1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Mar 1983 to 31 May 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

oestrus cycle and sperm parameters were not determined, data on sexual maturation were not evaluated, food consumption was only determined during pre-mating period, only brain, ovary and testes weights were determined.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: F0: 35 days
- Weight at study initiation: (P) Males: 104.8 g; Females: 108.4 g; (F1) Males: 36.8 – 48.1 g; Females: 33.7 – 43.1 g
- Housing: Individual, hanging, stainless steel, wire-bottom cages were employed during the quarantine period and all phases of the study, except during the mating, lactation, and gestation periods. Group housing in similarly designed galvanized steel caging was employed during the mating trials. These cages, equipped with solid-bottom stainless steel floorplates and nesting material were used during gestation and lactation. The floorplates and nesting material were supplied to gestating females on approximately the fifteenth day of gestation and were removed from the cages of lactating females when the progeny were approximately 7 days of age.
- Diet: rodent chow, ad libitum
- Water: ad libitum
- Acclimation period: Quarantine: 411 potential F0 parental animals were held in quarantine for 13 days following their arrival at this facility. During this time, the veterinary sciences staff examined the animals for health status. Initially, the animals were group-housed for their adaptation to the feed and the automated watering system. The animals were then housed individually for the remainder of the quarantine period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 25.5
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Mar 1983 to 31 May 1984

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was mixed with the basal diet using Hobart to achieve concentrations (w/w) of either 0, 100, 500, or 2500 ppm (active ingredient) test substance. Fresh diets were prepared at weekly intervals. Sufficient diet was offered to each animal to assure ad libitum feeding. Fresh diets were offered at weekly intervals with one exception: final diet preparation interval were fed 10 days.

Details on mating procedure:

- M/F ratio per cage: one male and two females
- Length of cohabitation: Males were rotated among the females at 10-day intervals. Each female was paired with a maximum of 2 different males.
- Proof of pregnancy: vaginal plug or sperm in vaginal
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually and supplied with nesting material at approximately 15 days of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The percent (w/w) of test substance in test diet was determined by analytical assay at approximately month 2 of the study and at monthly intervals thereafter, with one exception. During month 4 of the study, it was noted that the test article was crystallizing and had to be heated prior to diet preparation. To avoid heating the entire batch of test article each time the diet was prepared, an aliquot was obtained from the primary container to be maintained for diet preparation. A sample of this aliquot was analyzed to assure its integrity. Homogeneity and stability of prepared test diets were initiated prior to the start of the study. Analyses of the test article concentrations in prepared diets were conducted at monthly intervals. In addition, samples of the test article and the prepared test diets were sent to the Sponsor every 3 months (starting with the first month).

Duration of treatment / exposure:

12 weeks pre-mating and thereafter over two generations

Frequency of treatment:

Continuously

Details on study schedule:

See "Any other information on materials and methods incl. tables" section. Fifteen males and 30 females were selected from the population of progeny weaned from each treatment group to serve ss F1 parental animals. These selections were done randomly employing computer-generated random selection.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

Generation F0: males: 14.3 - 5.5 mg/kg bw; females: 15.6 - 6.7 mg/kg bw;
Generation F1: males: 15.8 - 6.0 mg/kg bw; females: 14.4. - 7.3 mg/kg bw;

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Generation F0: males: 73.4 - 11.5 mg/kg bw; females: 74.2 - 24.5 mg/kg bw;
Generation F1: males: 76.2 - 30.3 mg/kg bw; females: 73.1 - 39.9 mg/kg bw;

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

Generation F0: males: 297.6 - 144.7 mg/kg bw; females: 304.4 - 173.6 mg/kg bw;
Generation F1: males: 393.4 - 159.9 mg/kg bw; females: 393.4 - 200.4 mg/kg bw;

No. of animals per sex per dose:

15 males and 30 females

Control animals:

yes, concurrent vehicle

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: All animals were observed at least twice each day for mortality, morbidity, and overt signs of toxicity, with exception on two occasions the afternoon checks were inadvertently not recorded. Each female was observed daily during gestation and lactation. Any unusual observations noted during the periods of gestation and lactation were recorded.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Each animal was removed from its cage at least once each week and was thoroughly examined for the detection of physical change.

BODY WEIGHT:
- Time schedule for examinations: All F0 and F1 parental animals were weighed weekly for 12 weeks during the pre-mating period, males were then weighed at monthly intervals until their sacrifice. Those females which failed to conceive, deliver viable progeny, or retain progeny throughout the lactation period were weighed at the monthly intervals along with the males, until their termination. Final body weights were obtained for each animal at their termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: at weekly intervals for 12 weeks for F0 and F1 parental animals during the pre-mating period

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1a/F1b/F2a/F2b] offspring: The sexes and numbers of pups delivered , delivered viable, delivered stillborn, and found partially cannibalized were recorded for each litter of progeny on the day of parturition. The numbers of pups surviving to lactation days 4, 7, 14, and 21 (weaning) were also determined. Litters in excess of 8 pups were reduced to that number (employing computer-generated random selection for sacrifice) on lactation day 4. When possible, 4 males and 4 females were retained , however if less than 4 of either sex survived to day 4, all survivors of that sex were retained along with the appropriate number of pups of the other sex to obtain a total of 8 progeny.
Individual pup weights and sexes were determined on lactation days 0, 4, 7, 14, and 21 (weaning) for all surviving progeny of each litter obtained.
Each litter of progeny was examined twice daily for mortalities. Each pup was also examined thoroughly for developmental anomalies at birth and again at termination.

