litters according to the following formula:

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Offspring viability indices:

Viability and lactation indices were calculated according to the following formulae:

Viability index (%) = (number of live pups on day 4† after birth / number of liveborn pups on the day of birth) x 100
† Before standardisation of litters (i.e. before culling).

Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4† after birth) x 100
† After standardisation of litters (i.e. after culling).

The sex ratio was calculated for day 0 and day 21 after birth according to the following formula:

Sex ratio = (number of live male or female pups on day 0/21 / number of live male and female pups on day 0/21) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations for males and females (except gestation and lactation periods):
No clinical signs which might be attributed to the test material administered were detected in male or female F0 generation parental animals. The 3 doses (20, 100 and 500 ppm) administered as addition to the diet did not lead to disturbances of the general behavior in any of the F0 parental animals.
Only some spontaneous findings without any relation to dose occurred in a few animals of all groups including the controls. Mainly minor skin lesions, swelling of limbs or alopecia were found; furthermore, one control male showed a palpable mass at the flank towards the end of the study and for one 100 ppm male broken incisivi and chromodacryorrhea, which was also noted for one high dose female, were recorded.

Clinical observations for females during gestation:
There were no particular test material-related clinical findings in F0 females during gestation periods for F1a or F1b litters. Without any dose-response relationship insufficient/no nesting activity was observed for several dams of all groups during gestation (F1a and F1b). Moreover, during the gestation period for the F1b pups blood in bedding was found in one high dose female and haemophthalmia in one 100 ppm dam, which died subsequently.

Clinical observations for females during lactation:
There occurred no test material-related clinical findings in the dams during the lactation of F1a and F1b pups. Only some spontaneous findings were recorded: Dam No. 150 (20 ppm group) showed a skin lesion of the left hindlimb during the lactation period of F1a litter.
One animal of the 100 ppm group (No. 167) died during delivery of F1b pups. Moreover, after parturition, umbilical cords were not cut in some pups and/or placenta(e) were not consumed by dams of different groups without showing any dose-response relationship (F1a and/or F1b).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 2 F0 females (Nos. 158 and 167 - 100 ppm) which died spontaneously; these deaths are not associated with treatment. Animal No. 158 died during gestation of F1b litter (day 10 p.c.) and dam No. 167 died during the delivery of F1b pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the whole study the mean body weights/body weight gains of the males and of the females (including gestation and lactation periods) do not show any influence of the test material on these parameters. All differences between the controls and the animals of the test material-treated groups are assessed to be within the expected range of biological variation. This includes the few isolated statistically significant differences concerning body weight gain between the male controls and the 500 ppm males (week 9 - 10) and the female control and the 100 ppm group (week 4 - 5, lactation days 14 - 21 (F1a) and week 18 - 19).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences of biological relevance between the controls and the test material-treated groups concerning the food consumption of the F0 males during the premating period and the food intake of the F0 females during premating, gestation and lactation periods.
The amount of test material (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was determined.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material induced changes were found in the clinico-chemical examinations in both sexes of F0 parental animals.
There are some statistically significant inter-group differences in the results of the clinico-chemical data. These deviations are marginal, inconsistent or incidental. Accordingly, these changes are considered to be of no toxicological significance.
Furthermore, the statistically significant increase in alkaline phosphatase activity seen in the high-dose males of the F0 parental animals is not considered to be test material-related, because the value of this treatment group (5.42 μkat/L) is close to the upper limit of the range of historical control data of male Wistar rats after a study period of 6 months (4.19 μkat/L - 5.05 μkat/L) and no corresponding findings were observed in the other animals of this dose group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-induced changes were observed in the urinalyses in both sexes of F0 parental animals.
There are some statistically significant inter-group differences in the results of the urine data. These deviations are marginal, inconsistent or incidental. Accordingly, these changes are considered to be of no toxicological significance.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

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):

