Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-05-30, 1985-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
No assessment of the oestrus cycle and sperm parameters was performed. Upon pathology examinations no recordings of the number of implantation sites in the uterus, pituitary weight, thyroid weight, uterus weight, prostate weight and seminal vesicle weight
GLP compliance:
yes
Remarks:
statement of compliance missing
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: organic
- Physical state: solid
- Other: insoluble in water
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Sprague Dawley- derived [Tif:RAIf(SPF)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Limited, WST
- Age at study initiation: (P) 4 wks
- Weight at study initiation: (P) males and females: appr. 100 g
- Housing: prior to the initiation of the mating period the parent males and females (F0, F1) were kept 4 per cage in Macrolon cages equipped with a wire mesh top and a water bottle, saw dust (granulated form) serving as bedding material, thereafter one per cage
- Diet: ad libitum (Nafag No. 890)
- Water: ad libitum- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
admixed with pelleted diet

DIET PREPARATION
- Rate of preparation of diet (frequency): monthly
- Mixing appropriate amounts with: Nafag 890
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight- Proof of pregnancy: vaginal plug and / or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: transferred to individual cages and the day of delivery was determined.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test sample was extracted with ethyl acetate and the test substance was determined by HPLC using an external standard. The powdered sample was weighed (5.00 g)in a 50 mL flask and filled up with ethyl acetate to the mark, well shaken, and placed for 30 minutes in an ultra sonic bath.peak measurement: Perkin-Elmer Sigma 10 for peak area or manual measurement of peak heightscolumn: steel 316 Ti, length 220 mm, internal diameter 4 mm, packed with Nucleosil C18-10μcolumn temperature: room temperaturemobile phase: A, ethyl acetate; B, water:methanol (74:24)gradient: 10 % A -> 90 % A linear 20 minutes, purge 2 minutes (99 % A) equilibration time : 7 minutesflow rate: 2.0 mL/minretention time (typical value): test material = 16.55 minutes
Duration of treatment / exposure:
The parent animals (F0 and F1) received the test material during about 125 days, from one month of age (F0) onwards, or direct exposure commenced after weaning (F1). These periods of exposure to the test material included each one mating period of 12 days, starting 70 days after the initiation of dosing, i.e. at the animals' age of about 3 months. The F1 generation was additionally exposed to the test substance in utero and during lactation until weaning. Likewise, the F2 generation was exposed to the test substance from embryogenesis through weaning.
Frequency of treatment:
continuos
Details on study schedule:
- F1 parental animals not mated until 8 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 23 days of age.
- Age at mating of the mated animals in the study: 11 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
1 600 ppm
Dose / conc.:
4 000 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
28 (P) and 24 selected for breeding (F1)
Control animals:
yes, concurrent no treatment
Positive control:
none

Examinations

Parental animals: Observations and examinations:
During the periods of treatment general bodily condition, weight gain, food consumption (not determined during the mating periods), and symptoms were checked. Body weight and food consumption were determined twice a week (on Mondays and Thursdays) . During the periods of pregnancy and lactation these parameters were recorded for the females on days 6, 11, 16 and 21 post coitum and after birth of the young; body weight was also noted on "day 0 p.c." and "day 1 p.p." (=one day after birth of the young), respectively.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
The pups were weighed individually on days 4, 7, 14, 21, 28 and 35 post partum ( = "days p.p."). Developmental and behavioural abnormalities as well as spontaneous deaths were recorded. The main developmental parameters checked were as follows: pinna unfolding, incisor eruption, onset of hair growth, eye opening, descent of testicles or, respectively, opening of vagina.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were weaned.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
All the males and females from each test group selected to breed the following generation were autopsied soon after weaning of the F1 rats or F2 rats, respectively; they were submitted to a complete macroscopic pathological examination.
The following organs were weighed: brain, spleen, heart, thymus, liver, testicles / ovaries, kidneys, adrenal glands

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination: gross lesions; skin; mammary area; spleen; mesenteric lymph node; axillary lymph node; popliteal lymph node; sternum with bone marrow; femur with joint; skeletal muscle; trachea; lung; aorta; submandibular salivary gland; pancreas; oesophagus; stomach; small intestine; large intestine; urinary bladder; prostate; seminal vesicle; testicle; epididymis; uterus; vagina; ovary; pituitary gland; thyroid with parathyroid gland; peripheral nerve; spinal cord; eye with optic nerve; orbital gland; extraorbital lacrimal gland; zymbal gland; muzzle. The tissues indicated were prepared for microscopic examination and weighed: brain; adrenal glands; heart; spleen; kidneys; thymus; liver.