GROSS EXAMINATION OF DEAD PUPS:
- All pups found dead during the lactation period were subjected to a gross necropsy examination for the detection of gross malformations.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The surviving F0 parental males were sacrificed at 265 days of age having been fed their respective test/control diets for 230 days prior to sacrifice.
- Maternal animals: All surviving F0 generation females were sacrificed at 261-262 days of age having been fed their respective test/control diets for 226-227 consecutive days prior to sacrifice
- All F1 generation parental males were sacrificed at 232 to 251 days of age. The F1 generation females were 254 to 273 days of age at final sacrifice.

GROSS NECROPSY
- Gross necropsy examinations and organ weight determinations were performed on all parental animals surviving to final sacrifice. Additionally, any parental animals that were sacrificed in a moribund condition or died prior to final sacrifice were subjected to complete gross pathologic examinations.
- Gross necropsy consisted of the external body surface and all orifices, cranial cavity, external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs; and the carcass.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathologic examinations were performed upon all parental animals (sacrificed , found dead , moribund sacrifice). Slides were prepared for all tissues and organs listed below and a qualified pathologist evaluated these slides and reported all findings noted: liver, testes (with epididymides), ovaries (with corpus), uterus, prostate, vagina, seminal vesicles and all other tissues/organs appearing abnormal.
Absolute organ weights were determined for the brain (including brain stem) and ovaries or testes (with epididymides).

Postmortem examinations (offspring):

SACRIFICE
- 'a' Litter Progeny: Ten males and 10 females per treatment group were selected for gross pathologic study. These selections were done randomly employing computer-generated random selection. Additionally, any ' a' progeny that exhibited untoward developmental anomalies were subjected to pathologic studies. The remaining F1a/F2a pups were sacrificed and discarded.

- 'b' Litter Progeny:
F1b Progeny: Fifteen males and 30 females were selected from the population of progeny weaned from each treatment group to serve as F1 parental animals, selections were done randomly employing computer-generated random selection. Ten males and 10 females from each treatment group were then selected in a similar manner for pathologic study.
F2b Progeny: Ten males and 10 females from each treatment group were randomly selected for pathologic study. In addition, all 'b litter progeny which exhibited an untoward developmental anomaly were subjected to pathologic study. The remaining ' b' litter progeny were sacrificed and discarded.

GROSS NECROPSY
- Gross necropsy consisted of the external body surface and all orifices, cranial cavity, external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs; and the carcass.

HISTOPATHOLOGY / ORGAN WEIGTHS
Slides were prepared for all tissues and organs listed below and a qualified pathologist evaluated these slides and reported all findings noted: liver, testes (with epididymides), ovaries (with corpus), uterus, prostate, vagina, seminal vesicles and all other tissues/organs appearing abnormal.
Absolute organ weights were determined for the brain (including brain stem) and ovaries or testes (with epididymides).

Statistics:

Quantitative continuous variables i.e., body weights and food consumption, were analyzed by analysis of variance with significant differences described by that treatment further studied by multiple comparison (Tukey's or Scheffe's, dependent upon 'N' values).
Organ weight ratios were studied employing Kruskal-Wallis analyses. Chi-Square analysis and Fisher's Exact Test were performed where appropriate.

Reproductive indices:

Mating Index = (Number of Copulations/Number of Estrus Cycles Utilized) x 100
Fertility Index = (Number of Pregnancies/Number of Copulations) x 100
Gestation Index = (Number of Parturitions/Number of Pregnancies) x 100
Female Fertility Index = (Number of Pregnancies/Number of Females Mated) x 100
Male Fertility Index = (Number of Sires/Number of Males Mated) x 100

Offspring viability indices:

Born Viable = (No. Pups Delivered Alive/Total No. Pups Delivered) x 100
Born Dead = (No. Stillbirths/Total No. Pups Delivered) x 100
Born and Cannibalized = (No. Pups Found Partially Cannibalized/Total No. Pups Delivered) x 100
4 Day = (No. Pups Viable at Lactation Day 4/No. Pups Born Alive) x 100
7 Day = (No. Pups Viable at Lactation Day 7/No. Pups Retained at Lactation Day 4) x 100
14 Day = (No. Pups Viable at Lactation Day 14/No. Pups Retained at Lactation Day 4) x 100
21 Day = (No. Pups Viable at Lactation day 21/No. Pups Retained at Lactation Day 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three F0 parental females died prior to final sacrifice. The first female (100 ppm) was found dead during the pre-mating period at 92 days of age having been fed test diets for 57 days. The second female ( 500 ppm) was found dead during the rest period between the F1a and F1b litters at 175 days of age having been fed test diets for 140 days. The third female (2500 ppm), was found dead after delivering 14 viable and 2 stillborn F1b progeny; this female was 222 days of age and was fed respective test diet for 187 days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the the F0 generation, body weight data for males fed diets containing 500 and 2500 ppm were slightly less than the control males. However the extent of these reductions failed to indicate a direct relationship with the level of exposure to the test article and therefore appear to be the result of strain variance.
F0 parental females fed diets containing 2500 ppm exhibited reduced body weights when compared to the control females with statistically significant decreases noted at 13 of the 18 intervals. The pre-mating and total weight gains calculated for these females were also reduced.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males fed diets containing 2500 ppm test substance exhibited reduced food intake at weeks 1 and 7 of the F0 premating period. Food consumption values obtained for the 2500 ppm F0 parental females were reduced in comparison to the control females at 9 of the 13 food consumption intervals.

