Mecoprop

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Two-Generation Reproductive Toxicity: Hellwig (1992)

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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1984

Deviations:

no

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.

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.

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.

Dose / conc.:

20 ppm (nominal)

Dose / conc.:

100 ppm (nominal)

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

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

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: 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.

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

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

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

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

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: 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.

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

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

Critical effects observed:

no

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.

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:

not examined

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

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

Critical effects observed:

no

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.

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:

not examined

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.

Critical effects observed:

no

Reproductive effects observed:

no

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Single study conducted according to standardised guidelines and in compliance with GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

 Two-Generation Reproductive Toxicity: Hellwig (1992)

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.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity / teratogenicity (rabbit) range finding study: Tucker & Irvine (1978).

Under the conditions of this study, signs of toxicity were limited to the dose level of 100 mg/kg/day. At this dose the test material appeared to be toxic to the female rabbits but not to the surviving foetuses.

 

Pre-natal developmental toxicity / teratogenicity (rabbit): Irvine (1980).

Under the conditions of this study, there was no evidence of teratogenicity following treatment with the test material at the dose levels given.

 

Pre-natal developmental toxicity / teratogenicity (rat): Irvine (1980).

Under the conditions of this study, the test material showed no evidence of teratogenicity at the dose levels studied.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 1979 to 20 April 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The teratogenic potential of the test material by oral administration was evaluated in Sprague Dawley rats.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Female rats were nulliparous and aged between 10 and 12 weeks at the start of the study. Male rats were sexually mature.
- Housing: Prior to mating, the rats were housed in groups of five of the same sex in solid floor polypropylene cages with stainless steel lids (56 cm x 38 cm x 18 cm). During mating, one male was housed with four females in similar cages. Mated female rats were housed individually in solid floor polypropylene cages with stainless steel lids (38 cm x 25 cm x 18 cm).
- Diet: Commercial pelleted diet, ad libitum.
- Water: Mains water was available from glass bottles attached to each cage. The water was changed daily and the bottles replaced at approximately weekly intervals.
- Acclimation period: The animals were acclimatised to the laboratories for a minimum period of 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 ± 5.5 °C
- Humidity (%): 69.5 ± 20.5 %
- Air changes (per hr): The room was fully air-conditioned providing 18 - 20 complete air changes per hour.
- Photoperiod (hrs dark / hrs light): The lighting of the room was entirely artificial fluorescent lighting with a controlled cycle of 12 hours light (06.00 - 18.00 hours):12 hours dark.

Route of administration:

oral: gavage

Vehicle:

other: Methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared as a suspension in 1 % methyl cellulose and formulated so that a constant dose volume of 10 mL/kg was used.
- Each time that suspensions were prepared, separate aliquots of test material was weighed for each dosage level.
- Batches of test material suspensions were prepared at weekly intervals. The prepared suspensions were stored in opaque plastic containers at room temperature.

VEHICLE
- 1 % methyl cellulose.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Samples of each prepared batch of test and positive control material suspensions were stored in plastic containers at 4 °C for subsequent analysis, should this be required.

Details on mating procedure:

The rats were mated on a harem basis with four females per male. Vaginal smears were checked in the morning for the presence of sperm. The day on which sperm was observed was designated day 0 of gestation. Smearing was discontinued when sperm was found. The stage of oestrus at the time of mating was also recorded.
For mating, males and females were housed together for 23 days over a 39 day period.
The day on which sperm were observed in a vaginal smear was designated day 0 of gestation.

Duration of treatment / exposure:

The test and control materials were administered once daily from day 6 to day 15 of gestation (i.e. day 6 to day 15 after mating). The appropriate individual dose volumes administered were adjusted after each weighing i.e. on days 6, 9, 12 and 15, after mating.

Frequency of treatment:

Once daily.

Dose / conc.:

0 other: mg/kg/day

Remarks:

Group 1: Control

Dose / conc.:

20 other: mg/kg/day

Remarks:

Group 3

Dose / conc.:

50 other: mg/kg/day

Remarks:

Group 4

Dose / conc.:

125 other: mg/kg/day

Remarks:

Group 5

No. of animals per sex per dose:

Control: 30 female rats + 8 additional animals
Positive control: 12 female rats + 4 additional animals
Test material: 20 female rats + 8 additional animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the client, following examination of the data from a preliminary dose ranging study in the pregnant rat (report number 679R-277/6).
- Rationale for animal assignment: Immediately after mating, the female rats were allocated to treatment groups according to body weight, so that the mean weight and weight distribution were similar in all groups. and in a manner so as to reduce temporal bias. The position of the animals in the battery was determined using computer generated random number tables. At necropsy, the animals were killed in an order dictated by random number tables.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All mated females were examined at least once daily to determine their general health and behaviour. Any changes observed were recorded. Animals dying during the study were necropsied.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each mated female rat was recorded on days 0, 3, 6, 9, 12, 15, 18 and 21, after mating.

FOOD CONSUMPTION
- Food consumption of each mated female rat was recorded on days 3, 6, 9, 12, 15, 18 and 21, after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: The female rats were killed by cervical dislocation on day 21 after mating, dissected and subjected to gross necropsy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Numbers of corpora lutea.
- Numbers and positions of foetuses and intra-uterine deaths.

Intra-uterine deaths were classified as early or late. Late intra-uterine deaths showed embryonic or foetal tissue in addition to placental tissue, whereas early intra-uterine deaths showed decidual or placental tissue only.

Fetal examinations:

External examinations: Yes
- Individual foetal weights.
- Individual foetal crown/rump lengths.
- Sex of foetuses.
- The exterior of all the foetuses was examined.

Before being weighed and measured, the foetuses were killed by an intracardiac injection of Euthatal (pentobarbitone sodium solution, 200 mg/mL).

Visceral examinations: Yes
- One half of the foetuses from each litter was dissected under x2 magnification and the viscera examined.

Skeletal examinations: Yes
- The foetuses were then eviscerated and the carcasses placed in 1 % (w/v) aqueous potassium hydroxide containing 0.005 % Alizarin Red S to stain the ossified skeleton. The specimens were processed through ascending concentrations of glycerol/ distilled water. During this process (at the 30 - 50 % mole of glycerol/ distilled water stage) they were examined. They were preserved in absolute glycerol containing thymol crystals and stored in individual 30 mL plastic universal containers.

The remaining foetuses were placed in Bouin' s fixative for at least one week to allow fixation and partial decalcification. They were then transferred to 70 % alcohol, and free-hand transverse sections were made with a razor using Wilson's technique. The sections were examined under x6 magnification. The sections from each foetus were stored individually in plastic tubing containing Bouin's fluid.
Abnormalities were recorded as major (rare and/or probably lethal) or minor (common deviations from normal). Variations in the degree of ossification of the phalanges were also recorded at skeletal examination. Incompletely or non-ossified phalanges were classed as variants.
In calculation of the number of foetuses with defects, external/visceral defects and skeletal defects were considered separately because only half of the foetuses were examined for skeletal defects. Therefore, a foetus with both external/visceral defects and skeletal defects would be included twice in the calculation of defects.

Statistics:

Data were processed where appropriate to given mean values, group mean values and standard deviation.
Certain mean values were subscripted mean 1 and mean 2. Mean 1 values included data from those animals with functional corpora lutea in the ovaries on day 21 after mating. Mean 2 values included data from those animals with live foetuses. Group mean calculations were based on mean rather than individual data.

Standard statistical tests were applied to the data where appropriate, viz:-
- Foetal weights, foetal crown/rump lengths: Wilcoxon's rank sum test.
- Pre-implantation loss, implantations, intra-uterine deaths, foetal abnormalities: Fisher's two-sum randomisation (permutation) test with a Monte Carlo simulation for computation of significance levels.

With all statistical tests, the litter was considered as the experimental unit.

Indices:

Pre-implantation losses were calculated according to the formula:

% pre-implantation loss = (number of corpora lutea - number of implantations) / number of corpora lutea x 100

Sex ratios were calculated:

1: number of females / number of males

Historical control data:

In comparing data from this study with background data, the overall cumulative total (test facility and other laboratories, vehicle tested and animals from inactive treatments) was used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Group 3 (20 mg/kg/day test material):
- A clinical change was observed in only one animal. Number 98 (pregnant) showed red-brown discolouration of the hair on the head and neck from day 9 to day 21 after mating.

Group 4 (50 mg/kg/day test material):
- Two rats (numbers 228 and 588 - pregnant) showed red-brown discolouration of the hair on the head and neck from day 9 to day 21 after mating. No clinical changes were observed in any other animal.

Group 5 (125 mg/kg/day test material):
- A clinical change was observed in only one animal. In number 232 (pregnant), the eyes appeared pale from day 18 to day 21 after mating.
- One rat (number 203) littered on day 17 after mating. As the pups appeared normal for day 1 post-partum, it was presumed that mating actually occurred several days prior to the date recorded. The dam and progeny were killed, and the data from this animal were excluded from group totals and means.

Positive control (20 mg/kg/day aspirin)
- Two rats in this group showed clinical signs. In one (No. 31 - pregnant), hair loss was noted on the back from day 9 to day 21 after mating, and in the other (No. 72 - pregnant) difficulty in breathing prior to dosing was observed on day 11 after mating.
- One rat (No. 84 - not pregnant) was dosed in error for two days from day 3 after mating and was subsequently killed on day 15.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Group 1 (Control 1 % methyl cellulose)
- One rat died on day 14 after mating.
- No clinical changes were observed in any of the other animals.

Group 3 (20 mg/kg/day test material):
- One rat (number 136 - not pregnant) died on day 13 after mating.