None of the histopathological findings were considered to be associated with the administration of the test material. There were two premature decedents in test group 02 (100 ppm): Female No. 158 which had a malignant schwannoma and female No. 167 for which no cause of death could be established on histological examination. There was no evidence to suggest that either of these deaths were related to administration of the test material. Neither is the bilateral degeneration of the germinal epithelium and the subsequent dystrophic mineralisation in the testes, and the aspermia observed in the epididymides of male animal No. 44 in the lowest dose group (20 ppm) considered treatment-related, but rather as an incidental finding.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male Reproduction Data
For all F0 males (except No. 44, test group 01 - 20 ppm; mating for F1b pups) which were placed with females to generate F1a or F1b pups mating was confirmed so that the male mating index was 100 % in all groups (F1a and F1b) except for test group 01 (F1b) where it was 96 %.
Nearly all males (except 2 of the 20 ppm group) proved their fertility after the scheduled matings for F1a and/or F1b litters.
For 1 male of the 20 ppm group (No. 31) for which fertility could not be proven after mating for F1a or F1b litters, fertility was confirmed in the reevaluation of fertility; however, for another low dose male (No. 44) fertility could not be proven. The observable differences concerning the male fertility indices (for F1a and F1b) between the groups are finally assessed as being of spontaneous nature and not related to the test material administration. All values are in the range of the historical control.

Female Reproduction Data
The female mating indices calculated after the mating period for F1a and F1b litters was 100 % for all groups except for test group 01 (20 ppm), where it only reached 96 % for the second (F1b) mating period.
The mean duration until sperm could be detected (day 0 p.c.) varied between 2.4 and 2.7 days (F1a) or 2.1 and 2.8 days (F1b) without there being any differences of biological significance between the groups.
The female fertility indices, varied between 100 % and 88 % (concerning F1a) and between 100 % and 96 % (concerning F1b). However, for all dams except No. 166 (100 ppm) fertility was confirmed at least in one of the scheduled matings.
The mean duration of gestation (F1a and F1b) was similar in all groups.
Due to one dam of test group 02 (100 ppm) - No. 158 - which was pregnant, but died during the F1b gestation period, the gestation index calculated for test group 02 (100 ppm) was 96 %, while it was 100 % for all other groups (concerning F1a and F1b litters). The mean number of F1a and F1b pups delivered and the relevant live birth indices show no differences of biological relevance and no dose-related trend.

Re-evaluation of Fertility
2 males (Nos. 31 and 44 - 20 ppm) and 1 female (No. 166) of test group 02 (100 ppm) had to be re-evaluated. Fertility could be confirmed only for male No. 31.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

2 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation parental animals - Clinical observations for males and females
No clinical signs which might be attributed to the test material administered were obtained in male or female F1 generation parental animals of anyone test group during the relevant intervals (F1 males: Whole study period; F1 females: Premating and the interval after weaning of F2 pups until sacrifice: For clinical observations of the dams during gestation and lactation periods.
Only some spontaneous findings were recorded in single F1 males and females during premating like microphthalmia (male No. 217 - control group), chromodacryorrhea (male No. 272 - 100 ppm), malocclusion (males Nos. 247 - 20 ppm and 292 -500 ppm) and alopecia (female No. 378 - 500 ppm).

F1 generation parental animals - Clinical observations for females during gestation
No particular clinical findings were noted for F1 dams except no or insufficient nesting activity, which was recorded for several dams of all groups including the control and which occurred without any dose-response relationship.

F1 generation parental animals - Clinical observations for females during lactation
After parturition, only one dam (No. 358) of test group 12 (100 ppm) did not nurse its pups properly; all pups of this dam died until day 5 p.p..
No further clinical findings were seen.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the F1 parental animals died intercurrently or had to be sacrificed prematurely.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gains were not adversely affected by the administration of the test material to the parental animals of test groups 11, 12 and 13 (20, 100 and 500 ppm) during the whole study period including gestation and lactation periods of the dams for F2 litters. All differences between the controls and the test material-treated groups are regarded as spontaneous, including the sporadic and sometimes even statistically significantly increased or decreased body weight gains of the females in test groups 11 (20 ppm), 12 (100 ppm) and 13 (500 ppm) during premating, gestation and lactation, which occurred without any dose response relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No impairment of food consumption was recorded for the test material-treated F1 parental animals when compared to the controls, neither during the premating period nor during the gestation and lactation period of the F1 females. All differences between the groups are without any biological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test material induced changes were found in the clinico-chemical examinations in both sexes of F1 parental animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test material-induced changes were observed in the urinalyses in both sexes of F1 parental animals.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in the relative and absolute mean kidney weights were present in animals of both generations given 100 or 500 ppm. No morphological findings were present in the kidneys which could explain this organ weight change. Nonetheless the consistency of its occurrence suggests that it resulted from the administration of the test compound. Males of the F0 generation given the highest dosage had significantly lower mean relative liver weights than controls (p<0.05). However, this intergroup difference was attributed to chance since on light microscopy hepatic morphology was comparable and no corresponding change was seen in the F1 generation.