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

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The other treatment-related hepatic lesion was "clear-cell change" and was seen frequently in only the F0 males. The lesion consisted of both individual and small clusters of hepatocytes which had marked cytoplasmic vacuolation. These cells were scattered throughout the liver sections. Very little eosinophilic material remained in the cytoplasm of affected cells. The vacuolation sometimes consisted of numerous discrete cytoplasmic vacuoles, but in other cells nearly all the entire cytoplasm was clear without the presence of vacuoles. There was usually no nuclear displacement. The location of the affected cells varied within the hepatic lobules, and the overall severity of the change ranged from minimal to moderate within the sections examined.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of the F0 generation animals revealed a single benign neoplasm. It was found in the ear of a T-II female rat and was diagnosed as a fibroma. Although it occurred in a treated animal, such neoplasms can occur spontaneously in the ears or subcutaneous tissue of normal rats, and this tumor is not considered to be treatment-related.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Vaginal discharges were noted either during gestation or lactation of the F0 generation for 3 control (0 ppm), 1 T-I (100 ppm), 1 T-II (500 ppm ), and 5 T-III ( 2500 ppm ) females. One control and 1 T-I female resorbed their F1b litters and 2 T-III females resorbed their F1a litters. In addition, during the F1b litter T-III one female was found dead on lactation day 1 after delivering 14 viable and 2 stillborn pups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

100 ppm

Based on:

test mat.

Remarks:

approx. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No consistent treatment-related ante mortem observations were noted. One male control animal was sacrificed in a moribund condition during the F1b matings.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the F1 generation one male (500 ppm) was sacrificed during the F2b mating trials (205 days of age) in a moribund condition which apparently resulted from a cage injury. Two F1 generation females (2500 ppm) did not survive to final sacrifice. The first was found dead during parturition of its F2a litter (142 days of age) and the second was sacrificed in a moribund condition during parturition of its F2a litter (142 days of age).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the F1 generation, the 2500 ppm males exhibited reduced body weights when compared to the control males. This decrease began at the initiation of the F1 generation (week 0) and continued through the final sacrifice. All other body weight data obtained for the treated groups F1 parental males were comparable to the concurrent control males. F1 parental females fed diets containing 2500 ppm test substance exhibited reduced body weights when compared to the control females. Statistical evaluations of these data revealed significant reductions for 14 of the 19 intervals. In addition, the pre-mating and total weight changes for these females were reduced when compared to the control females. Significant body weight reductions were noted for the F1 generation females fed diets containing 500 ppm at weeks 10 and 11 and a significant reduction in the pre-mating weight gain was noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The F1 generation males fed diets containing 2500 ppm test substance exhibited reduced food intake at weeks 2, 6 and 10 of the pre-mating period. Females fed diets containing 2500 ppm test substance exhibited reduced food intake throughout the F1 pre-mating period. Food consumption data obtained for the 100 and 500 ppm males and females were considered to be comparable to the control animals. Statistical differences which were noted for these groups were sporadic in occurrence and therefore considered the result of strain variance.

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

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Three F1 generation animals died or were sacrificed in a moribund condition prior to final sacrifice. One male (500 ppm) was sacrificed in a moribund condition at 205 days of age and exhibited dried crusted blood in the nose and a maxilla that was fractured approximately 1 cm posterior to the incisors. One female (2500 ppm ) was found dead at 142 days of age and necropsy findings were limited to uterine counts. Necropsy findings for another 2500 ppm female that was sacrificed in a moribund condition were severe paleness of all body tissues, pale liver, multiple red areas in the lung, cannibalized pup remains in the stomach, 4 late resorptions in the left uterine horn, one pup involved in a torsion of the right uterine horn which obstructed the vagina, and a 180° left to right torsion of the vagina.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were found in the liver in F1 male and female rats. The most frequent change seen was "cellular swelling". This occurred in both periportal and centrilobular areas and consisted of a mild increase in the size of hepatocytes with an apparent increase in cytoplasmic volume and compression of adjacent hepatic sinusoids. The affected cells did not have the irregular vacuolation typical of glycogen which is frequently seen in normal hepatocytes from animals which had not been fasted. There sometimes was nuclear enlargement also present in these cells. The incidence and severity of cellular swelling increased with dose in both the F1 males and females. The incidence was over 95% in both sexes in the T-III group and the severity mild to moderate in the males and minimal to moderate in the females. The incidence of cellular swelling was also significant in the T-II group in both the males and females at the .05 level using Fisher 's Exact Test, This change was also evident in the liver of the T-III F2b weanlings, where it was present in 10/10 males and 9/ 10 females.
The other treatment-related hepatic lesion was "clear-cell change" and was seen frequently in the F1 males and females. The lesion consisted of both individual and small clusters of hepatocytes which had marked cytoplasmic vacuolation. These cells were scattered throughout the liver sections. Very little eosinophilic material remained in the cytoplasm of affected cells. The vacuolation sometimes consisted of numerous discrete cytoplasmic vacuoles, but in other cells nearly all the entire cytoplasm was clear without the presence of vacuoles. There was usually no nuclear displacement. The location of the affected cells varied within the hepatic lobules, and the overall severity of the change ranged from minimal to moderate within the sections examined. Three F1 animals died or were non-scheduled sacrifices prior to the scheduled sacrifice. One male fractured his maxilla and was sacrificed. One moribund female was sacrificed and found to have a uterine torsion. One female was found dead but the cause of death could not definitely be determined.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Four benign and one malignant neoplasms were found in the F1 generation animals. All these neoplasms occurred in T-I F1 generation rats. A fibroma occurred in one T-I male rat. The other neoplasms were each found in a different T-I female. The deciduoma found in the uterus of one animal consisted of a proliferation and persistence of decidual cells in the decidua basalis of the endometrium at an old implantation site. Such lesions, as well as the other neoplasms observed, do occur spontaneously in rats of this age range and are not considered treatment-related.