Group 4 (50 mg/kg/day test material):
- No deaths occurred.

Group 5 (125 mg/kg/day test material):
- No deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weights were calculated using only data from pregnant animals surviving to day 21 after mating.

Group 1 (control) animals showed a steady weight gain throughout gestation. Weight gain of group 3 animals (20 mg/kg/day test material) was similar to the group 1 controls. Group 4 animals (50 mg/kg/day test material) showed a slight retardation in weight gain between days 3 and 12 of gestation and a slightly lower overall percentage body weight gain than the group 1 controls. In group 5 (125 mg/kg/day test material) there was a retardation of weight gain in comparison with the group 1 controls from day 9 to day 21 after mating, and a lower overall body weight gain was observed. The retardation in group 5 was partly attributable to one pregnant animal (number 232) which lost weight over gestation and to another (number 42) which showed a very low weight gain. However, even if these two animals were discounted, the overall weight gain was still slightly lower than the group 1 controls.

There was a slight retardation of weight gain in group 2 (positive control) animals from day 6 to day 9 of gestation, but generally weight gain was similar to the group 1 controls.

The overall percentage weight gains (day 0 - 21 after mating) were 50.2 %, 46.2 %, 51.8 %, 43.7 % and 38.4 % in groups 1, 2, 3, 4 and 5, respectively.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food intakes were calculated using only data from pregnant animals surviving to day 21 after mating.

Mean food intake in the group 1 (control) animals increased steadily up to day 15 of gestation. Between day 15 and 18, a peak was observed. This was followed by a decrease towards the end of gestation.

Mean food intake levels in the test material treated groups were lower than in group 1 (control) before dosing commenced.

In group 3 (20 mg/kg/day test material), food intake was very slightly lower after the onset of dosing, but on days 12 to 15 it was higher than in group 1; thereafter it followed the control pattern.

In group 4 (50 mg/kg/ day test material), a reduction in mean food intake was observed at the start of the dosing period, then it increased slightly and returned to the control pattern from day 15 onward.

In group 5 (125 mg/kg/day test material), a reduction in food intake was observed between day 9 and day 15 of gestation and remained lower than in group 1 (control) throughout the post-dosing period.

In group 2 (positive control), a reduction in mean food intake was noted at the start of the dosing period (day 6 to day 9 of gestation) but then increased between days 12 and 15 and was greater than the control value. From day 15 onwards, it followed the control pattern.

The overall mean daily food intake was 25.5 g, 24.7 g, 25.3 g, 24.3 g and 22.5 g in groups 1, 2, 3, 4 and 5, respectively.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Group 1 (control 1 % methyl cellulose):
- One rat died on day 14 after mating. Necropsy revealsed slight opacity of the right eye and the lungs appeared moderately congested and showed evidence of oedema.
- At necropsy on day 21 after mating, the liver of one rat (pregnant) appeared fatty and in another the uterine meseneries appeared fatty.

Group 2 (positive control, 250 mg/kg/day aspirin)
- One rat (No. 84 - not pregnant) was dosed in error for two days from day 3 after mating and was subsequently killed on day 15. No abnormalities were detected at necropsy in this or an other animal.

Group 3 (20 mg/kg/day test material):
- One rat (number 136 - not pregnant) died on day 13 after mating. At necropsy, the lungs appeared slightly congested and there was partial collapse of all lobes.
- At necropsy on day 21 after mating, three rats (number 28 - pregnant and numbers 74 and 199 - not pregnant) showed excessive fat in the abdomen. No abnormalities were detected in any other animal.

Group 4 (50 mg/kg/day test material):
- At necropsy on day 21 after mating, one rat (number 14 - not pregnant) showed adhesion of the pericardial sac and the right lung lobes to the thoracic wall. In another rat (number 135 - pregnant), all the placentae appeared enlarged. No abnormalities were observed at necropsy in any other animal.

Group 5 (125 mg/kg/day test material):
- At necropsy on day 21 after mating, one rat (number 192 - not pregnant) had excessive fat in the abdomen. In another rat (number 42 - pregnant), two placentae appeared enlarged. No abnormalities were observed at necropsy in any other animal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Implantations
- Mean numbers of corpora lutea per dam were similar in group 1 (control) and in the test material treated groups. All values were higher than the cumulative normal mean for this strain of rat.
- Pre-implantation losses in groups 1 (control), 4 (50 mg/kg/day test material) and 5 (125 mg/kg/day test material) were similar but all values were higher than the cumulative normal mean. In group 3 (20 mg/kg/day test material), pre-implantation loss was significantly lower than in group 1 (control) (p<0.05: Fisher's test).
- The mean number of implantations per dam was similar to group 1 (control) in group 4 (50 mg/kg/day test material) and 5 (125 mg/kg/day test material) but was slightly higher in group 3 (20 mg/kg/day test material).
- In group 2 (positive control), pre-implantation loss was lower and the mean number of corpora lutea and implantations was higher than in group 1 (control).

Intra-uterine deaths
- Early intra-uterine deaths: Early intra-uterine deaths were observed in all test material treated groups and in group 1 (control). The incidence in groups 3 (20 mg/kg/day test material) and 4 (50 mg/kg/day test material) was similar to group 1 (control). The incidence in group 5 (125 mg/kg/day test material) was higher than in group 1 (control) but was not significantly different at the 95 % level of confidence (Fisher's test). The incidence in group 2 (positive control) was significantly higher than in group 1 (control) (p<0.01:Fisher's test).
- Late intra-uterine deaths: No late intra-uterine deaths were observed in groups 1 (control) or 3 (20 mg/kg/day test material) and, in group 4 (50 mg/kg/day test material), only one was noted. Five were observed in group 5 (125 mg/kg/day test material), but this number was not significantly different from the group 1 controls at the 95 % level of confidence (Fisher's test). Six late intra-uterine deaths were observed in group 2 (positive control) but the incidence was not significantly different from group 1 (control) at the 95 % level of confidence (Fisher's test).
- Total intra-uterine deaths (post-implantation loss): The incidence of total intra-uterine deaths was similar to that in group 1 (control) in groups 3 (20 mg/kg/day test material) and 4 (50 mg/kg/day test material), but significantly higher in group 5 (125 mg/kg/day test material) (p<0.05: Fisher's test). Post-implantation loss in group 2 (positive control) was significantly higher than in group 1 (control) (p<0.01:Fisher's test).

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Overall pregnancy incidences, including animals that died or were killed during the study were 65.8 %, 68.8 %, 71.4 %, 67.9 % and 75.0 % in groups 1, 2, 3, 4 and 5, respectively. The pregnancy incidences for the animals that survived to day 21 after mating were 64.9 %, 73.3 %, 74.1 %, 67.9 % and 74.1 % in groups 1, 2, 3, 4 and 5, respectively. Therefore, the number of mated females that became pregnant was unaffected by treatment.

Other effects:

not examined

Details on maternal toxic effects:

Body weight gain of animals treated at 125 mg/kg/day test material (group 5) was lower than in group 1 (control). The low group mean body weights were partly attributable to one dam which lost weight over gestation and to another which showed a very low weight gain. However, even if these two animals are discounted there was still a very slight retardation of weight gain in comparison with group 1 (control). As the weight gain in the dams given 50 mg/kg/day test material was also slightly retarded during pregnancy, these effects are considered to be a manifestation of maternal toxicity. There was no effect at the lowest dosage (20 mg/kg/day test material). These findings are consistent with the observed slight reductions in food intake among test material treated rats.

There was an indication of slight embryolethality at 125 mg/kg/day test material as post-implantation loss was statistically significantly higher than in group 1 (control). This was due to a high incidence of dams showing early intra-uterine deaths. There was no increase in post-implantation loss at the lower test material dose levels.

One rat (number 203) littered on day 17 after mating. As the pups appeared normal for day 1 post-partum, it was presumed that mating actually occurred several days prior to the date recorded. The dam and progeny were killed, and the data from this animal were excluded from group totals and means.

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Foetal weights:
- Mean foetal weight in group 5 (125 mg/kg/day test material) was slightly lower than in group 1 (control), but the difference was not significant at the 95 % level of confidence (Wilcoxon's test). Mean foetal weight in the other test material treated groups was similar to that in group 1 (control). All values were within the cumulative normal range, but were slightly lower than the cumulative normal mean. In group 2 (positive control), mean foetal weight was significantly lower than in group 1 (control) (p<0.01: Wilcoxon's test).

Foetal crown/rump lengths:
- Mean crown/rump length in group 5 (125 mg/kg/day test material) was significantly lower than in group 1 (control) (p<0.01: Wilcoxon's test). Mean crown/rump length in the other test material treated groups was similar to group 1 (control). All values were within the cumulative normal range, but were slightly lower than the cumulative normal mean. In group 2 (positive control), mean crown/rump length was significantly lower than in group 1 (control) (p<0.01: Wilcoxon's test).

Reduction in number of live offspring:

not examined

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The calculated sex ratios in the test material treated groups and in group 2 (positive control) did not differ significantly from group 1 (control) values (Wilcoxon's test - 95 % level of confidence). Group mean values were 1:0.90, 1:1.02, 1:0.92, 1:0.87 and 1:1.11 in groups 1, 2, 3, 4 and 5, respectively.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Number of foetuses:
- The mean number of foetuses per dam was similar in groups 4 (50 mg/kg/day test material) and 5 (125 mg/kg/day test material) and slightly higher in group 3 (20 mg/kg/ day test material) than in group 1 (control). All values were within the cumulative normal range and were higher than the cumulative normal mean.
- The mean number of foetuses per dam in group 2 (positive control) was slightly lower than in group 1 (control) as a result of higher post-implantation loss.