As with the F0 generation, mean kidney weights of animals given 100 or 500 ppm were significantly greater than control values. In males, statistically significant differences were attained in animals which had received 100 or 500 ppm for both absolute (p<0.05, p<0.01 respectively) and relative (p<0.01 for both groups) values while in females significant differences were noted at 100 and 500 ppm for relative weights (p<0.05, p<0.01 respectively) but only at 500 ppm for absolute weights (p<0.01).

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation parental animals - Male reproduction data
For all F1 males of all test groups, which were placed with females to generate F2 pups, mating was confirmed (i.e. the male mating index for all groups was 100 %). Whereas all control males and the males of test group 11 (20 ppm) proved to be fertile, the fertility of two males of the 100 and of one male of the 500 ppm group could not be proven during the scheduled matings for F2 litters.
The fertility indices were:
100 % for test group 10 (all males proved their fertility)
100 % for test group 11 (all males proved their fertility)
92 % for test group 12 (2 out of 25 males without proving their fertility)
96% for test group 13 (1 out of 25 males without proving its fertility)
However, all males in question - except male No. 262 (100 ppm group) - proved their fertility later. Therefore, the differences concerning the fertility indices of F1 males are regarded as incidental.


F1 generation parental animals - Female reproduction and delivery data
The female mating index reached 100 % for all test groups. The mean duration until sperm could be detected varied between 2.2 and 3.2 days.
The female fertility index calculated was 100 % for test group 10 and 11, 92 % for test group 12 and 96 % for test group 13.
However, the differences concerning the female fertility index between the groups are assessed as being of spontaneous nature, because all relevant females proved heir fertility later on.
The mean duration of gestation (F2) was similar for all groups.
All pregnant females gave birth to litters with liveborn pups. Consequently the gestation index was 100 % for all groups.
The mean number of delivered F2 pups and the percentage of liveborn F2 pups show no substantial differences between the control and the test material-treated groups. Consequently, the live birth index is not substantially influenced.

F1 generation parental animals - Reevaluation of fertility
Several males and females of test groups 12 and 13 (100 and 500 ppm) had to be reevaluated concerning their fertility. All of them proved to be fertile when mated with a fertile female or male except one 100 ppm - male (No. 262) for which fertility could not be proven.
The mating partner of this male showed a positive evidence of sperm, but did not become pregnant.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
No abnormal clinical symptoms were recorded for the F1a and F1b pups. Only some clinical findings (e.g. shortened tail, lesion of hindlimbs and traumatic lesion of cornea) were detected in very few F1a and F1b pups of different groups without a clear dose response relationship. These findings are finally assessed as spontaneous ones.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters: Pup number and status at delivery
The mean number of delivered F1a and F1b pups/dam and the percentages of liveborn and stillborn F1a and F1b pups do not show any differences of biological relevance between the groups.