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

During the F1 generation, vaginal discharges were noted either during gestation or lactation of the F1 generation for 3 control (0 ppm ), 2 T-I (100 ppm), 2 T-II (500 ppm) and 3 T-III (2500 ppm) females. One female from each of the control, T-I and T-II groups resorbed their F2a litters and 1 control, 2 T-I and 2 T-II females resorbed their F2b litters. On lactation days 0 and 1 of the F2b litter, T-III female exhibited pale eyes, ears, and appendages, was listless ( day 0), and had dried brown substance on fur (perianal). This female delivered a single pup that was cannibalized and still exhibited a tan vaginal discharge on lactation day 3 (3 days post parturition). In addition, during the F2a litter one T-III female was found dead after delivering 6 viable pups and another T-III female was sacrificed in a moribund condition on lactation day 1 after delivering 4 viable, 2 stillborn and 1 cannibalized pup.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the F1a litter, progeny obtained from dams exposed to 2500 ppm exhibited reduced body weights in comparison to the control progeny on lactation days 4, 7, 14, and 21. Statistical evaluations, utilizing the individual progeny body weight data, revealed these reductions to be statistically significant when compared to the control progeny; statistical evaluations utilizing the mean litter weight data, revealed significant reductions in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F1b progeny were reduced in comparison to the control progeny on lactation days 14 and 21. Statistically significant body weight reductions were noted for the 2500 ppm F1b progeny at lactation day 14 (analyses utilizing individual body weight data) and day 21 (analyses utilizing individual and mean litter weight data).

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:

no effects observed

Description (incidence and severity):

One 2500 ppm F1a anomalous pup (a partially opened left eyelid and a left eye which was smaller than normal), two 2500 ppm F1b anomalous pups (eyes which appeared smaller than normal; a left eye which was enlarged with an opacity) and one 2500 ppm F1b pup with an unopened eyelid (the eye was, however not missing, and apparently of normal size) were obtained from dams treated with the test substance.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared to the control males, the 2500 ppm F1a males exhibited significant reductions in mean body weight, brain weight, and testes with epididymides weight and a significant increase in their brain to body weight ratio. All other body weight and organ weight data obtained for F1a progeny were comparable to the control progeny. Body weight and organ weight data obtained for sacrificed F1b progeny were comparable to the control progeny.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Three F1a and 2 F1b anomalous progeny were obtained from the test substance treated dams. During the F1a litter, female's (100 ppm) pup was anurous and displayed clubbed limbs; another female's (100 ppm) pup had a cleft lip; and a female's (2500 ppm) pup displayed a partially opened left eyelid and a left eye which was smaller than normal was confirmed by necropsy examination.
During the F1b litter, a female's (2500 ppm) pup displayed a domed forehead and eyes which appeared smaller than normal, and another female's (2500 ppm) pup exhibited a left eye which appeared larger than normal and covered with a cloudy film. Gross pathologic examination of another pup revealed the brain was dilated, however no untoward findings were noted for the eyes. The necropsy examination of another pup of the same female revealed that the left eye was enlarged with an opacity. In addition, pup from a different 2500 ppm female exhibited an unopened eyelid , however gross pathologic examination revealed the eye was present and apparently normal in size.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Swelling of hepatocytes was found in the liver of the F1b weanlings. The incidence of this lesion was statistically significant in 2500 ppm males and females