Litter weights:
- Mean litter weight was higher in group 3 (20 mg/kg/day test material), slightly higher in group 4 (50 mg/kg/day test material) and slightly lower in group 5 (125 mg/kg/day test material) when compared with group 1 (control). None of the test material values was significantly different from group 1 (control) at the 95 % level of confidence (Wilcoxon's test).
- In group 2 (positive control), mean litter weight was significantly lower than in group 1 (p<0.05: Wilcoxon's test).

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Group 1, 3, 4 and 5 (negative control and treatment groups):
- Minor external/visceral defects: Foetuses showing minor external/visceral defects were observed in all test material treated groups and in group. 1 (control). The defects most frequently observed were dilated ureter, increased renal pelvic cavitation and subcutaneous haemorrhagic areas of the body. The incidence of foetuses showing only minor external/visceral defects was slightly, but not significantly, higher in the test material treated groups than in group 1 (control) (Fisher's test - 95 % level of confidence); the incidence was not dose related. Group percentage incidences were 8.2 %, 10.3 %, 15.4 % and 12.7 % in groups 1, 3, 4 and 5, respectively.

- Major external/visceral defects: In group 1 (control), one foetus showed a major defect. Foetus number 149/R2 showed encephalocele. No major defects were observed in group 3 (20 mg/kg/day test material). In group 4 (50 mg/kg/day test material), three foetuses from different litters showed major defects. Number 217/R6 showed encephalocele, number 228/R3 showed hydronephrosis and number 588/14 showed umbilical hernia. In group 5 (125 mg/kg/day test material), one foetus showed a major defect. Foetus number 260/R1 showed subcutaneous oedema and a short kinked tail. The percentage of foetuses showing major external/visceral defects was 0.3 %, 0.0 %, 1.4 % and 0.4 % in groups 1, 3, 4 and 5, respectively.
In group 2 (positive control) 16 of the 117 foetuses (13.7 %) showed major external and visceral defects. All the foetuses in group 2 (positive control) had variants and the number of litters affected was significantly higher than in group 1 (control) (p<0.05: Fisher's test). The nature and incidence of these defects confirms the susceptibility of this strain of rat to aspirin.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Minor skeletal defects: Minor skeletal defects were observed in all the test material treated groups and in group 1 (control). The defects observed were mainly incomplete sternebral ossification, bipartite thoracic vertebrae and incomplete ossification of the metatarsals. The incidence was higher in group 5 (125 mg/kg/ day test material) than in group 1 (control) but the difference was not significant at the 95 % level of confidence (Fisher's test). The increased incidence was largely due to a higher proportion of the foetuses showing retarded ossification. The percentage of foetuses showing minor skeletal defects was similar to, or lower than, group 1 ( control) in groups 3 (20 mg/kg/ day test material) and 4 (50 mg/kg/day test material). The percentage of foetuses in which the skeletons were examined, that showed minor skeletal defects was 38.8 %, 39.3 %, 29.8 % and 54.2 % in groups 1, 3, 4 and 5, respectively.

- Major skeletal defects: No major defects were observed in the test material treated groups or in group 1 (control). There were no significant differences between the control and test material treated groups in the incidences of major and minor external/visceral defects, major and minor skeletal defects and variants at the 95 % level of confidence (Fisher's test).

- Variants: The percentage of foetuses showing variants was similar to group 1 (control) in groups 3 (20 mg/kg/day test material) and 4 (50 mg/kg/day test material); the incidence was slightly but not significantly higher in group 5 (125 mg/kg/day test material) at the 95 % level of confidence (Fisher's test) .

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See 'External malformations'.

Details on embryotoxic / teratogenic effects:

There was evidence of slight foetotoxicity at the 125 mg/kg/day test material dose level, as mean foetal weight was slightly lower and mean crown/rump length was statistically significantly shorter than in group 1 (control). These effects were reflected in the higher proportion of foetuses showing delayed ossification although the incidence of minor skeletal defects and variants in the group treated at 125 mg/kg/day test material did not differ significantly from group 1 (control) (Fisher's test - 95% level of confidence).

Major defects were observed in the control and test material treated groups. There was no consistent pattern in the type of major defect observed and no treatment related effect was seen. The nature and incidence of major defects, the marked embryolethality and the marked test material in group 2 (positive control) proved the susceptibility of this strain of rat to aspirin.

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the test material showed no evidence of teratogenicity at the dose levels studied.

Executive summary:

The teratogenicity of the test material to the rat was assessed. 

Groups of sexually mature nulliparous Sprague Dawley-derived rats were dosed with the test material by oral gavage at dose levels of 20, 50 and 125 mg/kg daily (in a volume of 10 mL/kg) from day 6 to day 15 after mating. A group of animals receiving 1 % methyl cellulose (10 mL/kg) over the same period acted as the control group. In addition, a group of similar animals was dosed with aspirin (250 mg/kg/day) over the same period to serve as the positive control. (The vehicle control group and positive control group were common to a similarly designed, concurrent teratogenicity study), (report number 1996-277/7b).

There was no indication of treatment-related clinical changes in the dams.

Pregnancy incidence was unaffected by treatment with the test material. 

There was a slight retardation in the body weight gain of the group 5 dams (125 mg/kg/day test material) and a low overall body weight gain was observed. The group 4 dams (50 mg/kg/day test material) were also slightly affected, but the body weight gain of those in group 3 (20 mg/kg/day test material) was similar to that of the group 1 controls.

During the dosing period, all mecoprop treated groups showed some reduction in food intake; the effect was most pronounced in group 5 (125 mg/kg/day test material).

Pre-implantation loss and the mean number of live foetuses per litter were unaffected by treatment with the test material.

Post-implantation loss was significantly higher in group 5 (125mg/kg/day test material) (p<0.05: Fisher's test) than in group 1 (control), but was similar to the group 1 controls in the other test material treated groups.

In comparison with group 1 (control), mean foetal weight was slightly lower in group 5 (125 mg/kg/day test material).

Mean crown/rump length was similar to the group 1 controls in groups 3 (20 mg/kg/day test material) and 4 (50mg/kg/day test material) but significantly shorter in group 5 (125mg/kg/day mecoprop) (p<0.05: Wilcoxon's test).

The percentage of foetuses showing minor external and visceral defects in the test material treated groups was slightly, but not significantly, higher than in group 1 (control) (Fisher's test - 95 % level of confidence). The incidences were not dose-related.

The percentage of foetuses showing minor skeletal defects and variants was similar to, or lower than, the group 1 controls in groups 3 (20 mg/kg/day test material) and 4 (50 mg/kg/day test material). The incidence was higher in group, 5 (125 mg/kg/day test material) due largely to a higher proportion of foetuses showing retarded ossification. The incidence of major defects was unaffected by treatment with the test material.

In group 2 (positive control), findings were consistent with those expected following treatment with aspirin. These were increased post-implantation loss, reduced mean foetal weight and crown/rump length and an increased incidence of major foetal defects, in comparison with group 1 (controls).

Under the conditions of this study, the test material showed no evidence of teratogenicity at the dose levels studied.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 December 1976 to 27 January 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The teratogenic potential of the test material by oral administration was evaluated in Dutch Belted rabbits. This study was a preliminary dose ranging study.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

Dutch

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not specified, but sexually mature.
- Housing: The rabbits were housed in individual grid floor cages all in the same room.
- Diet: Commercial pellet diet, ad libitum.
- Water: Ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 10 %
- Air changes (per hr): Not specified.
- Photoperiod (hrs dark / hrs light): Natural lighting conditions which was supplemented by artificial lighting when personnel were working in the room.

Route of administration:

oral: gavage

Vehicle:

other: Methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared as suspensions in 1 % w/v methyl cellulose and formulated to a constant dose volume of 10 mL/kg. The suspensions were prepared at weekly intervals and stored in light proof plastic containers at room temperature.

VEHICLE
- 1 % w/v methyl cellulose

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Samples of sperm were collected each day from proven male Dutch Belted rabbits using an artificial vagina. The samples were maintained at 37 °C and each sample was checked for the presence of motile sperm before being pooled. The pooled semen sample (approximately 3 - 4 healthy samples) was diluted with 20 mL saline solution at 37 °C. Throughout the insemination procedure the dilute semen was maintained at 37 °C.

Each female rabbit was inseminated with 0.75 mL of dilute semen. Ovulation was induced by the intravenous administration of 25 I.U. chorionic gonadotrophin. The animals were fertilised over a 2 day period and allocated to the treatment groups in such a manner as to avoid temporal bias. The day of artificial insemination was taken as day 0.

Duration of treatment / exposure:

The suspensions were administered from day 1 to day 27 after artificial insemination.

Frequency of treatment:

Daily.

Duration of test:

The rabbits were killed by cervical dislocation on day 28 after insemination.

Dose / conc.:

100 other: mg/kg/day

Dose / conc.:

25 other: mg/kg/day

Dose / conc.:

0 other: mg/kg/day

Remarks:

Control

No. of animals per sex per dose:

5 animals in total for the treatment groups and 10 animals in total for the control group were tested.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 100 mg/kg/day and 25 mg/kg/day of test material were selected by the client.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The general condition of the animals was noted daily.

BODY WEIGHT: Yes
- Time schedule: The body weights were recorded every three days throughout the treatment period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day: The rabbits were killed by cervical dislocation on day 28 after insemination and subjected to a macroscopic examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Numbers of corpora lutea.
- Numbers and positions of foetuses and dead implantations.
In this report intra-uterine deaths have been divided into early and late on the basis that early intra-uterine deaths show implantation sites having placental or decidual tissue only, whereas late intra-uterine deaths have embryonic or foetal tissue in addition to placental tissue.

