5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2000 - Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

yes

Remarks:

only 10 P and F1 animals/sex were histopathological examined

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Deviations:

yes

Remarks:

only 10 P and F1 animals/sex were histopathological examined

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-630-3

EC Name:

-

Cas Number:

181587-01-9

Molecular formula:

C13H9Cl2F3N4OS

IUPAC Name:

5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethanesulfinyl)-1H-pyrazole-3-carbonitrile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: (P) approximately 6 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 151 - 175 g; Females: 126 - 150 g; (F1) Males: 62.6 - 71.8 g; Females: 56.1 - 68.0 g
- Housing: The animals were housed individually in solid-bottom polycarbonate cages with stainless steel wire lids with Sani-Chips® cage litter during the acclimation period and upon the initiation of the treatment period. Study animals were housed two per cage (one male:one female from the same dose level) during the mating period. Females were caged separately and individually once they were successfully mated (or at the end of the mating period).
- Diet: Purina Certified Ground Rodent Diet No. 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 20 Mar 2000
To: 28 Nov 2000

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: at least monthly; cageside stability: at least one week
- Mixing appropriate amounts with: Purina Certified Ground Rodent Diet No. 5002
- Storage temperature of food: frozen (-20°C)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For analysis of the dosed feeds, stock solutions (at 0.600 and 0.500 mg/mL) were prepared by dissolving the test substance in acetonitrile. Additional standard solutions were made by dilution of the stock solutions to achieve 0.300, 0.250, 0.210, 0.175, 0.120 and 0.080 mg/mL. The internal standard solution was 4 mg/mL of dimethylphthalate in acetonitrile. Vehicle standards were prepared from blank feed and acetonitrile. Aliquots of each dosed food formulation and control feed were weighed in triplicate à 10 g. An aliquot (50 mL) of acetonitrile was added and sonicated for five minutes without heat and shaken on a platform at low setting for 30 min. The feed was allowed to settle for at least five minutes. An extract from each sample was transferred to a separate one-dram amber vial containing 0.5 mL of the internal standard. The samples were mixed and analyzed by high performance liquid chromatography (HPLC) in a single injection (Waters HPLC with Waters 717 WISP autosampler, column: Supelco Lichrosphere RP-18 (250 x 4.6 mm ID, 5 µ particle size), mobile phase: acetonitrile/water (51:49), flow rate: 0.71 mL/min, injection volume: 20 µL, detector: UV Waters Model 490 at 277 nm). All dosed feed formulations used in the study had analytical values of 93.4 to 110% of target concentrations. Vehicle control feed formulations contained no test substance, with an estimated detection limit of 0.8 ppm.

Duration of treatment / exposure:

(P0) Males: 10 weeks before mating, 2 weeks during mating, 3 weeks during delivery period
(P0) Females: 10 weeks before mating, 2 weeks during mating, 3 weeks during gestation, 3 weeks during lactation until weaning
(P1) Males: 10 weeks before mating, 2 weeks during mating, 3 weeks during delivery period
(P1) Females: 10 weeks before mating, 2 weeks during mating, 3 weeks during gestation, 3 weeks during lactation until weaning

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13 - 15 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm (nominal)

Remarks:

for actual test substance intake, please refer to "Details on results"

Doses / concentrations 2

Dose / conc.:

75 ppm (nominal)

Remarks:

for actual test substance intake, please refer to "Details on results"

Doses / concentrations 3

Dose / conc.:

500 ppm (nominal)

Remarks:

for actual test substance intake, please refer to "Details on results"

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Chosen dose levels based on the results of the range-finding study (2000, report no. C020776, company study no. M-210173-01-2).
- Rationale for animal assignment: Animals were randomized by body weight, such that the body weights of all groups were homogeneous by study initiation.

Positive control:

not included

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: initially and weekly during mating (males and females); during gestation on gestation days (GD) 0, 7, 14 and 20 and on post-natal days (PND) 0, 4, 7, 14 and 21 (females)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

Oestrous cyclicity (parental animals):

Vaginal smears were taken for all females for the last three weeks before mating to evaluate presence, normalcy and duration of estrous cyclicity.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the completion of delivery of their litters.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including cranial cavity, carcass, external and cut surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities and their viscera and cervical tissues and organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights (all parental animals): spleen, adrenal glands, brain, liver, kidneys, thyroid, pituitary (fixed), prostate, testes, seminal vesicles (with coagulating glands and their fluids), epididymides with contents and fluids (total and caudal weight for either one or both), ovaries, uterus with oviducts and cervix

Histopathology (10 high dose animals/sex and 10 control F0 and F1 animals/sex): ovaries, uterus with oviducts and cervix, vagina, prostate, testis, seminal vesicles (with coagulating glands and their fluids), epididymides with contents (2, one intact and one minus the cauda), thyroid, liver, kidneys, other tissues with gross lesions identified as being potentially treatment related, reproductive organs of the animals from all groups suspected of reduced fertility
Based on histopathologic findings in the liver and thyroid of F0 females these organs (10/sex) from the low- and mid-dose groups were also submitted for histopathologic examination. Based on histopathologic findings in the thyroid and liver of the high dose F1 males and thyroid, liver, and kidneys of the high dose F1 females, the thyroid, liver, and kidneys from 10 F1 males and females each in the low- and mid-dose groups were also submitted for histopathology.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: A gross external examination was made on the F1 offspring not selected as parents or for necropsy and the F2 offspring not selected for necropsy. Any pup appearing moribound or dying during the study period was examined for gross internal abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including cranial cavity, carcass, external and cut surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities and their viscera and cervical tissues and organs. Three F1 and F2 weanlings/sex/litter (if possible) were selected for necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights: brain, liver, spleen, kidneys, thymus