F1a and F1b generation pups/litters: Pup viability/mortality
The number of pups, which died or were cannibalised from the day of birth until day 4 p.p. (before culling) was slightly increased in the 500 ppm group (F1a and 1b) and also marginally increased in the 100 ppm group (F1b) in comparison to the control group.
Consequently, the viability indices as indicators of the pups' viability during the first 4 days after birth were lowest in this/these test group(s), but still in the range of the historical control. Finally, the impaired pup viability in test groups 03 (500 ppm - F1a and F1b) and 02 (100 ppm - F1b) is assessed as test material-related. Similar effects were also found for the high dose F2 pups. The lactation index as an indicator of offspring survival between days 4 - 21 p.p., however, was not influenced by the substance administration (F1a and F 1 b).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
Only body weight gains of the high dose F1a pups were marginally impaired in comparison to the controls on days 7 - 14 p.p., which has to be attributed to the test material administration
The statistically significantly reduced weight gain of the 100 ppm F1b pups, however, is assessed as a spontaneous effect due to the missing dose-response relationship.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
The examination of stillborn pups, pups that died intercurrently, culled pups and surplus pups of F1a and F1b litters did not reveal any difference of biological relevance between the test groups either in the type or in the number of pup necropsy observations. The statistically significant increase of 20 ppm pups of the F1b generation, which showed findings at necropsy is mainly caused by a higher number of pups with dilated renal pelvis; however, this finding is also present in the historical control data at a comparable incidence. Only very few of the large number of examined pups of all groups showed some other spontaneous findings like incisors sloped, hydroureter and focal liver necrosis.

Results of the pathology examination indicated that the administration of the test material produced no morphological changes in animals of the F1 generation which could be attributed to the treatment with test material.
In comparison to controls, there was, in both generations, a statistically significant increase in the relative and absolute mean kidney weights of treated animals for which no correlating morphological finding was found at light microscopy.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All the histomorphological findings were considered to have occurred fortuitously and were not associated with the administration of the test compound.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters - Sex ratio
The sex distribution and sex ratios of live F1a and F1b pups on the day of birth and on day 21 p.p. did not show any substantial difference between controls and treated test groups; all differences observed are regarded as spontaneous.

F1a and F1b generation pups/litters - Development stages
As to the several morphological development stages monitored up to weaning there were no biologically relevant differences between the control and the test material-treated F1a or F1b pups. The statistically significantly lower number of F1b pups of test groups 02 and 03 (100 and 500 ppm) with pinna unfolding on time and the statistically significantly higher number of low dose F1b pups with eye opening on time are not assessed as test material-related effects due to missing dose-response relationship.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F1a and F1b generation pups/litters
Out of the different behavioral tests which the F1a and F1b pups had to undergo up to weaning, no remarkable differences, which could be assessed as test material-related, were noted.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

2

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup clinical observations
F2 generation pups did not show any clinical symptoms during rearing which could be attributed to the treatment. For a few pups some spontaneous findings like oedema of the hindlimbs, lesion of/or shortened tail and lesion of hind-and/or forelimbs were recorded without any dose-response relationship.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup number and status at delivery
The mean number of delivered F2 pups/dam and the percentages of liveborn and stillborn pups do not show any statistically significant differences between the groups. Although the number of stillborn pups seems to be increased in the test material-treated groups it is fully in the range of biological variation.

F2 generation pups/litters - Pup viability/mortality
The number of pups, which died or were cannibalised from days 0 - 4 p.p. (before culling) was increased in the 500 ppm group in comparison to the control group and as a consequence, the viability index as an indicator of the pups' viability during the first 4 days after birth was lowest in this test group; this has to be assessed as a test material-induced effect.
The statistically significantly increased number of cannibalised pups in the 20 ppm group, however, is regarded as an incidental finding, which was mainly caused by dam No. 337, which cannibalised already 7 out of the 16 pups cannibalised in total.
The lactation index as an indicator how pups were nursed during the rest of their rearing varied for F2 pups between 100 % (test group 11 - 20 ppm) and 97 % (test group 13 - 500 ppm) and did not show any substantial differences between the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup body weight data
Mean pup body weights of the 500 ppm group are statistically significantly lower in comparison to the relevant control values on days 14 and 21 p.p.; moreover, weight gain of these pups is impaired on days 4 - 7, 7 - 14 and 4 - 21 p.p., which has to be attributed to the test material administration. All other differences between the groups concerning pup body weight data of the F2 generation are of spontaneous nature.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup necropsy observations
All recorded pup necropsy observations (e.g. incisors sloped, cataract, dilated renal pelvis etc.) occurred without a clear dose-response relationship. They were recorded for a very few pups of different groups with or without involvement of the control group and are assessed as being of spontaneous origin.