Fetal examinations:

External examinations: Yes:
- Foetal weights, foetal crown/rump lengths and sex of foetuses were examined.
- Before being weighed and measured the foetuses were killed by an intracardiac injection of sagatal.
- All of the foetuses were examined externally.

Visceral examinations: Yes
- One half of the foetuses from each litter was then skinned, examined for visceral defects, sexed and eviscerated.

Skeletal examinations: Yes:
- The carcasses used for visceral examinations were placed in 1 % w/v aqueous potassium hydroxide containing 0.005 % w/v alizarin red S to stain and identify the ossified skeletons. These were subsequently examined and preserved by placing in ascending concentration of glycerol. They were finally stored in individual 250 mL glass Jars containing absolute glycerol with a few crystals of thymol to prevent bacterial growth.

The remaining foetuses were placed in Bouin's fluid for at least one week to allow fixation and partial decalcification. Transverse free hand razor sections were then prepared (using the Wilson free hand razor sectioning technique) and examined under x 6 magnification. The sections were preserved in plastic tubing containing Bouin's fluid.
Defects are classsed as either major or minor, with minor defects limited to the presence of supernumerary ribs either single or paired.

Statistics:

The data were analysed statistically by means of Student's 't' test and Fisher 2 sample test as appropriate. Student's 't' test was applied to foetal weight and foetal crown/rump lengths. Fisher 2 sample test was applied to intra-uterine deaths.
Mean values were either mean 1 or mean 2, where mean 1 values included data from those animals with functional corpora lutea on day 28 in the ovaries, whereas mean 2 was limited to data from those animals with live foestuses.

Indices:

Pre-implantation losses were calculated from the formula:

% pre-implantation loss = (No. of corpora lutea - No. of implantations) x 100 / No. of corpora lutea

The mean foetal weights and crown/rump lengths were calculated as weighted averages to allow for variations in litter sizes.

Sex ratios were calculated as 1: No. of females / No. of males

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 100 mg/kg/day group severe toxic reactions were observed. Two animals were noted to have reduced food intake (based on a subjective visual assessment) from day 12 (rabbit no. 6) and day 19 (rabbit no. 8). Their condition deteriorated.
No signs of toxicity were noted in the 25 mg/kg/day group.

One control animal (no. 19) showed clinical signs consistent with pneumonitis infection and died on day 19, and one (no. 36) showed signs of distress consistent with accidental intubation of the dosing material into the lungs after dosing on day 23, but survived to the end of the study. Both animals were found to be pregnant at necropsy.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No deaths were noted in the 25 mg/kg/day group.
In the 100 mg/kg group, rabbit no. 6 was killed on day 16 for humane reasons and rabbit no. 8 was found dead on day 23.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The 25 mg/kg/day group and the control group animals showed net weight gains of 14.4 % and 12.4 % respectively during pregnancy.
The majority of the pregnant animals in these groups showed steady weight gains during the treatment period. The pregnant animals in the 100 mg/kg/day group showed a net weight loss of 8.2 % during the treatment period.
Two animals (nos. 6 and 8) in the 100 mg/kg/day group lost weight rapidly over the first fifteen days of the dosing period and in both cases death resulted.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

In the 100 mg/kg/day group, no abnormalities were observed in either animal that died at necropsy and both animals were found to be pregnant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Only one animal (no. 9) treated with 100 mg/kg/day test material produced a live litter, with six corpora lutea and three implantations, giving a pre-implantation loss of 50.0 %. Two other animals in this group were pregnant (nos. 6 and 8) but neither animal survived the test. At necropsy animal no. 6 was found to have eight corpora lutea and seven implantations (all early intra-uterine deaths) and animal no. 8 had eight corpora lutea and eight implantation sites (all early intra-uterine deaths).
A total of 27 corpora lutea was found in the 25 mg/kg/day group giving a mean number per dam of 6.75. Twenty-three implantations were seen, giving a mean number per dam of 5.75. The pre implantation loss was 14. 8 %.

In the control group, 35 corpora lutea were found in the five surviving pregnant animals, and eight were seen in animal no. 19 which died on day 19. The mean number of corpora lutea per dam was 7.00. Twenty-six implantations were seen in the surviving pregnant dams, and eight in the animal that died. The mean number of implantations per dam was 5.20 giving a pre-implantation loss of 25.7 %.

Intra-uterine deaths
No intra-uterine deaths were seen in the surviving 100 mg/kg/day group litter, however in the two animals that died (nos. 6 and 8) a total of 15 early intra-uterine deaths were observed. One death, a late intra-uterine death was seen in the 25 mg/kg/day group, giving a mean number per dam of 0.25. One early intra-uterine death was noted in the surviving animals in the control group giving a mean number per dam of 0.20, and eight early intra-uterine deaths were seen in dam no. 19 which died during the study.
A total of 11 late intra-uterine deaths (nine in one litter - dam no. 36) was seen in the control group resulting in a mean value per dam of 2.20.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

The number of animals pregnant in each group was three out of five in the 100 mg/kg/day group, four out of five in the 25 mg/kg/day group and six out of ten in the control group.

Other effects:

not examined

Details on maternal toxic effects:

The administration of the test material by oral gavage to female Dutch Belted rabbits produced signs of toxicity at a dose level of 100 mg/kg/day. Two out of five of the female rabbits died and weight losses were observed in the survivors. At this dose the test material appeared to be toxic to the female rabbits.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Abnormalities:

effects observed, treatment-related

Localisation:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The single litter in the 100 mg/kg/day group contained three foetuses with a mean weight of 40 g and a mean crown/rump length of 98 mm.

The mean foetal weight of the 25 mg/kg/day group foetuses was smaller than that of the control group but the value of 32 g compared closely to the cumulative normal value for this strain of rabbit whereas the control 1 group value of 37 g was unusually high. However both values lay within the background range.

The same comment applies to the crown/rump lengths with mean values of 89 mm in the 25 mg/kg/day group and 94 mm in the control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

A total of 39 foetuses was examined all being alive when removed from the uterus.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio of the 25 mg/kg/day foetuses was 1:1.75 and the control group value was 1:0.75. All three foetuses in the 100 mg/kg/day group were males. However, the numbers involved were too small to attach any significance to the findings.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter size in the 25 mg/kg/day group was 5.50 foetuses per dam compared with the control group value of 2.80 foetuses per dam.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Minor defects occurred in one foetus from the 100 mg/kg/day group, four foetuses in the 25 mg/kg/day group and three foetuses in the control group. All the defects were of the supernumerary rib type. No major defects were observed in any of the groups.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The administration of the test material by oral gavage to female Dutch Belted rabbits produced no signs of toxicity to foetuses at a dose level of 100 mg/kg/day. The one litter which was produced was morphologically normal and the crown/rump length and weight of the foetuses were greater than those of the control group. At this dose the test material did not appear to be toxic to surviving foetuses.

The 25 mg/kg/day group produced no adverse effects and although the foetuses were both smaller in crown/rump length and weight than the controls they were considered to be of normal size for this strain of rabbit.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

no effects observed

Developmental effects observed:

no

Group Mean Body Weights: Pregnant Animals Only

Day	100 mg/kg Test Material	25 mg/kg Test Material	Controls
0	2.31 (3)	1.94 (4)	2.02 (6)
3	2.20 (3)	2.00 (4)	2.05 (6)
6	2.08 (3)	2.05 (4)	2.09 (6)
9	1.86 (3)	2.08 (4)	2.12 (6)
12	1.78 (3)	2.10 (4)	2.13 (6)
15	1.68 (3)	2.14 (4)	2.14 (6)
18	1.84 (2)	2.17 (4)	2.23 (5)
21	1.75 (2)	2.16 (4)	2.25 (5)
24	2.12 (1)	2.22 (4)	2.28 (5)
28	2.12 (1)	2.22 (4)	2.27 (5)
Bodyweight change	-8.2 %	+ 14.4 %	+ 12.4 %

Numbers in parentheses indicate number of animals weighed on each day.

 

Group Mean Data

Parameter	100 mg/kg Test Material	25 mg/kg Test Material	Controls
No. of animals pregnant (a)	3 / 5	4 / 5	6 / 10
No. of corpora lutea (b)	6	27	35
Mean 1 no. of corpora lutea per dam (c)	6.00	6.75	7.00
No. of implantations per dam (b)	3	23	26
Mean 1 no. of implantations per dam	3.00	5.75	5.20
% pre-implantation loss	50.0	14.8	25.7

(a): Includes pregnant animals that died during the study.

(b) Only included from surviving animals.

(c) Mean 1 values include data from those animals with functional corpora lutea in the ovaries.

 

Group Mean Data: Implantation Data

Parameter		100 mg/kg Test Material	25 mg/kg Test Material	Controls
Implantations	Total no.	3	23	26
	Mean 1 no. per dam	3.00	5.75	5.20
Foetuses	Total no.	3	22	14
	Mean 1 no. per dam	3.00	5.50	2.80
	% of implantations	100.0	95.7	53.8
Early intra-uterine deaths	Total no.	0	0	1
	Mean 1 no. per dam	0.00	0.00	0.20
	% of implantations	0.0	0.	3.8
Late intrauterine deaths	Total no.	0	1	11
	Mean 1 no. per dam	0.00	0.25	2.20
	% of implantations	0.0	4.3	42.3
Total intrauterine deaths	Total no.	0	1	12
	Mean 1 no. per dam	0.00	0.25	2.40
	% of implantations	0.0	4.3	46.2

 

Group Mean Data: Foetal Data

Parameter	100 mg/kg Test Material	25 mg/kg Test Material	Controls
No. of foetuses	3	22	14
Mean 1 no. of foetuses per dam	3.00	5.50	2.80
Mean 2 litter weight (g) (d)	121	174	128
Mean 2 foetal weight (g)	40	32	37
Mean 2 crown/rump length (mm)	98	89	94
No. of males	3	8	8
No. of females	0	14	6
Ratio of males: females	-	1:1.74	1:0.75
No. of foetuses showing minor defects	1	4	3
No. of foetuses showing major defects	0	0	0

(d) Mean 2 values include data from those animals with live foetuses.