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

During the F2a litter, the number of pups delivered, delivered viable and surviving to lactation day 4 were statistically reduced for the 2500 ppm group of dams exposed to the test substance when compared to the control dams. Statistically significant reductions were also noted for this group of dams during the F2b litter for progeny surviving to lactation days 7, 14 and 21. No other statistically significant differences were noted for delivery and population data during the F2a and F2b litters.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the F2a litter, progeny obtained from dams exposed to 2,500 ppm exhibited statistically reduced body weights in comparison to the control progeny on lactation days 4, 7, 14 and 21 utilizing the individual progeny body weight data. Statistical evaluations utilizing the mean litter weight data revealed significant reductions for this group in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F2b progeny were statistically reduced in comparison to the control progeny on lactation days 0 through 21 (utilizing individual body weight data) and on lactation days 4 through 21 (utilizing mean litter weight data).
The 500 ppm group' s F2a progeny revealed a statistically significant increase (utilizing individual progeny body weight data) on lactation day 0 and statistically significant reductions for the F2b progeny on lactation days 0, 14 and 21.
In general, statistical analyses of the 100 and 500 ppm progeny body weight data revealed sporadically occurring reductions that were either singular in occurrence or within the range that has been seen at this facility among control progeny. Additionally, although birth weights for the 100 ppm F2b progeny were reduced there was an increase in the mean numbers of pups per litter delivered by 100 ppm dams as compared to the control dams.

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:

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

When compared to the control males, the 2500 ppm F2a males exhibited significant reductions in mean final body weight, the testes with epididymides weight, and the testes with epididymides to brain weight ratio and a significant increase in their brain to body weight ratio.
A significant increase in the brain to body weight ratio was noted for the 2500 ppm F2b males and a significant reduction in brain weight was noted for the 2500 ppm F2b females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two F2a and 2 F2b anomalous progeny were obtained during the F1 generation. During the F2a litter, one control female's (0 ppm) pup displayed a raised area of skin on the abdomen and another female (500 ppm) delivered a stillborn pup which exhibited agnathia and possible exencephaly (dam cannibalized top of head). During the F2b litter, one female's (100 ppm) pup displayed clubbed limbs, shortened torso and a shortened tail, and another female's (500 ppm) pup kept the left eyelid closed. Gross pathologic examination of another pup from the 500 ppm female revealed the left eye was shrunken in size. The remaining anomalies noted were limited to runt pups and pups with hematomas and the incidence of these findings which occurred among the treated groups were similar to the control. Statistical analyses of the progeny anomaly data revealed no significant differences.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were found in the liver in F2b male and female rats. The most frequent change seen was "cellular swelling". This occurred in both periportal and centrilobular areas and consisted of a mild increase in the size of hepatocytes with an apparent increase in cytoplasmic volume and compression of adjacent hepatic sinusoids. The affected cells did not have the irregular vacuolation typical of glycogen which is frequently seen in normal hepatocytes from animals which had not been fasted. There sometimes was nuclear enlargement also present in these cells. The incidence and severity of cellular swelling increased with dose was evident in the liver of the T-III F2b weanlings, where it was present in 10/10 males and 9/ 10 females.

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: birth weights

Target system / organ toxicity (F2)

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

2 500 ppm

System:

male reproductive system

Organ:

testes

other: epididymides

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Dietary verification of doses

The dietary analyses results were generally within +10% of the theoretical diet concentrations. Homogeneity of the prepared diets and the stability of prepared diets through 14 days (diets stored frozen or ambient) were established by analytical methods.

Table 2. Test article consumption data in a two generation reproduction study in albino rats

Generation	Sex	Group (ppm)	Test article consumption: maximum-minimum week mean values (mg/kg)
F0 F0 F0 F0 F0 F0	M F M F M F	100 100 500 500 2500 2500	14.3 - 5.5 15.6 - 6.7 73.4 - 11.5 74.2 - 24.5 297.6 - 144.7 304.4 - 173.6
F1 F1 F1 F1 F1 F1	M F M F M F	100 100 500 500 2500 2500	15.8 - 6.0 14.4. - 7.3 76.2 - 30.3 73.1 - 39.9 393.4 - 159.9 393.4 - 200.4

 

Table 3. Body weight gain (g) of parental animal

Concentrations of test substance	Males				Females
	0 ppm	100 ppm	500 ppm	2500 ppm	0 ppm	100 ppm	500 ppm	2500 ppm
Pre-maiting weight gain (weeks 1-12)
F0	369.0	350.7	339.9	339.7	172.7	173.1	168.4	137.0**
F1	434.3	440.9	439.7	396.1	225.8	220.6	206.2**	191.6**
Total weight gain
F0	509.5	500.9	479.8	481.2	252.0	240.9	254.4	193.4**
F1	593.0	615.5	612.3	550.2	313.4	306.8	293.5	254.3**

** statistically significant difference P<0.01

 

Table 4. Body weight (g) of dams over gestation and lactation periods (week 13 forward)

	F0				F1
Concentration	0 ppm	100 ppm	500 ppm	2500 ppm	0 ppm	100 ppm	500 ppm	2500 ppm
Week (approximate)
16	296.0	305.4	318.4	269.6	284.3	285.3	286.3
20	311.9	308.0	312.5	271.3**	317.7	304.9	301.7	262.3
24	351.8	354.9	365.7	286.3	306.3	329.1	323.3	275.5
28	361.0	371.4	374.1	305.0	327.3	343.3	337.1	280.1*
32	-	-	-	-	336.7	337.7	322.2	283.7**
Final body weight	360.5	350.1	363.7	301.7**	353.1	350.1	336.6	287.9**

* statistically significant difference P<0.05, ** statistically significant difference P<0.01

 