Histopathological findings:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Sex ratio
The sex distribution and sex ratio of live F2 pups on the day of birth and on day 21 p.p. did not show any biologically relevant differences between controls and treated test groups. All differences observed are regarded as spontaneous.

F2 generation pups/litters - Development stages
As to the morphological development stages monitored up to weaning, the number of pups of test groups 11, 12 and 13 (20, 100 and 500 ppm) showing pinna unfolding on time was statistically significantly reduced in comparison to the control group; however, due to missing dose-response relationship and the unexpected high number of control pups with a positive test result, this is finally assessed as an incidental finding. This is also assumed for the lower number of pups of the intermediate dose group with a positive test result on eye opening.
The statistically significantly lower number of high dose pups, however, which showed auditory canal opening on time is probably connected with the retarded, test material-related growth of these pups.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F2 generation pups/litters - Behavioral tests
No substantial differences could be noted between the F2 pups of test groups 11 - 13 (20, 100, 500 ppm) and the control pups the observable differences are without any biological relevance.

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: Higher number of pups with delayed auditory canal opening.

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Absolute Weights: Mean Values Males F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	552.083	541.461	543.52	539.312
	SD	40.489	38.125	62.586	39.376
	n	23	23	25	25
Liver	M (g)	18.058	17.514	17.06	16.484
	SD	2.244	1.946	3.138	1.884
	n	24	23	25	25
Kidneys	M (g)	3.376	3.38	3.492	3.694**
	SD	0.287	0.337	0.281	0.231
	n	24	23	25	25
Testes	M (g)	3.796	3.706	3.667	3.73
	SD	0.305	0.289	0.367	0.267
	n	24	23	25	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Females F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	314.226	314.368	315.305	313.304
	SD	14.516	29.748	19.369	21.353
	n	23	25	22	25
Liver	M (g)	9.783	9.849	9.49	9.888
	SD	0.754	1.371	0.819	0.97
	n	23	25	22	25
Kidneys	M (g)	2.098	2.12	2.157	2.286**
	SD	0.146	0.186	0.142	0.166
	n	23	25	22	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Males F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	1000	100	100	100
	SD
	n	23	23	25	25
Liver	M (%)	3.244	3.232	3.124	3.053*
	SD	0.251	0.239	0.282	0.221
	n	23	23	25	25
Kidneys	M (%)	0.61	0.624	0.646*	0.687**
	SD	0.047	0.042	0.049	0.048
	n	23	23	25	25
Testes	M (%)	0.689	0.687	0.681	0.694
	SD	0.05	0.061	0.086	0.056
	n	23	23	25	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Females F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	23	25	22	25
Liver	M (%)	3.113	3.124	3.008	3.156
	SD	0.181	0.201	0.151	0.218
	n	23	25	22	25
Kidneys	M (%)	0.668	0.667	0.685	0.731**
	SD	0.043	0.054	0.039	0.051
	n	23	25	22	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Males F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	545.459	523.94	535.183	537.204
	SD	65.445	54.429	62.088	44.204
	n	22	25	23	24
Liver	M (g)	17.355	16.575	16.818	16.764
	SD	3.144	2.907	2.712	2.13
	n	22	25	23	24
Kidneys	M (g)	3.084	3.136	3.326*	3.562**
	SD	0.239	0.324	0.3	0.282
	n	22	25	23	24
Testes	M (g)	3.835	3.768	3.192	3.875
	SD	0.287	0.302	0.411	0.298
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Females F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	295.318	297.38	282.787	293.513
	SD	19.516	25.787	13.526	16.87
	n	22	25	23	24
Liver	M (g)	9.41	9.534	9.02	9.712
	SD	0.765	1.048	0.456	0.776
	n	22	25	23	24
Kidneys	M (g)	2.03	2.107	2.048	2.189**
	SD	0.16	0.201	0.157	0.181
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Males F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	22	25	23	24
Liver	M (%)	3.167	3.152	3.135	3.118
	SD	0.287	0.316	0.279	0.263
	n	22	25	23	24
Kidneys	M (%)	0.57	0.6	0.625**	0.664**
	SD	0.053	0.048	0.052	0.043
	n	22	25	23	24
Testes	M (%)	0.711	0.724	0.736	0.726
	SD	0.087	0.075	0.085	0.082
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Females F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	22	25	23	24
Liver	M (%)	3.19	3.209	3.191	3.309
	SD	0.222	0.263	0.12	0.184
	n	22	25	23	24
Kidneys	M (%)	0.688	0.71	0.725**	0.746**
	SD	0.043	0.048	0.051	0.045
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm. The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.