 

Nature and Incidence of Abnormalities

Parameter	100 mg/kg Test Material	25 mg/kg Test Material	Controls
No. of foetuses examined	3	22	14
No. of foetuses showing defects	1	4	3
No. of foetuses showing minor defects only	1	4	3
No. of foetuses showing major defects	0	0	0
Minor defects:
13 pairs of rubs	0	4	1
13 ribs on one side	1	1	2

Conclusions:

Under the conditions of this study, signs of toxicity were limited to the dose level of 100 mg/kg/day. At this dose the test material appeared to be toxic to the female rabbits but not to the surviving foetuses.

Executive summary:

The teratogenicity of the test material to the rabbit was assessed in a range-finding study. Dose levels of 100 mg/kg/day and 25 mg/kg/day of test material were administered by gavage to groups of five Dutch Belted rabbits from day 1 to day 27 after insemination. 

Signs of toxicity were limited to the 100 mg/kg/day group. Two animals treated at this level had a reduced food intake from day 12 after insemination. One animal (no. 6) was killed on day 16 and the other (no. 8) died on day 23; no abnormalities were seen at necropsy. Weight losses were observed in the surviving animals treated at this dose level. Only one animal in the 100 mg/kg/day group produced a litter, and this was morphologically normal. The litters of the 25 mg/kg/day group animals were normal by comparison with cumulative data on this strain of rabbit, but were smaller in both crown/rump length and weight than the control animals.

Under the conditions of this study, signs of toxicity were limited to the dose level of 100 mg/kg/day. At this dose the test material appeared to be toxic to the female rabbits but not to the surviving foetuses.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 1978 to 08 December 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The teratogenic potential of the test material by oral administration was evaluated in Dutch Belted rabbits.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

Dutch

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Female rabbits were sexually mature and were within the weight range 1.8 - 2.7 kg, with the exception of those inseminated on the last insemination day (day 15)when the weight range was extended to 1.8 - 3.1 kg.
- Housing: The rabbits were housed in individual galvanised steel cages with dimensions of 42 cm x 54 cm x 45 cm. The grid floors were constructed from 1.6 cm galvanised wire mesh steel excrement-removal trays. The cages were suspended 10 cm above stainless steel excrement-removal trays.
- Diet: Commercial pelleted diet ad libitum
- Water: Ad libitum
- Acclimation period: The animals were acclimatised to the laboratories for a minimum period of 18 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 ± 7 °C
- Humidity (%): 69. 5 ± 21. 5 %
- Photoperiod (hrs dark / hrs light): The lighting of the room was artificial fluorescent lighting with a controlled cycle of 14 hour light (06.00 - 20.00 hrs.): 10 hour dark.

Route of administration:

oral: gavage

Vehicle:

other: Methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared as suspensions in 1 % methyl cellulose and formulated so that a constant dose volume of 10 mL/kg was used. The prepared suspensions were stored in opaque glass containers at room temperature.
- Batches of test material suspensions were prepared at weekly intervals.
- Each time that suspensions were prepared, separate aliquots of test material were weighed for each dosage level.
- The appropriate individual dose volumes administered were adjusted after each weighing i.e. on days 6, 9, 12, 15 and 18 after insemination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each prepared batch of test and positive control material suspensions were stored in plastic bottles at 4 °C for subsequent analysis, should this be required.

Details on mating procedure:

The female rabbits were fertilised by artificial insemination. Samples of sperm were collected on each mating day, using an artificial vagina and a teaser doe, from male rabbits of proven fertility. Using a light microscope, each sample was examined, and those containing motile sperm were pooled. When sufficient samples had been obtained (approximately 5), the concentration of sperm in the pooled sample was assessed by standard dilution and observation and counting of the sperm in a known volume of saline using a haemocytometer. The sample was then diluted with 0.9 % physiological saline to a concentration of 16 million sperm/mL. Each female rabbit was inseminated with 0.75 mL of this dilute semen sample (approximately 12 million sperm).
On one day (insemination day 7) due to a calculation error, the sperm sample was prepared at a concentration of 15 million sperm/mL. The approximate sperm concentration administered to each femal was 11.25 million sperm/mL.

Throughout the insemination procedure, the sperm sample and saline solution were maintained at 37 °C in a thermostatically controlled water bath. Immediately following successful insemination, the female rabbits were injected intravenously via the marginal vein of the ear with 25 IU chorionic gonadotrophin to ensure ovulation.

The day of insemination was designated day 0 of gestation. Inseminations took place on fifteen days over a five week period.

Duration of treatment / exposure:

The test and control materials were administered once daily from day 6 to day 18 inclusive of gestation.

Frequency of treatment:

Daily.

Duration of test:

13 days.

Dose / conc.:

0 other: mg/kg/day

Remarks:

Group 1: Control

Dose / conc.:

12 other: mg/kg/day

Remarks:

Group 3

Dose / conc.:

30 other: mg/kg/day

Remarks:

Group 4

Dose / conc.:

75 other: mg/kg/day

Remarks:

GRoup 5

No. of animals per sex per dose:

Vehicle control: 30 female rabbits
Positive control: 10 female rabbits + 1 replacement
Test material group 3: 15 female rabbits + 1 replacement
Test material group 4: 15 female rabbits + 2 replacements
Test material group 5: 15 female rabbits

Replacement animals were included following the death or euthanasia of animals in these groups during the five week insemination period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the client, following examination of the data from a preliminary dose ranging study in the pregnant rabbit (683R-277/4).
- Rationale for animal assignment: The female rabbits were allocated to treatment groups according to body weight so that the mean weight and weight distribution were similar in all groups, and in a manner so as to reduce temporal bias. Immediately after each day's inseminations, the inseminated females were weighed, and the weights ranked in ascending order. Using random letter permutation tables with each letter corresponding to a group number, the females were allocated to groups in weight order. This process was followed on each insemination day until each group contained the requisite number of animals. The position of the animals on the battery was determined using the same random letter scheme. At necropsy, the animals were killed in an order dictated by the same random letter scheme.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All experimental female animals were examined at least once daily after insemination to determine their general health and behaviour. Any changes observed were recorded. Animals in extremis were killed immediately and necropsied. Similarly, animals dying during the study were necropsied.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of individual inseminated female rabbits were recorded on days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28 after insemination.

FOOD INTAKE: Yes
- Food intake of individual inseminated female rabbits was recorded on days 3, 6, 9, 12, 15, 18, 21, 24 and 28 after insemination.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: All animals surviving to day 28 after insemination were killed by cervical dislocation, dissected and subjected to gross necropsy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Numbers of corpora lutea.
- Numbers and positions of foetuses and intra-uterine deaths.

Intra-uterine deaths were classed as early or late. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue, whereas early intra-uterine deaths showed decidual or placental tissue only.

Fetal examinations:

External examinations: Yes
- Individual foetal weights.
- Individual foetal crown/rump lengths.
- Sex of foetuses.
- Before being weighed and measured, the foetuses were killed by an intracardiac injection of Euthatal (pentobarbitone sodium solution, 200 mg/mL).
- The exterior of all the foetuses was examined.

Visceral examinations: Yes
- One half of the foetuses from each litter was dissected and the viscera examined.

Skeletal examinations: Yes
- The foetuses were then eviscerated and the carcasses placed in 1 % (w/v) aqueous potassium hydroxide containing 0.005 % Alizarin Red S to stain the ossified skeleton. The specimens were processed through ascending concentrations of glycerol/ distilled water. During this process (at the 30 - 50 % mole of glycerol/ distilled water stage) they were examined. They were preserved in absolute glycerol containing thymol crystals and stored in individual glass 250 mL containers.

The remaining foetuses were placed in Bouin's fixative for at least one week to allow fixation and partial decalcification. They were then transferred to 70 % alcohol, and free-hand transverse sections were made with a razor using Wilson's technique. The sections were examined under x6 magnification. The sections from each foetus were stored individually in plastic tubing containing Bouin's fluid.

Abnormalities were recorded as major (rare and/or probably lethal) or minor (common deviations from normal).
In calculation of the number of foetuses with defects, external/visceral defects were considered separately. Therefore a foetus with both external/ visceral and skeletal defects is included twice in calculation of defects.
Foetuses were individually identified within a litter to enable each foetus to be matched to its weight and disposition in the uterine horn.

Statistics:

Data were processed where appropriate to give mean values, group mean values and standard deviations.
Mean values were subscripted mean 1, mean 2 and mean 3. Mean 1 values included data from those animals with functional corpora lutea in the ovaries on day 28 after insemination. Mean 2 values included data from those animals with live foetuses. Mean 3 values included data from those animals with foetuses examined for external/ visceral defects and skeletal defects. Group mean calculations were based on mean rather than individual data.

Standard statistical tests were applied to the data where appropriate:

- Foetal weights, foetal crown/rump lengths: Wilcoxon's rank sum test.
- Pre-implantation loss, implantations, intra-uterine deaths, foetal abnormalities: Fisher's two-sum randomisation (permutation) test with a Monte Carlo simulation for computation of significance levels.