Table 5. Selected litter data

Litter	Day	Mean number of live pups					Mean pup body weight (g)
		Concentration of test substance (ppm)
		0	100	500	2500	0		100	500	2500
F1a	0	12.2	11.7	11.2	11.8	6.1		6.0	6.1	6.2
	4	11.0	11.4	11.0	11.4	8.9		8.8	9.2	8.1**
	7	6.8	7.4	7.5	7.7	14.6		13.0**	15.2	12.3**
	14	6.8	7.2	7.5	7.4	28.2		25.4**	26.9*	21.9**
	21	6.8	7.2	7.4	7.4	48.0/46.1		44.1/42.5**	47.7/44.9	35.8/34.7**
F1b	0	11.4	12.7	13.2	12.4	6.1		6.0	6.4**	6.1
	4	10.3	12.3	12.9	12.1	8.8		9.2	9.5**	8.6
	7	6.4	7.7	7.8	7.4	13.4		14.5	14.5*	13.3
	14	6.3	7.7	7.7	7.2	25.7		27.7*	28.0**	22.9**
	21	6.3	7.7	7.7	7.2	44.3/40.1		46.1/43.5	47.7/43.5	35.6/32.5**
F2a	0	12.5	13.0	10.8	9.2**	5.4		5.3	5.7*	5.4
	4	12.0	11.6	10.4	8.1**	8.5		7.9**	8.3	7.5**
	7	7.8	7.2	7.2	6.5	13.7		13.3	13.0	10.8**
	14	7.8	7.2	7.0	6.2	25.6		25.4	25.2	20.0**
	21	7.8	7.1	7.0	6.2	43.8/41.1		42.5/40.4	42.5/39.8	31.5/30.2**
F2b	0	12.8	13.4	12.7	11.3	5.8		5.4**	5.5**	5.4**
	4	12.5	13.2	12.5	10.6	8.8		8.6	8.5	7.3**
	7	7.7	8.0	7.8	6.2*	14.6		14.7	13.8	10.9**
	14	7.7	8.0	7.8	5.7**	29.4		28.7	26.8**	21.9**
	21	7.7	8.0	7.8	5.7**	48.8/46.2		48.6/46.6	45.1/42.3**	36.7/33.3**

Day 21 pup body weights are males/females ** statistically significant difference P<0.01,

* P<0.05

Data for lactation days 7 and 14 not shown

 

Table 6. Histopathological changes in the liver of parental animals and progeny

Generation	Concentration of test substance
	Males				Females
	0 ppm	100 ppm	500 ppm	2500 ppm	0 ppm	100 ppm	500 ppm	2500 ppm
Hypertrophy
F0	7/15	3/15	13/15*	14/15*	4/30	3/29	6/30	29/30*
F1	0/15	1/15	5/15*	15/15*	0/30	2/30	15/30*	29/30*
F1b	2/10	1/10	2/10	10/10*	1/10	1/10	2/10	8/10*
F2b	0/10	0/10	2/10	10/10*	0/10	0/10	1/10	9/10*
F0	0/15	2/15	3/15	14/15*	1/30	1/29	1/30	1/30
F1	2/15	5/15	8/15*	11/15*	2/30	4/30	7/30	10/30*
F1b	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
F2b	0/10	0/10	0/10	1/10	1/10	0/10	0/10	0/10

* statistically significant difference P<0.05

 

Table 7. Mean testes weights (% of control group)

Generation	Dietary concentration of the test substance
	Males
	0 ppm	100 ppm	500 ppm	2500 ppm
Mean absolute testes weight (with epididymides) (% of control group)
F0	100	101	97.1	109
F1a	100	96.4	93.5	66.2*
F1b	100	107	109	96.7
F2a	100	93.7	94.8	73.0*
F2b	100	106	98.5	84.8
Mean relative testes (with epididymides) / body weight (% of control group)
F0	100	103	102	114
F1a	100	101	99.7	100
F1b	100	108	103	112
F2a	100	94.7	96.2	98.2
F2b	100	107	101	108

* statistically significant difference P<0.05

 

Table 8. Mean brain weights (% of control group) in male rats

Generation	Dietary concentration of the test substance
	Males
	0 ppm	100 ppm	500 ppm	2500 ppm
Mean absolute brain (% of control group)
F0	100	101	101	101
F1a	100	98.0	99.4	88.9**
F1b	100	98.3	104	97.1
F2a	100	101	99.4	95.0
F2b	100	102	103	96.1
Mean relative brain / body weight (% of control group)
0	100	102	106	106
F1a	100	107	113	137*
F1b	100	103	97.1	116
F2a	100	107	101	129**
F2b	100	103	105	121**

* statistically significant difference P<0.05

** statistically significant difference P<0.01

 

Table 9. Mean brain weights (% of control group) in female rats

Generation	Dietary concentration of the test substance
	Females
	0 ppm	100 ppm	500 ppm	2500 ppm
Mean absolute brain (% of control group)
F0	100	99.1	101	101
F1a	100	102	102	97.4
F1b	100	102	103	96.3
F2a	100	102	103	95.5
F2b	100	102	101	92.4*
Mean relative brain / body weight (% of control group)
F0	100	102	101	120**
F1a	100	98.9	96.1	122
F1b	100	94.6	89.3	110
F2a	100	117	103	115
F2b	100	95.0	101	108