All the females which died perinatally were carefully necropsied and, as far as possible, the contents of the uteri examined and the numbers of implantation sites determined. Those females without litters were autopsied about 5 days after the expected day of delivery. The uterine horns were also placed in ammonium sulfide to detect possible occurrence of abortion and/or resorption sites. To determine the numbers of implantation sites the uterine horns of all females were placed in the aqueous solution of ammonium sulfide.
Postmortem examinations (offspring):
SACRIFICE
One randomly selected weanling of each sex from each litter and group was autopsied on day 23 p.p. following bleeding under ether anaesthesia. For the control and high-dose animals the tissues were processed and examined, for the low and intermediate dose animals the tissues were retained for possible further reference. The following organs were weighed: brain, adrenal glands, heart, spleen, kidneys, thymus, liver.
The weanlings not selected for autopsy or breeding the following generation were killed by COz-asphyxiation, submitted to macroscopic pathological examination and discarded from day 35 post partum, i.e. the last registration of body weight.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination: skin; spleen; trachea; lung; aorta; pancreas; oesophagus; stomach; small intestine; large intestine; urinary bladder; pituitary gland; thyroid with parathyroid gland; eye with optic nerve.The tissues indicated were prepared for microscopic examination and weighed: brain; adrenal glands; heart; spleen; kidneys; thymus; liver.
Statistics:
Mean values were calculated for body weight of parent animals and pups, food consumption, daily intake of test material, number of implantation sites, initial litter sizes and organ-weight ratios; standard deviation was determined, in addition, for numbers of implantation sites and litter sizes. For organ-weight ratios location and/or dispersion between the control group and the experimental groups was calculated. Whenever feasible, further statistical evaluation of data was performed. The main tests applied were:
- Chi2 test, incl. correction of Yates, for comparing e.g. death ratios of pups and male-to-female sex ratios of progeny.
- Student's t-test, one-tailed or two-tailed, for comparing implantation rates, litter sizes as well as random samples of body weights or food intake.
- Fisher's exact test to compare mating and/or fertility rates.
- Jonckheere test for comparing organ-weight ratios.
Reproductive indices:
The fertility index of the parent rats was determined on the basis of the numbers of males used for mating and the numbers of females bearing implantation sites.
Offspring viability indices:
Litter size, sex ratios, and litter mortality were determined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of intolerability of the test substance were noted throughout the period of exposure.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Female no. 50 (P control group) died on the third day of the mating period by an accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In the three experimental groups of the male animals, particularly the group exposed to 1600 ppm, the bodyweight gain was found to be superior to that of the control group.
In contrast to the males, the females showed a reduction in bodyweight gain during week 6 at 10000 ppm. Parallel to this finding, food intake was also depressed to some extent during several weeks. In both the other experimental groups, body-weight gain and food consumption showed a tendency of decrease in comparison with the control. In consideration of the differences of average initial body weights on "day 0" of pregnancy, the body-weight gain during pregnancy was reduced in the experimental groups, particularly the group exposed to 10000 ppm. During the period of lactation body weight was also lower, to some extent, in the experimental groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males the average food intake was also found to be increased at some extent through several weeks in the experimental groups. The marked increase in food consumption of the parent females (P, F1) during the lactation periods resulted in an also marked increase in the daily uptake of the test material.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All microscopical changes observed in some control and test animals were incidental in nature and unrelated to the treatment with the test article.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Most of the females were mated during the first half of the mating period, i.e. at least in the course of one oestrus cycle. The mating rates were determined at 88.9 % for the control group, 89.3 % for the 1,600 ppm group, 96.4 % for the 4,000 ppm group and 82.1 % for the 10,000 ppm group. For 4 females each of the 1600 ppm and 4000 ppm groups as well as 8 females of the control group the day of copulation was undeterminable, i.e. neither spermatozoa nor a vaginal plug were found. These females were also allowed to litter and raise their offspring.
The fertility indices were found to be similar for the control group and the groups exposed to 1,600 ppm and 4,000 ppm, respectively. At 10,000 ppm the fertility index was apparently reduced; there was not found, however, a statistical difference from the control by applying Fisher's exact test (P = 0.1011).
Implantation rate was similar for the control group and the experimental groups exposed 1600 ppm and 4000 ppm, respectively. A slightly depressed implantation rate was observed at 10000 ppm; no statistically significant difference was found, however, by applying the one-tailed t test of Student (P > 0.05).