Statistics:

Treatment groups were compared to the concurrent control group using either parametric ANOVA under the standard assumptions or robust regression method which do not assume homogeneity of variance or normality. The homogeneity of variance assumption was examined via Levene's Test. If Levene's Test indicated lack of homogeneity of variance (p<0.05), robust regression methods were used to test all treatment effects. The presence of linear trends was analyzed by GLM procedures for homogenous data or by robust regression methods for nonhomogenous data. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS® 6.12 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to control via Dunnett's Test. For the litter-derived percentage data (e.g., periodic pup survival indices), the data were arcsine square root transformed prior to analysis, since percentage derived from litters tend to have unequal variances. The transformed data were then analyzed using ANOVA weighted by litter size in order to further stabilize the variances. A test for linear trend was also performed on the transformed data weighted by litter size. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group. Frequency data such as reproductive indices were not transformed. All indices were analyzed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. Acquisition of developmental landmarks as well as F2 newborn anogenital distance, were analyzed by Analysis of Covariance (ANCOVA) using body weight at acquisition or measurement as the covariate. For all statistical tests, the significance limit of 0.05 (one- or two-tailed) was used as the criterion for significance.

Reproductive indices:

Females:
Mating Index (%) = (No. of sperm-positive females / No. of females paired) * 100

Fertility Index (%) = (No. of pregnant females / No. of sperm-positive females) * 100

Gestational Index (%) = (No. of females with live litters / No. of pregnant females) * 100

Males:
Mating Index (%) = (No. of males impregnating females / No. of males paired) * 100

Fertility Index (%) = (No. of males siring litters / No. of males impregnating females) * 100

Pregnancy Index (%) = (No. of pregnant females / No. of males impregnating females) * 100

Offspring viability indices:

Live Birth Index (%): (No. of live pups at birth / total no. of pups born) * 100

4-Day Survival Index (%): (No. of pups surviving 4 days (precull) / total no. of live pups at birth) * 100

7-Day Survival Index (%): (No. of pups surviving 7 days / total no. of live pups at 4 days (postcull)) * 100

14-Day Survival Index (%): (No. of pups surviving 14 days / total no. of live pups at 7 days) * 100

21-Day Survival Index (%): (No. of pups surviving 21 days / total no. of live pups at 14 days) * 100

Lactation Index (%): (No. of pups surviving 21 days / total no. of live pups at 4 days (postcull)) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

General clinical observations in males and females at all time points were alopecia, sores, chromodacryorrhea, rust-colored fur (due to grooming of chromodacryorrhea discharge over head and neck), piloerection and indicated no treatment-related effects. During the prebreed and mating period aggressive behavior, resolution, squinting eyes, kinked tail, audible sniffing and teeth chattering in males were observed. Teeth chattering was also observed in one male at control group during the post-mating exposure period. In females, occasional findings of red ears, skin missing on the tail and blood present in vaginal smear or urine were observed during the prebreed period through weaning.
In summary, the findings were not considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: decreased body weight of P0 females; increased body weight change of P0 females

The body weight and body weight change of all males and females at control group, 10 and 75 ppm were not affected by treatment during the 10-week prebreed exposure period. The males were held after mating until the delivery of all F1 litters (SD 84-105). During this period, male body weights were equivalent across all dose groups and the body weight change was significantly decreased only for SD 91-98 in the 75 ppm group.
The body weight of the females was significantly lower at 500 ppm at week 10 and their body weight change was significantly reduced for the entire prebreed period (SD 0-70). The body weight and body weight change of the females was not affected by treatment during gestation. The maternal lactational body weights were significantly reduced at 500 ppm for PND 0, 4 and 7, with a significant decreasing trend across dose groups for PND 0, 4, 7 and 14. Lactational body weight change was significantly reduced at 500 ppm for PND 14-21 and significantly increased at 75 ppm for PND 7-14. For the entire lactation period (PND 0-21), the body weight change was significantly increased at 500 ppm and not affected at 75 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

500 ppm: In females, food consumption (g/day) during the ten-week prebreed exposure period was significantly reduced for weeks 1 through 4 (SD 0-7, 7-14, 14-21 and 21-28) and was accompanied by a significant overall trend for reduced feed consumption.
75 ppm: In females, feed consumption in g/kg bw/day was significantly increased for week 6.

The female feed consumption (g/day) during the ten-week prebreed exposure period were significantly reduced at 500 ppm for weeks 1 through 4 (SD 0-7, 7-14, 14-21 and 21-28) and were accompanied by a significant overall trend for reduced feed consumption. There were also significant differences (when the data were expressed as g/kg bw/day) at 500 ppm for weeks 1, 2 and 4. At 75 ppm, feed consumption in g/kg bw/day was significantly increased for week 6. There were no effects on feed consumption at 10 ppm and control group. During gestation, the feed consumption of females expressed as g/day or as g/kg bw/day, exhibited a significant upward trend across dose groups for GD 14-20 and was slightly but significantly higher than the control value at 500 ppm for g/kg bw/day. Overall, maternal feed consumption was considered to be similar across all groups during the gestation period. During lactation, the maternal food consumption, expressed as g/day or g/kg bw/day, was statistically equivalent across all groups and time points.
The male feed consumption (g/day) was significantly lower at 75 ppm for the 10-week prebreed period (SD 0-70), especially for week 2 (SD 7-14) and week 9 (SD 56-63). When the data were expressed as g/kg bw/day, there were significant differences between the 75 and 500 ppm treatment group for week 2 (SD 7-14) and at 75 ppm for week 3 (SD 14-21) when compared to controls. Nevertheless, feed consumption expressed as g/kg bw/day was equivalent across all groups for the overall 10-week prebreed exposure period (SD 0-70). After the mating period, the feed consumption data (g/day) of males showed no treatment-related effect. When expressed as g/kg bw/day, feed consumption on SD 91-98 was significantly increased at 500 ppm.

Food efficiency:

no effects observed

Description (incidence and severity):

The percentual food efficiency was not affected.

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):

not examined

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):

500 ppm: In the females, hepatocyte hypertrophy was observed in 8/10 livers and thyroid follicular cell hypertrophy in 5/10 thyroid glands.

There were no treatment- or dose-related histopathologic findings in any F0 male organs examined, including reproductive organs, kidneys, liver and thyroid gland. No treatment-related findings were observed in any other female group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: 500 ppm: reduced food efficiency in P0 females; increased food efficiency in P0 females during lactation (see details on results)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

All females evaluated in each group were cycling. In the control group and in the 10, 75 and 500 ppm groups, 2 (6.7%), 5 (16.7%), 0 (0%), and 1 (3.3%) females, respectively, exhibited an abnormal estrous cycle. The cycle length was 4.15 days in the control group, 4.44 days at 10 and 75 ppm and 4.56 days at 500 ppm. At 75 and 500 ppm, the mean cycle length was slightly but significantly longer than the mean control value. The mean value at 10 ppm was not statistically significantly different from the control value due to a larger variance. It is notable, however, that the cycle length at 500 ppm was equal to the control value of another study run concurrently for this sponsor (unpublished report) and values for all groups were within the performing laboratory's historical control values for this strain of rat (based on eight multigeneration studies under the present testing guidelines). Therefore, it was concluded that there were no toxicologically-relevant differences in estrous cycle length among groups.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences among groups for any andrological parameters, including percent motile sperm, percent progressively motile sperm, epididymal sperm concentration (10E5), testicular spermatid head concentration (10E5), daily sperm production (DSP) per testis, efficiency of daily sperm production, and percent abnormal sperm. At control group, 10, 75 and 500 ppm, respectively, 7.10, 4.16, 5.28, and 6.34% abnormal sperm were estimated. These mean values in all groups are higher than usual (historical control values are 2.0-3.3% in four recent studies in the performing laboratory) and are due to one control male with a high incidence of amorphous and pin head sperm, that was paired with a female who was sperm positive but not pregnant. At 10 ppm, the number of males with increased incidence of blunt hook, pin head, and head only was slightly higher overall. At 75 ppm, one male had a high incidence of pin head and head-only sperm, and was paired with a female, who was found sperm positive and delivered a live litter. At 500 ppm, one male had a high incidence of amorphous, pin head, and head-only sperm and was paired with a female who became sperm positive but did not deliver a litter and had no implant sites at necropsy.

Reproductive performance:

no effects observed

Description (incidence and severity):

In the control group and the 10, 75 and 500 ppm groups, the number of detected sperm-positive, confirmed pregnant females was 25, 27, 25 and 25, respectively. All confirmed pregnant females (29, 29, 29 and 26 at control and 10, 75, and 500 ppm, respectively) delivered live litters.

Details on results (P0)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The intake of the test substance, expressed as mg/kg bw/day across all male dose groups, averaged approximately 0.66, 4.77 and 32.33 mg/kg bw/day at 10, 75 and 500 ppm, respectively, for the entire prebreed period (SD 0-70). During the post-mating exposure of males, the intake of the test substance was approximately 0.5 (first week, days 84-91) - 0.48 (last week, days 98-105) mg/kg bw/day at 10 ppm, 3.73-3.50 mg/kg bw/day at 75 ppm and 25.38-24 mg/kg bw/day at 500 ppm. The percentual food efficiency was not affected.
For females, average intakes of the test substance were approximately 0.78, 5.82 and 37.36 mg/kg bw/day at 10, 75 and 500 ppm, respectively, for the 10-week prebreed period. The percentual food efficiency was significantly reduced at 500 ppm for the ten-week prebreed period (SD 0-70). During gestation (GD 0-20), the intake of the test substance was approximately 0.65, 5.03 and 34.12 mg/kg bw/day at 10, 75 and 500 ppm, respectively. Percent food efficiency was not affected at any time interval measured. The maternal lactational intake of the test substance was approximately 1.59, 12.18 and 79.05 mg/kg bw/day at 10, 75 and 500 ppm, respectively, confounded by pups self-feeding starting in the second week of postnatal life. During lactation, the percentual food efficiency was significantly increased at 10, 75 and 500 ppm for PND 7-14, and was significantly decreased at 500 ppm for PND 14-21. For the total lactational period (PND 0-21), the food efficiency was significantly increased at 500 ppm.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: corresponds to 4.77 and 5.82 mg/kg bw/day for males and females, respectively

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed (highest dose tested)

Remarks on result:

other: corresponds to 32.33 and 37.36 mg/kg bw/day for males and females, respectively

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In P1 males, clinical observations during the prebreed period consisted of alopecia seen in all groups; chrornodacryorrhea (one, two, and one males) in the 0, 75, and 500 ppm dose groups, respectively; rust-colored fur seen in one male each in the 10, 75, and 500 ppm groups; and scabs (one male at 75 ppm) and sores predominately on the upper body (four, two, and four males at 0, 75, and 500 ppm, respectively).
Clinical observations of P1 males during the postmating holding period included alopecia in two males at 0 ppm and in one male each at 10, 75, and 500 ppm, respectively. Chromodacryorrhea was seen in one male at 0 ppm, and rust-colored fur was seen in one male each at 10, 75, and 500 ppm, respectively. One male at 75 ppm had a sore on his shoulder throughout the postmating holding period. None of these findings were considered treatment related.

Clinical observations of the P1 females during the prebreed and mating periods indicated alopecia (two females in the control group and three at 75 ppm); blood at eartag (two females at 0 ppm and one female at 75 ppm); chromodacryorrhea (one female at 0 ppm and two females at 10 ppm); red vaginal discharge (one female at 75 ppm on study day 65 and blood in her vaginal smear on study day 69 and 70; probably due to irritation from lavage); one female each with rust-colored fur, a scab on neck, and a sore on the face at 75 ppm; and one female at 0 ppm with scab and sore. One female in the control group was found with a prolapsed right eye and lacerated tail on study day 8 after inadvertently being dropped during cage transfer. The eye was surgically enucleated by the attending veterinarian, and subsequent healing was uneventful.
Clinical observations of the P1 females during the postmating holding period were limited to alopecia in one female each at 1, 10, and 75 ppm, respectively.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: In P1 females, weekly body weights were significantly reduced for nine (weeks 1-8 and 10) of the ten weeks of prebreed. P1 female body weight change was significantly reduced for sd 0-7 and 42-49.
500 ppm: P1 male body weights were significantly reduced through sd 28, with a significant trend but not pairwise reduction on sd 35 and 42. Body weight gain was also significantly reduced only at 500 ppm for sd 0 to 7 and 7 to 14. Body weight change was significantly increased at 500 ppm for sd 49 to 56. During the total ten-week prebreed period, body weight change was equivalent across all groups.
P1 males were held after mating until delivery of all F2 litters (sd 84-105), with dietary exposures continuing. Body weight was equivalent across groups. Body weight change, however, was significantly reduced in all doses for sd 84-91, with no dose-response relationship. Body weight change was also significantly reduced only at 500 ppm for sd 98-105.
The data from retained P1 females (females who were not pregnant were held through sd 105) during the postmating holding period (two to six per group) were not analyzed statistically but are presented for completeness. P1 female body weights and weight changes are presented in Table 56. Body weights appear to be reduced at 500 ppm, and body weight changes are variable across all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

P1 female feed consumption values, expressed as g/day, were significantly reduced at 75 and 500 ppm for weeks 1, 2, 3, 5, and 6, and also at 500 ppm for week 4. Feed consumption in
g/day for the entire prebreed period (sd 0-70) was significantly lower only at 500 ppm. When feed consumption was expressed as g/kg/day, there were no effects at any dose for any interval.

In P1 males, there were no significant differences in feed consumption at any time interval during the entire prebreed period when expressed as g/kg/day. P1 male feed consumption was unaffected when expressed as g/day or g/kg/day at any dose, for any interval measured during the postmating holding period.

In P1 males, feed consumption, expressed as g/day, was significantly reduced at 500 ppm for sd 0 to7 (week 1) , 7 to 14 (week 2), 14 to 21 (week 3), and 21 to 28 (week 4), and for the entire prebreed period (sd 0 to 70), with a significantly downward trend for sd 28 to 35 (week 5) and 35 to 42 (week 6).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In P1 males, percent food efficiency was significantly lower at 75 ppm but only for sd 7 to 14. However, it was significantly higher at 500 ppm for weeks 5 and 8 and for the entire prebreed period. There was a significant trend for increased percent food efficiency expressed on prebreed weeks 3 to 5, 7 to 9, and for the entire ten-week period.
In P1 males, percent food efficiency was significantly reduced at 10, 75, and 500 ppm for sd 84-91 but for no other interval measured.
In P1 females, percent food efficiency was significantly higher at 75 ppm for week 3 and at 500 ppm for weeks 2 and 3, but similar across all groups for the entire ten-week prebreed exposure period.

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):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: For P1 males, absolute pituitary weights were reduced, absolute thyroid and liver weights were significantly increased, absolute brain weights were significantly decreased, relative liver and thyroid weights were increased, relative pituitary weight was significantly reduced. In P1 females, absolute spleen and brain weights were significantly decreased, absolute and relative liver weights were significantly increased, absolute pituitary weights were significantly decreased, relative thyroid weight was significantly increased, relative pituitary weight was significantly decreased.
75 ppm: For P1 males, absolute pituitary weights were reduced, absolute brain weights were significantly decreased, relative pituitary weight was significantly reduced. In P1 females, absolute and relative liver weights were significantly increased, absolute pituitary weights were significantly decreased.
10 ppm: For P1 males, absolute brain weights were significantly decreased. For P1 females, absolute pituitary weights were significantly decreased, relative pituitary weight was significantly decreased.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: For P1 males, apparent increase in the incidence of hydronephrosis of the right kidney of 20% compared to 0% in controls. For P1 females, dark brown liver (in 25/30; 83%) and dark brown kidney bilateral (in 16/30; 53%) were noted, as well as left adrenal with two dark red areas in one female

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: For P1 males, follicular cell hypertrophy in nine of ten thyroids versus none in any other groups were observed, hepatocyte cytoplasmic alterations were also observed in three of ten male livers versus none in any other group. In P1 females, hepatocyte hypertrophy in eight often female livers, and follicular cell hypertrophy in nine often P1 female thyroids and nephropathy in four of ten kidneys (one, two, and one of ten each at 0, 10, and 75 ppm, respectively).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The test substance intake for the postmating holding period was approximately 0.54 (first week) - 0.51 (last/third week) mg/kg/day at 10 ppm, 4.14 - 3.80 mg/kg/day at 75 ppm, and26.77 - 24.65 mg/kg/day at 500ppm, in P1 males.
The test substance intake for the postmating holding period in mg/kg/day was 0.86 (week 1)- 0.53 (week 3) at 10ppm, 5.01 - 4.77 at 75ppm, and 31.40 - 32.09 at 500 ppm, with zero to four P1 females per group per interval.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Vaginal cyclicity was evaluated for P1 females for 21 consecutive days during the last three weeks of the prebreed period. All females in all groups were cycling; 3, 5, 8, and 4 females
in the 0, 10, 75, and 500 ppm groups, respectively, had abnormal cycles. Cycle length was equivalent across all groups, with a mean duration of 4.30, 4.58, 4.83, and 4.76 days at 0, 10, 75, and 500 ppm, respectively.
At necropsy, paired ovarian primordial follicle counts (ten females each at 0 and 500 ppm) were equivalent between the two groups. The stage of the estrous cycle at demise was statistically equivalent across all groups, although the percentage of females in proestrus in controls (32.1%) was higher than in the treatment groups (11.5, 13.8, and
11.1% at 10, 75, and 500 ppm, respectively). The number of control females in metestrus (25.6%) was correspondingly lower than in the treatment groups (46.2, 62.1, and 51.8% at 10, 75, and 500 ppm, respectively). There was no dose-related pattern to these findings.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Absolute and relative paired testes, paired epididymides, prostate, seminal vesicles with coagulating gland weight, and relative brain weights were unaffected by treatment. There were no significant differences among groups for percent motile or progressively motile sperm, epididymal sperm concentrations, testicular spermatid head concentration, daily sperm production (DSP),
efficiency of DSP, or percent abnormal sperm. The mean values for percent abnormal sperm were 3.14, 1.96, 1.73, and 2.27% at 0, 10, 75, and 500 ppm, respectively.

Reproductive performance:

no effects observed

Description (incidence and severity):

P1 male and female mating, fertility, and gestational indices were equivalent across all dose groups. The precoital interval (in days) was also equivalent across all groups. Gestational length in days was equivalent across groups, with mean values of 22.0, 22.1, 22.0, and 22.0 days at 0, 10, 75, and 500 ppm, respectively. The percent prenatal (postimplantation) loss was significantly increased at 75 ppm (but not at 500 ppm); this finding was not considered treatment related. The number of implantation sites per dam was equivalent across all groups.

Details on results (P1)

TEST SUBSTANCE INTAKE
For P1 males, test substance intake, expressed as mg/kg/day, exhibited the expected increases across groups, averaging approximately 0.80, 6.03, and 39.63 mg/kg/day at 10, 75, and 500 ppm, respectively, for the entire prebreed period (sd 0 —70). P1 male test substance intake also exhibited the expected decreases over time within groups: 1.50 (first week) - 0.60 (last week)
mg/kg/day at 10 ppm, 11.22 - 4.46 mg/kg/day at 75 ppm, and 74.47 - 29.79 mg/kg/day at 500 ppm.
For P1 females, test substance intake during the ten-week prebreed period was approximately 0.91, 6.76, and 45.20 mg/kg/day at 10, 75, and 500 ppm, respectively. Test substance intake also exhibited the expected decreases over time within groups: 1.48 (week 1) - 0.69 (week 10) mg/kg/day at 10 ppm, 10.97-5.28 mg/kg/day at 75 ppm, and 74.61-34.70 mg/kg/day at 500 ppm.

GESTATION - P1 FEMALES FOR F2 LITTERS
The number of females which were detected sperm positive, confirmed pregnant were 23, 26, 25, and 27 at 0, 10, 75, and 500 ppm, respectively. P1 maternal body weights were significantly reduced at 500 ppm for gd 0, 7, and 14 but not for gd 20. P1 maternal body weight change was significantly reduced at all dose levels for gd 0-7 with no dose-response relationship. FP1female body weight gain was significantly lower for the gestational period (gd 0-20) only at 500 ppm.
Maternal gestational feed consumption, expressed at g/day, was significantly reduced at 500 ppm for gd 0-7, 7-14, and for the entire gestational period (gd 0-20). When expressed as g/kg/day, there were no effects of treatment at any dietary dose for any gestational interval. Food efficiency was significantly decreased on gd 0-7 at 10, 75, and 500 ppm. Maternal gestational intake of the test substance (gd 0-20) was approximately 0.68, 5.38, and 34.68 mg/kg/day for 10, 75, and 500 ppm, respectively.
Clinical observations of Fl dams during gestation included alopecia in one, four, and two dams in the 10, 75, and 500 ppm groups, respectively. Chromodacryorrhea was seen in one dam at 0 ppm and rust-colored fur in one dam at 0 ppm and two dams at 75 ppm. Sore(s) at multiple sites (one dam) and on the neck (one dam) were observed at 500 ppm.

LACTATION - F1 FEMALES FOR F2 LITTERS
The number of females which were confirmed pregnant and delivered live litters were 25, 28, 29, and 28 at 0, 10, 75, and 500 ppm, respectively. Maternal lactational body weights were statistically significantly reduced at pnd 0, 4, 7, and 14 at 500 ppm. Maternal lactational body weight change was significantly reduced for pnd 0-4 at 500 ppm. Maternal lactation feed consumption as g/day was significantly reduced at 500 ppm for pnd 4-7, pnd 14-21, and for the entire lactational period (pnd 0-21). When expressed as g/kg/day, there were no differences at any time interval for any group. Maternal lactational food efficiency was significantly lower than the control at 500 ppm only for the pnd 0-4 interval. Maternal lactational intake of the test substance for the entire lactational period (pnd 0-21) was approximately 1.67, 12.77, and 80.58 mg/kg/day at 10, 75, and 500 ppm, respectively, confounded by the pups self feeding beginning during the second week of postnatal life.
Clinical observations of the P1 dams during lactation included alopecia in two,four, five, and four dams at 0, 10, 75, and 500 ppm,respectively. Bloody discharge the day of or after delivery was observed in one dam each at 10 and 75 ppm. Chromodacryorrhea (one dam each at 0, 10, and 500 ppm) and rust-colored fur (two, two,three, and two dams each at 0, 10, 75, and 500 ppm, respectively) were also observed. These findings were considered non-treatment related.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: corresponds to 6.03 and 6.76 mg/kg bw/day for males and females, respectively

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed (highest dose tested)

Remarks on result:

other: corresponds to 39.63 and 45.20 mg/kg bw/day for males and females, respectively.

Target system / organ toxicity (P1)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations of F1 pups during the lactation period indicated no treatment- or dose-related incidences or severities and included found dead pups (and euthanized moribund, missing, and presumed dead), pale or hypothermic pups, failure to nurse (no milk band), chewed extremities (presumably by dam), hematomas, missing left eye (one pup at 500 ppm on pnd 21), and delayed development (one female at 75 ppm with both eyes not yet open). These are all common findings in the lactation period. The total number of pups found dead (or missing and presumed dead) through pnd 4 were 26, 19, 20, and 30, and from pnd 5-21 were 1, 6, 7, and 9 at 0, 10, 75, and 500 ppm, respectively

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1: There were no treatment-related increases in pup losses during lactation. Through PND 4, the total number of dead, missing and presumed dead pups was 26, 19, 20 and 30 in the control, 10, 75 or 500 ppm group, respectively. Between PND 5 and 21, 1, 6, 7 and 9 pups were found dead or missing and presumed dead at control, 10, 75 and 500 ppm, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Beginning on pnd 4 and for pnd 7, 14, and 21, pup body weights per litter (for all pups and separately by sex) were significantly reduced at 500 ppm. F1 weanling male body weight was significantly reduced (by approximately 19%) at 500 ppm.
F1 weanling female body weight was significantly reduced at 75 and 500 ppm (by approximately 6% and 18%, respectively).

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:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: delay of sexual maturation in F1 male. In females, the age of acquisition was not delayed, but the body weight at acquisition was significantly reduced at 500 ppm.

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):

In males, relative spleen and paired kidney weights were significantly decreased at 500 ppm. Relative liver and brain weight were significantly increased at 500 ppm.
In females, relative paired kidney weight was decreased at 500 ppm. Relative brain and liver weights were significantly increased at 500 ppm (indicating that these findings were related to the decreased body weight at that dose)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hydronephrosis (bilateral or right) was seen in one to two females per dose group and in two to five males per dose group. One female in the 75 ppm group had a missing eye, barely open eyelids, and enlarged Harderian glands (bilateral). One male at 500 ppm had a missing left eye, accompanied by an incomplete eyelid and enlarged Harderian gland. One male at 10 ppm had a pale liver, and one female pup at 500 ppm had multiple red foci on the thymus.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio (% males per litter) and average number of pups per litter on pnd 0, 4, 7, 14, and 21 were statistically equivalent across all groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

75 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:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations of F2 pups during the lactational period (pnd 0-21) included found dead, missing, presumed dead, hematomas, pale, no milk band, bite wound or cut on leg, bite wound on forehead, misshapen eye, closed and wounded eye, and walking flat footed on left rear leg. The number of pups found dead, missing and presumed dead on pnd 0 through 4 were 25, 12, 20 and 33 at 0, 10, 75 and 500 ppm, respectively. The number of pups with the same criteria on pnd 5 through 21 were 4, 12, 1, and 2 at 0, 10, 75, and 500 ppm, respectively. There were no treatment-related increases in F2 pup lactational losses.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

At weaning, on pnd 21, there was no change in the number of live litters from pnd 0. The survival indices for pnd 0-4 were 94.8% (0 ppm), 98.9% (10 ppm), 98.6% (75 ppm), and 96.7% (500 ppm).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean F2 body weights by litter for all pups or separately by sex were unaffected by treatment on pnd 0, 4, and 7. However, F2 pup body weights per litter were significantly reduced when analyzed as all pups or separately by sex at 500 ppm for pnd 14 and 21. There were no effects on pup body weight at 10 or 75 ppm for any interval measured during lactation.
At necropsy, F2 male weanling body weight was significantly reduced at 500 ppm (87% of the control value). At necropsy, F2 female weanling body weight was significantly reduced at 500 ppm (87% of the control value).

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):

no effects observed

Description (incidence and severity):

The delay in acquisition of puberty for P1 males and females resulted in the measurement of anogenital distance (and body weight) of F2 pups at birth (pnd 0), a "triggered" endpoint (U.S. EPA OPPTS, 1998). Average F2 male anogenital distance was equivalent across all groups. When the male anogenital distance was statistically analyzed by Analysis of Covariance (ANCOVA), with each male body weight on pnd 0 as the covariate, there were also no statistically significant differences among groups. Average F2 female anogenital distance was also equivalent across all groups, per se, and when adjusted for body weight (by (ANCOVA).

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 ppm: In F2 males, absolute male thymus, spleen, paired kidney, and brain weights were also slightly but significantly reduced, most likely due to the reduced body weight at this dietary dose. Relative liver and brain weight was significantly increased. Relative paired kidney weight was significantly decreased. In F2 females, absolute thymus, spleen, paired kidney and brain weights were significantly decreased. Relative paired kidney weight was significantly reduced, and relative liver and brain weight was significantly increased
75 ppm: In F2 males, relative liver weight was increased

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 male weanlings exhibited missing left eye (one male at 500 ppm) and hydronephrosis of right kidney in two, six, two, and one F2 male weanlings at 0, 10, 75, and 500 ppm, respectively. One female F2 weanling in the 75 ppm group had an irregular white mass of the right eye, and two females in the 500 ppm group has a missing left (one) or right (one) eye. Also seen was hydronephrosis, right or bilateral, in three, five, and two F2 weanlings at 10, 75, and 500 ppm, respectively, with none seen in the control group. One female (who also exhibited missing right eye) at 500 ppm also had a 4 mm unossified area in the midline of the skull. None of these findings were considered to be related to treatment.

Gross necropsy of F2 pups which died and were available for examination included 21 (13 with abdominal organs too autolyzed to evaluate and two cannibalized and unable to be evaluated) at 0 ppm, 14 (three with abdominal organs too autolyzed to evaluate) at 10 ppm, 18 (three with abdominal organs too autolyzed to evaluate, and one cannibalized and unable to be evaluated) at 75 ppm, and 27 (three with abdominal organs too autolyzed to evaluate) at 500 ppm. Most of the findings included patent (open) ductus arteriosus, no air in lungs, no milk in stomach, and abdominal organs autolyzed (especially early in lactation). These are usual findings in dead pups during the lactational period. One pup at 75 ppm exhibited left testis with petechial hemorrhages on pnd 0, and one pup at 500 ppm exhibited anal atresia. These findings were not considered treatment related.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

The number of litters on pnd 0 were 24, 28, 29, and 28 at 0, 10, 75, and 500 ppm, respectively. F2 pup sex ratio (percent males per litter) was equivalent across all groups throughout lactation. The mean number of live pups per litter was significantly reduced on pnd 0 at 75 and 500 ppm; however, they were equivalent across all dose groups for the remainder of the lactational period and well within the historical control range

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F2

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: P0 reproductive indices

 

	Control	dose groups [ppm]
		10	75	500
No. of males started on study	30	30	30	30
No. of females started on study	30	30	30	30
No. of females paired	30	30	30	30
No. of males paired	30	30	30	30
No. of females that mated	30	30	30	29
No. of males that mated	30	30	30	29
Mating Index [%]	100	100	100	96.7
No. of pregnant females	29	29	29	27
Fertility Index [%]	96.7	96.7	96.7	93.1
No. of females with live litters (PND 0)	29	29	29	26
Gestational Index [%]	100	100	100	96.3
No. of males siring litters	29	29	29	27
Pregnancy Index [%]	96.7	96.7	96.7	93.1
Days until sperm-positive (days)	2.6	2.4	2.5	2.2
	± 0.3	± 0.2	± 0.3	± 0.3
	n=26	n=28	n=26	n=27
Gestational length (days)	22.1	22.0	22.2	22.0
	± 0.1	± 0.1	± 0.1	± 0.1
	n=25	n=27	n=25	n=24
No. of live litters on PND 0	29	29	29	26
No. of live litters on PND 4	28	29	29	26
No. of live litters on PND 21	28	29	29	26
No. of Implantation Sites per litter	14.83	15.69	16.62	15.70
	± 0.68	± 0.57	± 0.47	± 0.71
	n=29	n=29	n=29	n=27
Post-implantation loss per litter [%]	9.47	10.02	7.58	13.49
	± 2.73	± 1.92	± 1.34	± 3.99
	n=29	n=29	n=29	n=27

Table 2: F1 lactational indices

 

	Control	dose groups [ppm]
		10	75	500
No. of live pups on PND 0	14.1	14.4	15.4	14.7
	± 0.8	± 0.6	± 0.4	± 0.5
No. of dead pups on PND 0	0.6	0.3	0.3	0.8
	± 0.3	± 0.1	± 0.2	± 0.3
Total No. of pups on PND 0	14.7	14.8	15.8	15.5
	± 0.7	± 0.6	± 0.4	± 0.5
Stillbirth index [%]	3.9	2.2	2.0	5.0
	± 2.3	± 0.9	± 1.1	± 1.9
	n=29	n=29	n=29	n=26
Livebirth index [%]	96.1	97.8	98.0	95.0
	± 2.3	± 0.9	± 1.1	± 1.9
	n=29	n=29	n=29	n=26
4 Day survival index [%]	95.6	97.4	97.9	97.5
	± 3.4	± 1.3	± 0.9	± 0.8
	n=29	n=29	n=29	n=26
7 Day survival index [%]	99.6	99.3	99.0	98.5
	± 0.4	± 0.5	± 1.0	± 0.9
	n=28	n=29	n=29	n=26
14 Day survival index [%]	99.6	98.5	98.7	98.5
	± 0.4	± 1.5	± 1.0	± 0.9
	n=28	n=29	n=29	n=26
21 Day survival index [%]	100.0	99.7	99.7	100.0
	± 0.0	± 0.3	± 0.3	± 0.0
	n=28	n=29	n=29	n=26
Lactational index [%]	99.3	97.6	97.6	96.9
	± 0.5	± 1.8	± 1.8	± 1.2
	n=28	n=29	n=29	n=26
Percent males per litter on PND 0	46.5	51.1	53.7	55.1
	± 3.0	± 2.6	± 2.2	± 2.2
	n=29	n=29	n=29	n=26
Percent males per litter on PND 21	46.3	50.1	51.4	50.1
	± 2.6	± 2.1	± 1.2	± 1.4
	n=28	n=29	n=29	n=26

Table 3: F1 reproductive indices

 

	Control	dose groups [ppm]
		10	75	500
No. of males started on study	30	30	30	30
No. of females started on study	30	30	30	30
No. of females paired	30	30	30	30
No. of males paired	30	30	30	30
No. of females that mated	27	29	30	29
No. of males that mated	27	29	30	29
Mating Index [%]	90.0	96.7	100.0	96.7
No. of pregnant females	25	28	29	28
Fertility Index [%]	92.6	96.6	96.7	96.6
No. of females with live litters (PND 0)	25	28	29	28
Gestational Index [%]	100	100	100	100
No. of males siring litters	25	28	29	28
Pregnancy Index [%]	92.6	96.6	96.7	96.6
Days until sperm-positive (days)	2.8	2.4	2.7	2.3
	± 0.5	± 0.3	± 0.5	± 0.3
	n=25	n=27	n=26	n=28
Gestational length (days)	22.0	22.1	22.0	22.0
	± 0.1	± 0.1	± 0.1	± 0.1
	n=23	n=26	n=25	n=27
No. of live litters on PND 0	25 X	28	29	28
No. of live litters on PND 4	24	28	29	28
No. of live litters on PND 21	24	28	29	28
No. of Implantation Sites per litter	15.08	16.57	15.59	15.32
	± 0.71	± 0.41	± 0.59	± 0.53
	n=25	n=28	n=29	n=28
Post-implantation loss per litter [%]	4.68#	7.51	11.91**	9.33
	± 1.42	± 1.80	± 2.15	± 2.30
	n=24	n=28	n=29	n=28

 

X: PND 0 date was missed for one female in the control group and therefore none of her litter data could be included since PND 0 was not known. She was included as a sperm-positive, pregnant female with a live litter and her number of implantation sites was also included.

 

#: ANOVA Test; p<0.05

**: Dunnett’s Test; p<0.01

Table 4: F2 lactational indices

 

	Control	dose groups [ppm]
		10	75	500
No. of live pups on PND 0	15.6#	15.4	13.9*	14.0*
	± 0.5	± 0.4	± 0.5	± 0.6
	n=24	n=28	n=29	n=28
No. of dead pups on PND 0	0.1	0.3	0.4	0.7
	± 0.1§	± 0.1	± 0.2	± 0.3
	n=24	n=28	n=29	n=28
Total No. of pups on PND 0	15.7	15.7	14.3	14.8
	± 0.5	± 0.4	± 0.6	± 0.5
	n=24	n=28	n=29	n=28
Stillbirth index [%]	0.8	1.6	2.9	5.3
	± 0.5§	± 0.8	± 1.0	± 2.0
	n=24	n=28	n=29	n=28
4 Day survival index [%]	94.8	98.9	98.6	96.7
	± 3.5	± 0.6	± 0.7	± 2.1
	n=24	n=28	n=29	n=28
7 Day survival index [%]	99.6	98.6	99.7	99.6
	± 0.4	± 0.8	± 0.3	± 0.4
	n=24	n=28	n=29	n=28
14 Day survival index [%]	98.6	96.7	100	99.6
	± 1.4	± 1.9	± 0	± 0.4
	n=24	n=28	n=29	n=28
21 Day survival index [%]	100	99.6	100	99.6
	± 0	± 0.4	± 0	± 0.4
	n=24	n=28	n=29	n=28
Livebirth index [%]	99.2	98.4	97.1	94.7
	± 0.5§	± 0.8	± 1.0	± 2.0
	n=24	n=28	n=29	n=28
Lactational index [%]	98.3#	95.4	99.7	99.3
	± 1.7	± 2.3	± 0.3	± 0.5
	n=24	n=28	n=29	n=28
Percent males per litter on PND 0	47.4	50	49.1	53.9
	± 2.5	± 2.0	± 3.3	± 2.6
	n=24	n=28	n=29	n=28
Percent males per litter on PND 21	49.6	50.4	48.2	52.4
	± 1.2	± 1.1	± 2.6	± 2.0
	n=24	n=28	n=29	n=28

 

#: ANOVA Test; p<0.05

§: Test for Linear Trend; p<0.05

*: Dunnett’s Test; p<0.05

Applicant's summary and conclusion

Conclusions:

Under the conditions chosen in this two-generation reproductive toxicity study performed according to OECD 416, there were no histopathologic findings in P1 females at 0, 10, or 75 ppm. Therefore, the no observable adverse effect level (NOAEL) for P0 and P1 parental animals for systemic toxicity is 75 ppm, based on the likely adaptive and transient histopathologic findings at 500 ppm. The NOAEL for reproductive toxicity for P0 and P1 parental animals was considered to be 500 ppm, the highest dose tested. Based on the decreases in F1 and F2 pup body weights at 500 ppm during lactation (beginning on pnd 4 for F1 and on pnd 14 for F2 offspring) and into the postwean/prebreed period, with associated delays in acquisition of puberty at this dietary dose, the offspring toxicity NOAEL is 75 ppm.