* statistically significant difference P<0.05

** statistically significant difference P<0.01

Applicant's summary and conclusion

Conclusions:

Swelling of hepatocytes in areas of the liver, diagnosed as cellular swelling, was found in the liver of the male and female F0, F1b, F1 and F2b rats in a dose-related manner and appears to be due to the administration of the test substance. The incidence of this lesion was statistically significant at 500 and 2500 ppm in the F0 and F1 parents and at 2500 ppm in the F1b and F2b weanlings. The incidence of clear-cell change in the liver also increased with increasing doses of the test substance in the male F0 rats and the male and female F1 rats and appears also to be related to administration of the test article.
Statistical analysis of the incidence of both hepatocellular swelling and of clear-cell change in rats receiving 100 ppm of the test substance revealed the incidence of these lesions was not significantly different from those of the controls and therefore the lesions in this dose level are not felt to be treatment-related.
Therefore, the NOAEL for parental toxicity in this two generation reproduction study in rats in compliance with GLP and following an method equivalent to OECD 416 guideline used dietary administration of the test substance was 100 ppm (app. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females). The NOAEL for reproduction and offspring toxicity was 500 ppm (48.8 mg/kg bw for males and 43.7 mg/kg bw in females).

Executive summary:

The study was conducted according to draft Guidelines of U.S:FIFRA Subdiv. F § 83-4 and it complies with OECD 416 and directive 87/302 EEC Part B guidelines. The final Guidelines were not yet released when the study was conducted. The study was performed according to the principles of GLP.

A two-generation reproduction study was conducted to ascertain the potential effects of the test substance upon the growth, reproductive performance, and offspring of 2 consecutive generations of albino rats. Ad libitum feeding of diets containing either 0 (VC), 100 (T-I), 500 (T-II), or 2500 (T-III) ppm (active ingredient) the test substance was employed as the route of administration.

The results obtained during this study are summarized as follows. Four F0 generation parental animals died or were sacrificed in a moribund condition: a 0 ppm male was sacrificed in a moribund condition during the F1b mating trials; a 100 ppm female was found dead during the pre-mating period; a 500 ppm female was found dead during the rest period between the F1a and F1b litters; and a 2500 ppm female was found dead during parturition of her F1b litter.

During the second generation, a 500 ppm male was sacrificed in a moribund condition during the F2b mating trials (apparently the result of a cage injury) and two 2500 ppm females died during parturition of their F2a litters (one was found dead and one was sacrificed in a moribund condition ). The remaining parental animals (F0 and F1 generation) survived to final sacrifice.