Details on results (P0)

The mean duration of pregnancy was determined at 21.1 days for the control group and the 1600 ppm group and 21.2 days for the 4000 ppm and 10000 ppm groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
4 000 ppm (nominal)
Sex:
female
Basis for effect level:
other: reduced body weight
Dose descriptor:
NOAEL
Effect level:
4 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: reduced fertility index (not statistically significant)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No spontaneous deaths were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males the average body-weight gain was comparable for all groups.
In fermales the average body-weight gain was comparable for the control group and the groups exposed to either 4,000 ppm or 10,000 ppm until the initiation of mating. At 1,600 ppm the body
weight was significantly reduced during the 8th and 9th weeks of direct exposure to the test substance. During pregnancy, body weight was significantly reduced in the 1,600 ppm-group on day 6 post coitum (FIG. 5.2). By consideration of the latter finding, the course of body weight during lactation was roughly comparable for all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake was also roughly comparable for all groups for males. Parallel to the reduction in body weight on several days, the average food consumption was also depressed to some extent in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Description (incidence and severity):
For female no. 440 (control) opacity of the right eye was recorded to occur at the age of two months.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Description (incidence and severity):
MALES
Statistically significant deviations from the control were consistent with:
- elevated brain-to-body weight ratio at 1600 ppm;
- reduced absolute liver weight as well as liver-to-body weight ratio at 1600 ppm;
- reduced kidneys-to-body weight ratio at 4000 ppm;
- elevated testicles-to-brain weight ratio at 4000 ppm;
- reduced spleen-to-brain weight ratio at 10000 ppm.

FEMALES
statistically significant deviations from the control were consistent with:
- elevated kidney-to-body weight ratio at 1600 ppm and 10000 ppm;
- elevated adrenals-to-brain weight ratio at 4000 ppm;
- reduced ovaries-to-brain weight ratio at 1600 ppm;
- reduced absolute spleen weight as well as spleen to-body weight ratio at 4,000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic pathological changes were recorded for the males autopsied after weaning of the F2 rats
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All microscopical changes observed in some control and test animals were incidental in nature and unrelated to the treatment with the test article.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The mating rates of F1 were determined to range between 83.3 % (1600 ppm) and 100 % (control, 4000 ppm); a rate of 91.7 % was calculated for the 10000 ppm-group.
For one female F1 of the 4000 ppm group and two females F1 each of the other groups the day of copulation was indeterminable. These females were also allowed to litter and raise their offspring.
The fertility indices are considered to be comparable for all groups. The average implantation rate was similar for all groups.
The mean duration of pregnancy was determined at 21.0 days for the 10,000 ppm-group and 21.1 days for the other groups.
Two females of the control group (nos. 402 and 406) as well as one female each of the 1,600 ppm group (no. 516) and the 10,000 ppm group (no. 728) lost their whole litter immediately after parturition . For a further female of the 10,000 ppm group
(no. 714) death of all pups was recorded to occur up t o day 7 p o s t partum; the dam littered one day prior to expected delivery . To maintain continuity of data through gestation and lactation these females were exluded from determination of average
body weight.