With all statistical tests, the litter was considered as the experimental unit.

Indices:

Pre-implantation losses were calculated according to the formula:

% pre-implantation loss = ((number of corpora lutea - number of implantations) / number of corpora lutea) x 100

Sex ratios were calculated:

1: number of females / number of males

Historical control data:

In comparing data from this study with background data, the overall cumulative total (test facility and other laboratories; vehicle tested animals and animals from inactive treatments) was used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Group 1 control (1 % methyl cellulose)
Sero-purulent discharge was noted in two non-pregnant animals (nos. 208 and 197).

Group 2 positive control (200 mg/kg/day thalidomide)
One animal in this group (no. 312 - pregnant) died on day 19 after insemination having shown laboured respiration on day 18 and a red-brown fluid on the nose on days 17 and 18.
No clinical observations were noted for the surviving animals.

Group 3 12 mg/kg/day test material
- In one animal (number 311 - pregnant) a clinical observation of red-brown fluid round the nose was noted on day 21 after insemination.

Group 4 30 mg/kg/day test material
- One animal (number 192 - pregnant) died having shown laboured breathing and a ser-purulent nasal discharge.
- One animal (number 376 - not pregnant) died on day 11 after insemination having shown no clinical abnormalities.
- No clinical observations were noted in the surviving animals, though five animals showed abnormalities at necropsy on day 28.

Group 5 75 mg/kg/day test material
- On day nine after insemination, animal number 210 (pregnant) had a sero-purulent nasal discharge.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Group 1 control (1 % methyl cellulose)
No deaths were pbserved in this group.

Group 2 positive control (200 mg/kg/day thalidomide)
One animal in this group (number 312 - pregnant) died on day 19 after insemination.
Euthanasia was performed on one animal (number 361 - pregnant) after apparent vertebral dislocation on day nine after insemination.

Group 3 12 mg/kg/day test material
- No deaths were observed in this group.
- Euthanasia was performed on one animal (number 282 - pregnant) with bilateral conjunctivitis on day seven after insemination.

Group 4 30 mg/kg/day test material
- Two animals in this group died. One animal (number 192 - pregnant) died on day ten having shown laboured breathing and a sero-purulent nasal discharge.
- The other animal (number 376 - not pregnant) died on day 11 after insemination having shown no clinical abnormalities.

Group 5 75 mg/kg/day test material
- No deaths were observed in this group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weights were calculated using only data from pregnant animals surviving to day 28 after insemination.

Group 1 (control animals) showed a slight body weight loss at the beginning of the treatment period (days 6 - 9 after insemination), thereafter a weight gain was observed. Similar weight changes to the group 1 controls were observed in group 3 (12 mg/kg/day test material) and group 5 (75 mg/kg/day test material). Group 4 animals (30 mg/kg/day test material) showed a weight gain throughout gestation. The overall weight gains (days 0 - 28 after insemination) for the group 1, 3, 4 and 5 animals were 10.6 %, 10.3 %, 9.9 % and 9.8 %, respectively.

The mean body weight of the group 2 (positive control) animals was lower than the other groups on day 0 after insemination, and fluctuations in weight were observed throughout gestation. The overall weight gain (day 0 - 28 after insemination) was only 2.4 %.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Group 1 control (1 % methyl cellulose)
At necropsy on day 28 after insemination, one animal (number 263) which was not pregnant showed extensive fat deposition in the abdomen and around the heart. In one pregnant animal (number 300), the right lung appeared consolidated, and there were numerous cysts on the mesenteries. Three animals (number 291 - pregnant and numbers 379 and 383 - not pregnant) showed lesions on the liver and, in two pregnant animals (numbers 240 and 301), the liver appeared fatty.

Group 2 positive control (200 mg/kg/day thalidomide)
One animal in this group died on day 19. At necropsy, the left lung appeared congested and had fibrous adhesions and the right lung appeared as a cream/ red mass with adhesions to the thoracic wall, diaphragm and the pericardium.
Euthanasia was performed on one animal (number 361- pregnant) after apparent vertebral dislocation on day nine after insemination. At necropsy, no signs of haemorrhage were observed on the back, but the lungs of this animal appeared congested.
For the two surviving animals, two showed abnormalities at necropsy on day 28. In animal number 241 (pregnant), the lungs appeared congested, the gall bladder appeared enlarged and all the implantations were early intra-uterine deaths. In the other animal (number 270 - pregnant), the lungs appeared consolidated, there were fluid filled cysts on the mesenteries and there was a small abscess in the pelvic region of the abdomen.

Group 3 12 mg/kg/day test material
- Euthanasia was performed on one animal (number 282 - pregnant) with bilateral conjunctivitis on day seven after insemination. At necropsy, no other abnormalities were noted.
- At necropsy on day 28, one animal (number 311 - pregnant) showed compaction of the stomach with hair, irritation of the mucosal surface of the stomach, gaseous distension of the intestines, and all the implantations were early intra-uterine deaths.
- One other animal showed abnormalities at necropsy. In animal number 304 (pregnant), the lungs appeared congested and there was extensive fat deposition in the abdomen.

Group 4 30 mg/kg/day test material
- One animal (number 192 - pregnant) died on day 10. At necropsy, the lungs appeared black and congested; there was blood coloured fluid in the thoracic cavity, and the animal appeared to show pleurisy and pericarditis.
- In the other animal (number 376 - not pregnant) at necropsy, the lungs appeared congested; there was a large quantity of fluid in the thoracic cavity; the right lung lobe was deep purple in colour and covered with a fibrous material with attachments to the thoracic cavity wall, and the pericardium appeared thickened and was adhered to the epicardium.
- In animal number 244 (pregnant), the gall bladder appeared very small (approximately 5 mm in diameter). Animal number 238 (pregnant) had lesions on the liver and the surface of the kidneys appeared pitted. In animal number 392 (pregnant), there were fluid filled cysts on the liver. Animal number 373 (pregnant) showed extensive fat deposition in the abdomen and, in animal number 231 (pregnant), the right lung appeared consolidated, the liver appeared fatty and the surface of the kidneys appeared pitted.

Group 5 75 mg/kg/day test material
- Abnormalities at necropsy on day 28 were observed only in nonpregnant animals. Two animals (numbers 351 and 214) showed extensive fat deposition in the abdomen. In one animal (number 266), fluid filled cysts were noted on the uterine mesenteries and, in the other animal (number 353), numerous abscesses were observed in the right uterine horn.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Food intake was recorded throughout gestation. However, because of excessive wastage by some of the does, food intake could not be estimated, and the data generated were considered to be inconsequential.
In general, food intake appeared to be similar in the test material treated groups and the group 1 controls. Group 2 animals however, showed a lower level of food intake than the group 1 animals.

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean numbers of corpora lutea per doe in groups 2 (positive control), 3 (12 mg/kg/day test material) and 5 (75 mg/kg/day test material) were lower than the group 1 controls with values of 8.92, 7.88, 8.21 and 8.00 corpora lutea per doe in groups 1, 2, 3 and 5, respectively. In group 4 (30 mg/kg/day test material), the mean number of corpora lutea per doe (9.21) was greater than the group 1 controls. All mean values were within the cumulative normal range.

The pre-implantation loss in group 4 (25.6 %) was higher than in the group 1 controls and the other groups, and was higher than the cumulative normal for this strain of rabbit. Pre-implantation loss in the other test material treated groups and in group 2 (positive control) was lower than the group 1 controls.

The mean number of implantations per doe was slightly lower in the test material treated groups and in group 2 (positive control) than in the group 1 controls. Mean values ranged from 6.50 implantations per doe in group 2 (positive control) to 7.29 implantations per doe in group 1 (control). All mean values were within the cumulative normal range .

Intra-uterine deaths:
Intra-uterine deaths were observed in animals surviving to day 28 after insemination from all groups including controls.

No early intra-uterine deaths were observed in group 5 (75 mg/kg/day test material), and only one early intra-uterine death was observed in each of group 1 (control) and group 4 (30 mg/kg/day test material). In group 3 (12 mg/kg/day test material ), early intra-uterine deaths were observed in three does. In one of these does (number 311), all six of the implantations were early intra-uterine deaths. The group mean percentage of implantations that were early intra-uterine deaths was 8.2 %.

In group 2 (positive control), early intra-uterine deaths were observed in five does. Seven early intra-uterine deaths were seen in one doe (number 241) and the only implantation of doe number 307 was an early intra-uterine death. The group mean percentage of implantations that were early intra-uterine deaths was 23.1 %. The number of does showing early intra-uterine deaths in this group was significantly greater than the group 1 controls (p<0.01), Fisher's test).

Single late intra-uterine deaths were observed in group 1 (control) and group 3 (12 mg/kg/ day test material). In group 5 (75 mg/kg/ day test material), two does each had one late intra-uterine death and in group 4 (30 mg/kg/day test material), a total of four late intra-uterine deaths was observed. Two of these were seen in one doe, and two other does each had one late intra-uterine death.

In group 2 (positive control), three does each had one late intra-uterine death and another doe had five late intra-uterine deaths. The group mean percentage of implantations that were late intra-uterine deaths was 15.4 %. The number of does showing late intra-uterine deaths in this group was significantly greater than the group 1 controls (p<0.01, Fisher's test).