During the F0 generation, body weight data obtained for the treated males did not reveal any reductions which were considered to be the result of the test substance ingestion. During the F1 generation, the 2500 ppm males exhibited reduced body weight when compared to the control males. All other body weight dataobtained for the treated F1 parental males were comparable to the concurrent control males. F0 and F1 parental females fed diets containing 2500 ppm exhibited reduced body weights throughout their respective generations. In addition, the pre-mating and overall weight gains for the F0 and F1 2500 ppm females were decreased when compared to the concurrent control females. The F1 generation females fed diets containing 500 ppm exhibited significant reductions at 2 of the 12 pre-mating intervals and in the pre-mating weight gain when compared to the control females, other significant body weight reductions were noted. F0 generation males fed diets containing 2500 ppm exhibited reduced food intake at weeks 1 and 7 of the F0 pre-mating period; No F1 generation males fed diets containing 2500 ppm exhibited reduced food intake at weeks 2, 6 and 10 of the pre-mating period. All other food consumption data obtained for parental males during both generations were comparable to the control males during the pre-mating periods. Food consumption values obtained for the 2500 ppm F0 and F1 parental females were reduced in comparison to the control females. Food consumption data obtained for the 100 and 500 ppm females were considered to be comparable to the control females. No consistent antemortem observations were noted which appeared to be the result of the test substance ingestion. Reproductive indices calculated for the groups of rats fed the test substance were comparable to the concurrent control group. Vaginal discharges were noted either during gestation or lactation of the F0 generation for 3 control (0 ppm), 1 T-I ( 100 ppm), 1 T-II ( 500 ppm), and 5 T-III ( 2, 500 ppm) females. One control and 1 T-I female resorbed their F1b litters and 2 T-III females resorbed their Fla litters. In addition, during the F1b litter a T-III female was found dead on lactation day 1 after delivering 14 viable and 2 stillborn pups. During the F1 generation, vaginal discharges were noted either during gestation or lactation of the F1 generation for 3 control (0 ppm), 2 T-I ( 100 ppm), 2 T-II ( 500 ppm) and 3 T-III (2,500 ppm) females. One female from each of the control, T-I and T-II groups resorbed their F2a litters and 1 control, 2 T-I and 2 T-II females resorbed their F2b litters. On lactation days 0 and 1 of the F2b litter, a T-III female exhibited pale eyes, ears, and appendages, was listless ( day 0), had dried brown substance on fur ( perianal). This female delivered a single pup which was cannibalized and still exhibited a vaginal discharge (tan) on lactation day 3 (3 days post parturition). In addition , during the F2a litter a T-III female was found dead after delivering 6 viable pups and a T-III female was sacrificed in a moribund condition on lactation day 1 after delivering 4 viable, 2 stillborn and 1 cannibalized pups. Delivery and population data obtained for the groups of dams exposed to the test substance were comparable to the control dams during both the F1a and F1b litters. the number of pups delivered, delivered viable and surviving to During the F2a litter , lactation day 4 were statistically reduced for the 2500 ppm dams when compared to the control dams. Statistically significant reductions were also noted for this group of dams during the F2b litter for progeny surviving to lactation days 7, 14 and 21. No other significant differences were noted for delivery and population data during the F2a and F2b litters. Calculated indices of progeny survival were considered to be comparable for treated and control groups during the F1a and F1b litters. During the F2b litter, progeny survival indices at lactation days 7, 14 and 21 were statistically reduced for the 2500 ppm group dams. No other significant differences were noted for progeny survival during the F2a and F2b litters. During the F1a litter, progeny obtained from dams exposed to 2500 ppm exhibited reduced body weights in comparison to the control progeny on lactation days 4, 7, 14, and 21. Statistical evaluations, utilizing the individual progeny body weight data, revealed these reductions to be statistically significant when compared to the control progeny; statistical evaluations utilizing the mean litter weight data, revealed significant reductions in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F1b progeny were reduced in comparison to the control progeny on lactation days 14 and 21. Statistically significant body weight reductions were noted for the 2500 ppm F1b progeny at lactation day 14 (analyses utilizing individual body weight data) and day 21 ( analyses utilizing individual and mean litter weight data). During the F2a litter, progeny obtained from dams exposed to 2500 ppm exhibited statistically reduced body weights in comparison to the control progeny on lactation days 4, 7, 14 and 21 utilizing the individual progeny body weight data. Statistical evaluations utilizing the mean litter weight data, revealed significant reductions for this group in comparison to the control progeny beginning at lactation day 7 and continuing through weaning ( lactation day 21). Body weight data obtained for the 2500 ppm F2b progeny were statistically reduced in comparison to the control progeny on lactation days 0 through 21 (utilizing individual body weight data) and on lactation days 4 through 21 (utilizing mean litter weight data). Body weight data obtained for the 100 and the 500 ppm progeny did not reveal any consistent alterations which appeared to be a direct result of the test article. Examinations of progeny structural development did not reveal any aberrant findings which were considered to be related to the test substance exposure. Statistical evaluation of the organ weight data obtained for F0 generation animals revealed the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females and significant reductions in mean final body weight, brain weight, and testes with epididymides weight and a significant increase in their brain to body weight ratio for the 2500 ppm F1a male progeny. During the F1 generation, the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females. When compared to the control males, the 2500 ppm F2a males exhibited significant reductions in mean final body weight, the testes with epididymides weight, and the testes with epididymides to brain weight ratio and a significant increase in their brain to body weight ratio. Mean final body weight data obtained for the 2500 ppm F2a female progeny were somewhat less than the control females, however, no statistically significant differences were noted. The 2500 ppm F2b male and female progeny exhibited significant reductions in mean final body weights. A significant increase in the brain to body weight ratio was noted for the 2500 ppm F2b males and a significant reduction in brain weight was noted for the 2500 ppm F2b females. No other statistically significant differences were noted in organ weight data obtained for the the test substance animals when compared to their concurrent control. Gross pathologic examination of the parental animals from both generations (sacrifice, found dead, moribund sacrifice) revealed no untoward findings which appeared to be the result of the test substance ingestion. Necropsy examination conducted upon sacrificed progeny and progeny which died during the lactation period revealed no untoward findings which were considered the result of the test substance exposure. The results of the microscopic examinations conducted by the third party laboratory revealed the exposure of male and female albino rats to 100, 500 or 2500 ppm of the test substance in the diet during gestation and for two reproductive cycles did not produce any changes in the reproductive tract that appear to be treatment-related. Swelling of hepatocytes in areas of the liver, diagnosed as cellular swelling, was found in the liver of the male and female F0, F1b, F1 and F2b rats in a dose-related manner and appears to be due to the administration of the test substance. The incidence of this lesion was statistically significant at the .05 level using Fisher's Exact Test, at 500 and 2500 ppm in the F0 and F1 parents and at 2500 ppm in the F1b and F2b weanlings. The incidence of clear-cell change in the liver also increased with increasing doses of the test substance in the male F0 rats and the male and female F1 rats and appears also to be related to administration of the test article. The incidence of clear-cell change was similarly statistically significant for at least one group and one sex of rats at both the 500 and 2500 ppm levels. Statistical analysis of the incidence of both hepatocellular swelling and of clear-cell change in rats receiving 100 ppm of the test substance revealed the incidence of these lesions was not significantly different from those of the controls and therefore the lesions in this dose level are not felt to be treatment-related. No other lesions found in this study were considered treatment-related.

Therefore, the NOAEL for parental toxicity in this two generation reproduction study in rats in compliance with GLP and following an method equivalent to OECD 416 guideline used dietary administration of the test substance was 100 ppm (app. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females). The NOAEL for reproduction and offspring toxicity was 500 ppm (48.8 mg/kg bw for males and 43.7 mg/kg bw in females).