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The general development and behaviour of the young were not impaired in the experimental groups on the comparison with the control group. No abnormal nesting behaviour (nursing, suckling, creeping) was noted. Pinna unfolding, incisor eruption, onset of hair growth, descent of testicles and, respectively, opening of the vagina followed their normal course.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Postpartum mortality occurred at similar incidences in all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body-weight gain of the young rats (F1) were found to be comparable for the control group and the groups exposed to 4000 and 10000 ppm, respectively; at 1600 ppm the body-weight gain appeared to be slightly reduced.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Statistically significant deviations from the controls were consistent with:
- Reduced absolute thymus weight in males and females of the 1600 ppm group;
- Reduced absolute spleen weight as well as spleen-to brain weight ratio in the females exposed to 1600 ppm
- Spleen-to-body weight ratio was reduced in the females at 4000 ppm and 10000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F2) Neither for those young (F1, F2) selected for histopathology and killed short time after weaning nor for those ones killed after day 35 post partum, macroscopic pathological changes of the organs were recorded.
Histopathological findings:
no effects observed
Description (incidence and severity):
All microscopical changes observed in some control and test animals were incidental in nature and unrelated to the treatment with the test substance.
Description (incidence and severity):
The average initial litter sizes determined immediately after birth of the F1 young were similar for all groups. The male-to-female sex ratios were comparable for the control group and the experimental groups exposed to 1,600 ppm and 10,000 ppm, respectively. At 4,000 ppm this ratio was found to be changed in favour of the females (Chi* test incl. Yates' correction, P < 0.05).

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
The general devlopment and behaviour of the young were not impaired in the experimental groups on the comparison with the control group. No abnormal nesting behaviour (nursing, suckling, creeping) was noted. Pinna unfolding, incisor eruption, onset of
hair growth, descent of testicles and, respectively, opening of the vagina followed their normal course.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Concerning postpartum mortality of the young rats, similar percentages were observed in all groups until about two weeks after weaning . At necropsy the dead pups did not show macroscopic pathological changes apart from occasional postmortem
alterations of organs.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 10,000 ppm the body-weight gain of the young rats (F2) was slightly reduced. In the view of variations among the litters of each group, the mean body-weight gain was not considered to be altered.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
statistically significant deviations from the control were consistent with:
- elevated brain-to-body weight ratio at 10000 ppm (males);
- elevated heart-to-body weight ratio in males exposed to 10000 ppm at a "positive" trend from the control to this high concentration;
- heart-to-body-weight ratio also elevated in the females of the 4000 ppm group;
- reduced absolute heart weight in the females exposed to 10000 ppm;
- reduced absolute liver weight as well as liver-to brain weight ratio in the males exposed to 10000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither for those young (F1, F2) selected for histopathology and killed short time after weaning nor for those ones killed after day 35 post partum, macroscopic pathological changes of the organs were recorded.
Histopathological findings:
no effects observed
Description (incidence and severity):
All microscopical changes observed in some control and test animals were incidental in nature and unrelated to the treatment with the test substance.
Other effects:
no effects observed
Description (incidence and severity):
The average initial litter sizes were comparable for all groups. The male-to-female sex ratios for the sucklings of each group were similar and not different from the controls.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Approximate daily intake of test material by F0 and F1 adults:

F0 parent animals

 Group (ppm)  Males  Females
 1600  120.1 ( 86.8 - 213.5)  165.7 ( 88.6 - 413.6)
 4000  292.6 (192.0 - 509.2)  411.0 (226.9 - 1030.4)
 10000  743.9 (517.2 - 1278.4)  1030.0 (573.9 - 2473.7)

F1 parent animals

 Group (ppm)  Males  Females
 1600  112.1 ( 88.3 - 155.3)  165.4 ( 85.3 - 388.8)
 4000  279.0 (211.8 - 391.9)  412.0 (207.8 - 934.6)
 10000  702.6 (535.6 - 988.7)  1034.9 (506.1 - 2333.3)

Applicant's summary and conclusion

Conclusions:
The foregoing results on the test article were considered to indicate some slight toxicity in the F0 parent females exposed to 10000 ppm as primarily expressed by a transient reduction in body weight. Although the fertility rate of the F0 adults of this group was not significantly altered from the statistical point of view, this finding was also suggested to reflect some slight toxicity; the reduced "fertility index" was consistent with an increased number of mated but non-pregnant females.