The overall percentage of implantations that were intra-uterine deaths was 1.1 %, 38.5 %, 9.2 %, 5.2 % and 2.6 % in groups 1, 2, 3, 4 and 5 respectively. The number of does showing intra-uterine deaths in group 3 (12 mg/kg/day test material) was significantly greater than the controls (p<0.05; Fisher's test) and, in group 2 (positive control), the number of does showing intra-uterine deaths was also significantly greater than the controls (p<0.01; Fisher's test). The group 2 value was greater than the cumulative normal for this strain of rabbit, though the group 3 value was lower.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Overall pregnancy incidences, including animals that died or were killed during the study were 80.0 %, 90.9 %, 93.8 %, 88.2 % and 73.3 % in groups 1, 2, 3, 4 and 5, respectively.
The pregnancy incidences for the animals that survived to day 28 after insemination were 80.0 %, 88.9 %, 93.3 %, 93.3 % and 73.3 % in groups 1, 2, 3, 4 and 5, respectively.

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weight in group 4 (30 mg/kg/day test material), group 5 (75 mg/kg/day test material) and the positive control group was slightly lower than the group 1 controls. The mean foetal weight of group 3 (12 mg/kg/day test material) was the same as the group 1 controls. The mean foetal weight was 34.2 g, 33.6 g, 34.2 g, 33.8 g and 33.2 gin groups 1, 2, 3, 4 and 5, respectively. None of the mean values were significantly different from the group 1 controls at the 95 % level of confidence (Wilcoxon's test), and all the mean foetal weight values were greater than the cumulative normal for this strain of rabbit.

None of the mean values was significantly different from the group 1 controls at the 95 % level of confidence (Wilcoxon's test).

Reduction in number of live offspring:

not examined

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The sex ratios of group 1 (control), group 2 (positive control), group 3 (12 mg/kg/day test material) and group 4 (30 mg/kg/day test material) were 1:1.16, 1:1.29, 1:1.12, and 1:1.22, respectively. In group 5 (75 mg/kg/day test material) the value of 1:1.53 males:females was slightly different from the other groups.

However, the intergroup variation in sex ratios was within the cumulative background range for this strain of rabbit and a treatment effect is not suspected.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The mean number of foetuses per doe in the test material treated groups and the positive control group was lower than the group 1 controls. In group 2 (positive control), the mean number of foetuses per doe was 4.00 and, in group 3 (12 mg/kg/day test material), 4 (30 mg/kg/day test material) and 5 (75 mg/kg/day test material), the mean numbers of foetuses per doe were 6.36, 6.50 and 6.91, respectively. For comparison, the group 1 (control) mean value was 7.21 foetuses per doe. All values were within the background range, and the test material values were similar to the cumulative normal mean.

Mean litter weights in the test material treated groups and the positive control group were lower than the group 1 controls. The mean litter weight was 243.7 g, 169.8 g, 220.7 g, 208.1 g and 226.5 gin groups 1, 2, 3, 4 and 5, respectively.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Groups 1, 3, 4 and 5 (negative control and test material treated groups)
- Minor external/visceral defects: Foetuses showing minor external/visceral defects were observed in all test material treated groups and the group 1 controls. The defect most frequently observed was post caval lung lobe agenesis. .The incidence of foetuses showing minor external/visceral defects was similar in the group 1 controls and in groups 3 (12 mg/kg/day test material) and 4 (30 mg/kg/day test material); it was lower in group 5 (7 5 mg/kg/day test material) than the group 1 controls. The percentage incidences were 4.0 % in the group 1 controls, 4.5 % in group 3, 4.4 % in group 4, and 1.3 % in group 5.
- Major external/visceral defects: In group 1 (control), three foetuses from different litters showed major defects. In litter 235, one foetus showed rectal stenosis, in litter 301, one foetus showed arthrogryposis in one forelimb and, in litter 396, one foetus showed omphalocele. In group 3 (12 mg/kg/day test material), only one foetus showed major defects. This foetus, from litter 366, showed unilateral kidney agenesis and hydronephrosis of the existing kidney. No major defects were observed in group 4 (30 mg/kg/day test material). In group 5 (75 mg/kg/day test material), two foetuses from different litters showed major defects. One foetus from litter 316 showed enlargement of the lateral ventricles and, in litter 303, one foetus showed multiple major and minor defects consisting of thoracogastroschisis, omphalocele, unilateral forelimb amelia, unilateral forelimb hemimelia, muscle flexure and post caval lung lobe agenesis.
- The percentage of foetuses showing major external/visceral defects was 1.7 % in the group 1 controls, 1.1 % in group 3 (12 mg/kg/day test material), 0.0 % in group 4 (30 mg/kg/day test material) and 2.6 % in group 5 (75 mg/kg/day test material).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Minor skeletal defects: Minor skeletal defects were observed in all test material treated groups and the group 1 controls. The defects observed were mainly supernumerary ribs or retarded ossification. Incidences of foetuses showing minor skeletal defects were similar in the test material treated groups and the group 1 controls. The percentage of foetuses, in which the skeletons were examined, showing skeletal defects, was 43.7 % in the group 1 controls, 37.8 % in group 3 (12 mg/kg/day test material), 45.7 % in group 4 (30 mg/kg/day test material), and 43.6 % in group 5 (75 mg/kg/day test material).
- Major skeletal defects: No major skeletal defects were observed in group 1 (control), group 3 (12 mg/kg/day test material) or group 4 (30 mg/kg/day test material). In group 5 (75 mg/kg/day test material), the major external/visceral defects of unilateral forelimb hemimelia and unilateral forelimb amelia observed in one foetus from litter number 303 were also seen at skeletal examination. In group 5, the percentage of foetuses, in which the skeletons were examined, that showed major skeletal defects was 2.6 %.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See 'External malformations'.

Other effects:

not examined

Description (incidence and severity):

Mean foetal crown/rump length showed little intergroup variation. Mean foetal crown/rump length was slightly greater than the group 1 controls in group 3 (12 mg/kg/day test material), slightly shorter than the group 1 controls in groups 2 (positive control) and 4 (30 mg/kg/day test material), and the same as the group 1 controls in group 5 (75 mg/kg/day test material). The mean foetal crown/ rump length was 94. 6 mm, 93. 9 mm, 95. 3 mm, 94. 4 mm and 94.6 mm in groups 1, 2, 3, 4 and 5, respectively.
All the mean crown/rump length values were greater than the cumulative normal for this strain of rabbit.

Details on embryotoxic / teratogenic effects:

- Statistical evaluation of defects: The incidence of foetuses showing defects, both external/visceral and skeletal (minor or major), in the test material treated groups was not significantly different from the group 1 controls at the 95 % level of confidence (Fisher's test).

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Group Mean Body Weights (kg): Pregnant Animals Surviving to day 28 After Insemination

Days After Insemination	Group 1 Control (1 % Methyl Cellulose)	Group 2 Positive Control (200 mg/kg/Day Thalidomide)	Group 3 (12 mg/kg /Day Test Material)	Group 4 (30 mg/kg /Day Test Material)	Group 5 (75 mg/kg /Day Test Material)
0	2.26	2.05	2.23	2.33	2.24
3	2.29	2.08	2.27	2.34	2.26
6	2.31	2.12	2.29	2.39	2.27
9	2.30	2.08	2.28	2.42	2.26
12	2.32	2.10	2.29	2.42	2.26
15	2.37	2.11	2.33	2.43	2.29
18	2.40	2.10	2.36	2.47	2.32
21	2.41	2.05	2.40	2.53	2.35
24	2.45	2.08	2.42	2.56	2.38
28	2.50	2.10	2.46	2.56	2.46
% bodyweight change	+10.6	+2.4	+10.3	+9.9	+9.8

 

 

Group Mean Data

Parameter	Group 1 Control (1 % Methyl Cellulose)	Group 2 Positive Control (200 mg/kg/Day Thalidomide)	Group 3 (12 mg/kg /Day Test Material)	Group 4 (30 mg/kg /Day Test Material)	Group 5 (75 mg/kg /Day Test Material)
Total number of pregnancies (a)	24/30	10/11	15/16	15/17	11/15
Total number of pregnancies %	80.8	90.9	93.8	88.2	73.3
Total number of pregnancies (does surviving to day 28 after insemination)	24/30	8/9	14/15	14/15	11/15
Total number of pregnancies (does surviving to day 28 after insemination)%	80.0	88.9	93.3	93.3	73.3
Number of corpora lutea (b)	214	63	115	129	88
Mean 1 number of corpora lutea per doe (c)	8.92	7.88	8.21	9.21	8.00
Number of implantations (b)	175	52	98	96	78
Mean 1 number of implantations per doe (c)	7.29	6.50	7.00	6.86	7.09
% pre-implantation loss	18.2	17.5	14.8	25.6	11.4

(a) = Includes pregnant animals that died during the study.

b) Only included from pregnant animals surviving to day 28 after insemination.

(c) Mean 1 values include data from those animals with functional corpora lutea in the ovaries on day 28 after insemination.

 

Implantation Data

Parameter		Group 1 Control (1 % Methyl Cellulose)	Group 2 Positive Control (200 mg/kg/Day Thalidomide)	Group 3 (12 mg/kg /Day Test Material)	Group 4 (30 mg/kg /Day Test Material)	Group 5 (75 mg/kg /Day Test Material)
Implantations	Total number (b)	175	52	98	96	78
	Mean 1 number per doe (c)	7.29	6.50	7.00	6.86	7.09
Foetuses	Total number (b)	173	32	89	91	76
	Mean 1 number per doe (c)	7.21	4.00	6.36	6.50	6.91
	% of implantations	98.9	61.5	90.8	94.8	97.4
Early intra-uterine deaths	Total number (b)	1	12*	8	1	0
	Mean 1 number per doe (c)	0.04	1.50	0.58	0.07	0.00
	% of implantations	0.6	23.1	8.2	1.0	0.0
Later intra-uterine deaths	Total number (b)	1	8*	1	4	2
	Mean 1 number per doe (c)	0.04	1.00	0.07	0.29	0.18
	% of implantations	0.6	15.4	1.0	4.2	2.6
Total intra-uterine deaths (post-implantation loss)	Total number (b)	2	20*	9*(1)	5	2
	Mean 1 number per doe (c)	0.08	2.50	0.64	0.36	0.18
	% of implantations	1.1	38.5	9.2	5.2	2.6

*:Significantly different from the control group (p<0.01 – Fisher’s test).