Executive summary:

The reproductive toxicity of the test material was assessed according to OECD Test Guideline 416, JMAFF, EPA/FIFRA and European Commission guidelines and in compliance with GLP.

The test material was administered to groups of 25 male and 25 female immature rats (F0 parental generation) as a constant homogeneous addition to the food in different dose levels (0, 20, 100 or 500 ppm). At least 70 days after the beginning of treatment, F0 animals were mated to produce a first litter (F1a) and subsequently re-mated to produce a second litter (F1b retained only until weaning). Groups of 25 males and 25 females selected from F1a pups as F1 parental generation were offered diets containing 0, 20, 100 and 500 ppm of the test material post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of F2 weanlings and F1 adult animals. Test diets containing the test material were offered continuously throughout the study.

Food consumption of the F0 and F1 parents was determined regularly during premating (once weekly) and additionally during gestation and lactation periods. In general, body weights of F0 and F1 parents were determined once weekly. However, during gestation and lactation females were weighed on days 0, 7, 14 and 20 of gestation, on the day of parturition, and on days 4, 7, 14 and 21 after delivery. Pups were weighed on the day after birth and on days 4, 7, 14 and 21 thereafter.

The parents' and pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Pups were sexed and monitored with respect to their development stages and their behaviour in certain tests. Their viability was recorded. All pups were examined macroscopically at necropsy; if necessary, certain pups were additionally inspected for any organ/skeletal findings.

Blood and urine samples were taken from 12 F0 and 12 F1 males and females of each test group towards the end of the relevant study period for clinico-chemical examinations.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system.

The amount of test material administered (during premating (F0 and F1 parental animals)) was of an average of about 2 mg/kg body weight/day in the 20 ppm group, of about 10 mg/kg body weight/day in the 100 ppm group, and of about 50 mg/kg body weight/day in the 500 ppm group.

 

The following findings were obtained and assessed as test material-related:

500 ppm group (ca. 50 mg/kg body weight/day):

F0 parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F1a and F1b pups

Clinical examinations: Slightly increased number of F1a and F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability indices; marginal impairment of F1a pup body weight gain between days 7 - 14 p.p.

F1parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F2 pups

Clinical examinations: Increased number of pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index; lower mean pup body weights in comparison to the controls on days 14 - 21 p.p.: clear impairment of pup body weight gain between days 4 - 7, 7 - 14 and 4 - 21 p.p.; higher number of pups with delayed auditory canal opening.

 

100 ppm group (ca. 10 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased relative kidney weights (males only).

F1a and F1b pups: Slightly increased number of F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index.

F1parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased absolute (males only) and relative (both sexes) kidney weights.

F2 pups: Nothing abnormal detected.

 

20 ppm groups (ca. 2.0 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F1a and F1b pups: Nothing abnormal detected

F1 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F2 pups: Nothing abnormal detected.

 

It can be said in conclusion that the dietary administration of the test material to rats in doses of 500 ppm (ca. 50 mg/kg body weight/day) caused signs of systemic toxicity in the parental animals of both generations and their progeny. At this dose level, however, no indications for a test material-induced impairment of the reproductive function were present.

100 ppm (ca. 10 mg/kg body weight/day) was still marginally toxic, but there were no indications of a disturbed reproductive function of the F0 or F1 parents.

20 ppm (ca. 2 mg/kg body weight/day) was tolerated by the parental animals of both generations and by all litters (F1a, F1b and F2) without any test material-related adverse effects.

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm. (equivalent to ca. 40 mg/kg bw/day).

 The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.