*(1): significantly different from the control group (p.0.05 - Fisher’s test).

(b): Only included from pregnant animals surviving to day 28 after insemination.

(c): Mean 1 values included data from those animals with functional corpora lutea in the ovaries on day 28 after insemination.

 

Foetal Data

Parameter	Group 1 Control (1 % Methyl Cellulose)	Group 2 Positive Control (200 mg/kg/Day Thalidomide)	Group 3 (12 mg/kg /Day Test Material)	Group 4 (30 mg/kg /Day Test Material)	Group 5 (75 mg/kg /Day Test Material)
Number of foetuses(b)	173	32	89	91	76
Mean 1 number of does (c)	7.21	4.00	6.36	6.50	6.91
Mean 2 litter weight (g)(e)	243.7	169.8	220.7	208.1	226.5
Mean 2 foetal weight (g)(e)	34.2	33.6	34.2	33.8	33.2
Mean 2 crown/rump length (mm)(e)	94.6	93.9	95.3	94.4	94.6
Number of males	80	14	42	41	30
Number of females	93	18	47	50	46
Ratio male: female	1:1.16	1:1.29	1:1.12	1:1.22	1:1.53

(e): Mean 2 values include data only from those animals with live foetuses in utero on day 28 after insemination.

(b): Only included from pregnant animals surviving to day 28 after insemination.

(c): Mean 1 values include data from those animals with functional corpora lutea in the ovaries on day 28 after insemination.

Conclusions:

Under the conditions of this study, there was no evidence of teratogenicity following treatment with the test material at the dose levels given.

Executive summary:

The teratogenicity of the test material to the rabbit was assessed. 

Groups of sexually mature artificially inseminated Dutch Belted rabbits were dosed with the test materialby oral gavage at dose levels of 12, 30 and 75 mg/kg daily from day 6 to day 18 after insemination. A group of animals receiving 10 mL/kg 1 % methyl cellulose over the same period acted as the vehicle control group. In addition, a group of similar rabbits was dosed with 200 mg/kg thalidomide daily over the same period to serve as the positive control. (The vehicle control group and the positive control group were common to a similarly designed concurrent teratogenicity study, report number 1737R-277/5. 

In group 2 (positive control), the findings were consistent with those expected following treatment with thalidomide. These were: low overall maternal body weight gain, low levels of maternal food intake, increased pre- and post-implantation loss, low mean numbers of foetuses per doe, slightly reduced mean foetal weight and crown/rump length and a statistically significantly increased incidence of major foetal defects in comparison with the group 1 controls.

There were no clinical changes or deaths in the does which could be related to test material treatment.

Pregnancy incidence was unaffected by treatment with the test material.

Maternal body weight gain in the test material treatment groups was similar to the group 1 controls.

Administration of the test material had no effect on pre- and post-implantation loss.

The mean number of foetuses per doe was lower in the test material treated groups than in group 1 (controls), although the differences were only slight.

In the test material treated groups, mean litter weight was lower than the group 1 controls, which may be a reflection of the lower number of foetuses per doe.

There was no effect of treatment with test material on either mean foetal weight or mean foetal crown/rump length.

There was no increase in the incidence of foetuses showing major or minor external and visceral or skeletal defects in the test material treated groups in comparison with the group 1 controls.

Under the conditions of this study, there was no evidence of teratogenicity following treatment with the test material at the dose levels given.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Pre-Natal Developmental Toxicity/ Teratogenicity Range Finding Study (Rabbit): Tucker & Irvine (1978).

The teratogenicity of the test material to the rabbit was assessed in a range-finding study. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Dose levels of 100 mg/kg/day and 25 mg/kg/day of test material were administered by gavage to groups of five Dutch Belted rabbits from day 1 to day 27 after insemination. 

Signs of toxicity were limited to the 100 mg/kg/day group. Two animals treated at this level had a reduced food intake from day 12 after insemination. One animal (no. 6) was killed on day 16 and the other (no. 8) died on day 23; no abnormalities were seen at necropsy. Weight losses were observed in the surviving animals treated at this dose level. Only one animal in the 100 mg/kg/day group produced a litter, and this was morphologically normal. The litters of the 25 mg/kg/day group animals were normal by comparison with cumulative data on this strain of rabbit, but were smaller in both crown/rump length and weight than the control animals.

Under the conditions of this study, signs of toxicity were limited to the dose level of 100 mg/kg/day. At this dose the test material appeared to be toxic to the female rabbits but not to the surviving foetuses.

 

Pre-Natal Developmental Toxicity/ Teratogenicity (Rabbit): Irvine (1980a).

The teratogenicity of the test material to the rabbit was assessed. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of sexually mature artificially inseminated Dutch Belted rabbits were dosed with the test materialby oral gavage at dose levels of 12, 30 and 75 mg/kg daily from day 6 to day 18 after insemination. A group of animals receiving 10 mL/kg 1 % methyl cellulose over the same period acted as the vehicle control group. In addition, a group of similar rabbits was dosed with 200 mg/kg thalidomide daily over the same period to serve as the positive control. (The vehicle control group and the positive control group were common to a similarly designed concurrent teratogenicity study, report number 1737R-277/5. 

In group 2 (positive control), the findings were consistent with those expected following treatment with thalidomide. These were: low overall maternal body weight gain, low levels of maternal food intake, increased pre- and post-implantation loss, low mean numbers of foetuses per doe, slightly reduced mean foetal weight and crown/rump length and a statistically significantly increased incidence of major foetal defects in comparison with the group 1 controls.

There were no clinical changes or deaths in the does which could be related to test material treatment.

Pregnancy incidence was unaffected by treatment with the test material.

Maternal body weight gain in the test material treatment groups was similar to the group 1 controls.

Administration of the test material had no effect on pre- and post-implantation loss.

The mean number of foetuses per doe was lower in the test material treated groups than in group 1 (controls), although the differences were only slight.

In the test material treated groups, mean litter weight was lower than the group 1 controls, which may be a reflection of the lower number of foetuses per doe.

There was no effect of treatment with test material on either mean foetal weight or mean foetal crown/rump length.

There was no increase in the incidence of foetuses showing major or minor external and visceral or skeletal defects in the test material treated groups in comparison with the group 1 controls.

Under the conditions of this study, there was no evidence of teratogenicity following treatment with the test material at the dose levels given.

 

Pre-Natal Developmental Toxicity/ Teratogenicity (Rat): Irvine (1980b).

The teratogenicity of the test material to the rat was assessed. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of sexually mature nulliparous Sprague Dawley-derived rats were dosed with the test material by oral gavage at dose levels of 20, 50 and 125 mg/kg daily (in a volume of 10 mL/kg) from day 6 to day 15 after mating. A group of animals receiving 1 % methyl cellulose (10 mL/kg) over the same period acted as the control group. In addition, a group of similar animals was dosed with aspirin (250 mg/kg/day) over the same period to serve as the positive control. (The vehicle control group and positive control group were common to a similarly designed, concurrent teratogenicity study), (report number 1996-277/7b).

There was no indication of treatment-related clinical changes in the dams.

Pregnancy incidence was unaffected by treatment with the test material. 

There was a slight retardation in the body weight gain of the group 5 dams (125 mg/kg/day test material) and a low overall body weight gain was observed. The group 4 dams (50 mg/kg/day test material) were also slightly affected, but the body weight gain of those in group 3 (20 mg/kg/day test material) was similar to that of the group 1 controls.

During the dosing period, all mecoprop treated groups showed some reduction in food intake; the effect was most pronounced in group 5 (125 mg/kg/day test material).

Pre-implantation loss and the mean number of live foetuses per litter were unaffected by treatment with the test material.

Post-implantation loss was significantly higher in group 5 (125 mg/kg/day test material) (p<0.05: Fisher's test) than in group 1 (control), but was similar to the group 1 controls in the other test material treated groups.

In comparison with group 1 (control), mean foetal weight was slightly lower in group 5 (125 mg/kg/day test material).

Mean crown/rump length was similar to the group 1 controls in groups 3 (20 mg/kg/day test material) and 4 (50mg/kg/day test material) but significantly shorter in group 5 (125mg/kg/day mecoprop) (p<0.05: Wilcoxon's test).

The percentage of foetuses showing minor external and visceral defects in the test material treated groups was slightly, but not significantly, higher than in group 1 (control) (Fisher's test - 95 % level of confidence). The incidences were not dose-related.

The percentage of foetuses showing minor skeletal defects and variants was similar to, or lower than, the group 1 controls in groups 3 (20 mg/kg/day test material) and 4 (50 mg/kg/day test material). The incidence was higher in group, 5 (125 mg/kg/day test material) due largely to a higher proportion of foetuses showing retarded ossification. The incidence of major defects was unaffected by treatment with mecoprop.

In group 2 (positive control), findings were consistent with those expected following treatment with aspirin. These were increased post-implantation loss, reduced mean foetal weight and crown/rump length and an increased incidence of major foetal defects, in comparison with group 1 (controls).

Under the conditions of this study, the test material showed no evidence of teratogenicity at the dose levels studied.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity.