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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity (OECD 416), rat:

NOAEL (reproduction/fertility P0, P1): 500 ppm (corresponding to 32.33 mg/kg bw/day for P0 males and 37.36 mg/kg bw/day for P0 females; 39.63 mg/kg bw/day for P1 males and 45.2 mg/kg bw/day for P1 females)

Link to relevant study records

Reference

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

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

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

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

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

Dose / conc.:

10 ppm (nominal)

Remarks:

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

Dose / conc.:

75 ppm (nominal)

Remarks:

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

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

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

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: 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.

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.

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

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

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

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: 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.

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.

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

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.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

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

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

Critical effects observed:

no

Reproductive effects observed:

no

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity

In a preliminary range-finding study, 10 Sprague-Dawley rats per sex and group were treated with the test substance at doses of 10, 50, 250 and 1000 ppm in the diet (corresponding to 0.7, 3.6, 17.9 and 68.1 mg/kg bw/day for P0 males and 0.8, 3.8, 20.6 and 71.9 mg/kg bw/day for P0 females during mating; 0.7, 3.6, 18.4 and 72.3 mg/kg bw/day for P0 females during gestation and 1.8, 9.5, 43.4 and 172.6 mg/kg bw/day for P0 females during lactation as well as 1.5, 7.3, 36.2 and 150.1 mg/kg bw/day for F1 males and 1.6, 8.4, 38.0 and 161.7 mg/kg bw/day for F1 females during the post-weaning period, respectively) (M-210173-01-2, 2000). All animals received the test substance during pre-mating and mating. The females were additionally treated during gestation and weaning until post-natal Day (PND) 21. The F1-generation received the test substance for 7 weeks during weaning and growth into adult hood.

No treatment-related deaths occurred during the study period. In the P0-generation, the NOAEL for systemic toxicity is considered to be 50 ppm due to increased liver weights in males and females and enlarged livers in females. The parental reproduction parameters (including mating index, fertility index, gestational index, pregnancy index and number of live pups) were not affected by test substance indicating a NOAEL of 1000 ppm for fertility. In the F1-generation, a delay in vaginal patency and preputial separation of the high dose animals was observed. Therefore, the NOAEL for developmental toxicity is considered to be 250 ppm. No effects on viability were observed. The body weights of the pups of the 1000 ppm group were decreased during lactation and the post-wean period.

Based on the outcome of this study, a two-generation reproductive toxicity study was performed according to OECD 416 and under GLP conditions (M-240522-01-2, 2001). Thirty male and female rats per group were mated on a one-to-one basis. The test substance was administered in doses of 10, 75 and 500 ppm (corresponding to 0.7, 4.8 and 32.3 mg/kg bw/day for P0 males and 0.8, 5.8 and 37.36 mg/kg bw/day for P0 females during the pre-breed period; 0.7, 5.0 and 34.1 mg/kg bw/day for P0 females during gestation and 1.6, 12.2 and 79.1 mg/kg bw/day for P0 females during lactation as well as 0.8, 6.0 and 39.6 mg/kg bw/day for P1 males and 0.9, 6.8 and 45.2 mg/kg bw/day for P1 females during the pre-breed period; 0.7, 5.4 and 34.7 mg/kg bw/day for P1 females during gestation and 1.7, 12.8 and 80.6 mg/kg bw/day for P1 females during lactation, respectively) with the diet to the animals of the P0 - and P1-generation during pre-mating, mating, gestation, lactation until weaning on PND 21.

In the P0-generation, increased liver and thyroid weights in males and females and decreased body weight as well as increased adrenal weights in the females of the 500 ppm group were observed. Furthermore, brown-colored livers and kidneys were observed in females of the 500 ppm group and histopathological examinations in this group revealed liver hypertrophy and thyroid follicular cell hypertrophy. In the adult P1 males, the pituitary weights were reduced at 75 and 500 ppm and the liver and thyroid weights were increased at 500 ppm. In adult females, liver weights were significantly increased at 75 and 500 ppm and the thyroid weights were increased at 500 ppm. Necropsy revealed an increase in hydronephrosis in males and brown-colored livers and kidneys in females of the 500 ppm group. Due to these findings, the NOAEL for systemic toxicity is considered to be 75 ppm (corresponding to 4.77 mg/kg bw/day for P0 males and 5.82 mg/kg bw/day for P0 females as well as 6.03 mg/kg bw/day for P1 males and 6.76 mg/kg bw/day for P1 females, respectively).

No treatment-related findings on reproduction were observed in both sexes of the P0 - and P1-groups. Therefore, the NOAEL for reproductive toxicity (fertility) is considered to be 500 ppm (corresponding to 32.33 mg/kg bw/day for P0 males and 37.36 mg/kg bw/day for P0 females as well as 39.63 mg/kg bw/day for P1 males and 45.2 mg/kg bw/day for P1 females, respectively).

The F1 male and female weanling body weights were reduced in the 500 ppm group and the female weanling body weights were even reduced at 75 ppm. Associated with this observation, the age of acquisition of preputial separation was significantly delayed in the males of the 500 ppm group. In females, the age of acquisition was not delayed, but the body weight at acquisition was significantly reduced at 500 ppm. In this dose group, also a decrease in spleen and kidney weights was observed in males, while the brain and liver weights of both sexes were increased.

The NOAEL for postnatal, systemic toxicity for F1 was considered to be 75 ppm.

In the male and female weanlings of the F2-generation, a reduction in body weight was observed on PND 14 and PND 21 at 500 ppm. At necropsy, F2 weanling body weight was significantly reduced at 500 ppm (87% of the control value). Further, a decrease in relative kidney weight and increases in relative liver and brain weights were observed at 500 ppm. The NOAEL for postnatal, systemic toxicity for F2 was considered to be 75 ppm.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414), rat:

NOAEL (maternal systemic toxicity): 10 mg/kg bw/day

NOAEL (fetal developmental toxicity): 10 mg/kg bw/day (developmental effects as a secondary non-specific consequence of maternal toxicity effects)

Prenatal developmental toxicity (OECD 414), rabbit:

NOAEL (maternal systemic toxicity): 0.5 mg/kg bw/day

NOAEL (maternal developmental toxicity): 0.5 mg/kg bw/day

NOAEL (developmental toxicity): 2 mg/kg bw/day (developmental effects as a secondary non-specific consequence of maternal toxicity effects)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 1999 - Feb 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no data reported on dam thyroid weight and histopathological assessment, dam thyroid hormones and fetal anogenital distance

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Draft 1998 (for updating guideline of 1981)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

current version adopted in 2018

Deviations:

yes

Remarks:

no investigation of dam thyroid weight and histopathological assessment, dam thyroid hormones, fetal anogenital distance

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F., Japan, No. 4200

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: New Zealand White Crl: Kbl/BR (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Saint Aubin les Elbeuf, France
- Weight at study initiation: 2.48 - 3.69 kg
- Housing: individually, in plastic cages on a perforated cage floor
- Diet: Certified Rabbit Pellet diet UAR 110 C-10, 250 ± 5 g
- Water: filtered water, ad libitum
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29 Aug 1999
To: 09 Dec 1999

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution of 0.5% methylcellulose 400 (w/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: An appropriate amount of the test substance was suspended (w/v) in the vehicle and stored at approximately 5 °C (± 3 °C). Homogeneity of the suspensions was checked for all concentrations. Stability of the test substance in suspension was determined before the begin of the study at concentrations of 62.5, 75 and 15000 mg/L and was found acceptable within a period of 21 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aqueous methylcellulose samples were diluted with acetonitrile. The quantification was performed by HPLC using UV detection at 277 nm and external standardization. Test substance formulations in aqueous methylcellulose were demonstrated to be stable over a 21-day period at 5°C ± 3°C. Concentrations checks were in a range of [90-106%] of the nominal values.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day of insemination referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Day 6 - 28 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

29 days

Dose / conc.:

0.25 mg/kg bw/day (actual dose received)

Dose / conc.:

0.5 mg/kg bw/day (actual dose received)

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Dose / conc.:

4 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses based on results obtained in a previous range-finding study (1999) in rabbits.
- Rationale for animal assignment (if not random): The rabbits were allocated to the different groups using a body weight dependent procedure to ensure that the females inseminated with the same male were distributed among the different groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon); on weekends and public holidays once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: full feeder weights were measured on GD 1 and daily thereafter through GD 28; empty feeder weights were measured on GD 2 and every day through GD 29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29
- Organs examined: macroscopic examination of the visceral organs, recording of the number of ribs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- individual body weights of live foetuses

Statistics:

For maternal and litter parameters, Dunnett's test was used to compare exposed groups to controls when the overall effect was statistically significant (p<0.05 via the ANOVA F-test). The Generalized Estimating Equations (GEE) linear regression model was used for analyzing fetal body weight and fetal death status, and the GEE logistic model was used for analysing fetal sex. Although fetal death status is a binary outcome (dead vs. live foetus), this parameter was still analysed via a linear regression model because the distribution of the variable was too sparse for computations required for logistic regression. All GEE regression models assumed exchangeable intralitter correlations and used a robust variance estimator for regression parameters. Individual t-tests for pairwise comparisons to control were evaluated when the overall treatment effect was statistically significant (p<0.05 via a Wald chi-square test).

Indices:

Percentage of pre-implantation loss: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] * 100

Percentage of post-implantation loss: [(no. of implantations - no. of live foetuses) / no. of implantations] * 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs were those commonly observed, recorded once only or distributed between different groups and were not considered to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the study period, no treatment-related deaths were observed. In the control group, 3/60 females were found dead on GD 10, 12 and 21. The necropsy revealed a gavage error in two cases and in the other case no abnormal findings were observed. In the 0.25 mg/kg bw/day group, 2/30 females died on GD 8 and 10 due to a gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4 mg/kg bw/day, the mean body weight changes for all intervals during the dosing period were significantly lower than the corresponding control values. From GD 6 through 18, a body weight loss of -0.27 kg was observed compared to a gain of 0.21 kg in the control group. At 2 mg/kg bw/day, the body weight changes were reduced for many intervals, showing statistical differences for GD 6-8, 6-14, 6-18, 6-22 and 6-26. At 0.25 and 0.5 mg/kg bw/day, the body weight changes were considered to be unaffected by treatment when compared to the controls. The corrected body weight change did not show any statistical differences between treated and control groups.
Tables for maternal body weights and weight gains can be found in attached documents.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption showed similar significant effect when expressed in g/day or g/kg/day. At 4.0 mg/kg bw/day, mean food consumption was significantly reduced during each interval from GD 6 to GD 22 and significantly increased for GD 26 to 29. At 2.0 mg/kg bw/day, food consumption was significantly reduced for GD 10 to 14 and for GDI 4 to 18, At 0.25 and 0.5 mg/kg bw/day, mean food consumption was unaffected by treatment.
Tables for food consumption can be found in attached documents.

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):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterine weight did not show significant differences between groups (please refer to the table in the attached document).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 4 mg/kg bw/day, accentuated lobular pattern of the liver was observed in females that aborted (10/11 females with abortions).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

In the 4 mg/kg bw/day group, 11/28 pregnant females aborted between GD 21 and 27. Nearly all of this females (10/11) had pale and/or accentuated lobular pattern of the liver. In the 2 mg/kg bw/day group, 2/24 pregnant females aborted on GD 25 and 28.
All females aborted after reduced food consumption and body weight loss indicating maternal toxicity as cause of the observed abortions. No abortion was observed in any other dose group.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 4.0 mg/kg bw/day, the mean post-implantation loss percentage was increased (not statistically significant) when compared to the control group value. It was linked to an increased number of late resorptions (statistically significant for females with live foetuses) and an increased percentage of dead foetuses (no statistically significant).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

At 4.0 mg/kg bw/day, two females were observed with resorptions only.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 4.0 mg/kg bw/day, an increased number of late resorptions was observed (statistically significant for females with live foetuses).

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 4.0 mg/kg bw/day, an increased percentage of dead foetuses was observed (not statistically significant).

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The pregnancy rate was between 80 and 97% across all groups. At least 20 females per group treated with up to 2 mg/kg bw/day had live foetuses at cesarean section (see table 1). In the 4 mg/kg bw/day group, 13 females had live foetuses.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

0.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

development

Effect level:

0.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

development

Effect level:

2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

abortions starting at 2 mg/kg bw/day; increased post-implantation loss and late resorptions at 4 mg/kg bw/day. However, effects were regarded as secondary to the reduced food consumption of dams.

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

At 4.0 mg/kg bw/day, the number of live foetuses was slightly but not statistically significantly reduced when compared to control group value.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The number of runt foetuses (body weight < 28.0 g) was not affected by treatment. No foetal external findings were considered to be related to treatment as all findings concerned one foetus only per group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

No malformations showed any dose-related increase. No marked ossification delay was noted but a few findings were slightly increased at 2.0 and 4.0 mg/kg bw/day (incomplete ossification of pubis, metacarpal and/or middle phalanges). All the other variations and anomalies were considered to be randomly distributed between all groups.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The examination of heads of half of the number of foetuses did not reveal any treatment-related findings.
At visceral examination, a few malformations were observed but none of them was considered to be related to treatment except the two diaphragmatic hernia recorded at 4 mg/kg bw/day. All variations and anomalies were considered to be randomly distributed between treated and control groups without any treatment-related increase.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall findings (reduction in number of fetuses (non-significant), diaphragmatic hernia (low incidence (2 fetuses in 2 litter)) and incomplete ossification (slight severity)

Dose descriptor:

NOEL

Effect level:

0.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: overall findings (reduction in number of fetuses (non-significant), diaphragmatic hernia (low incidence with 2/80 fetuses in 2 litter) and incomplete ossification (slight severity)

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

4 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Animal status and pregnancy rate

 

	Control	dose groups [mg/kg bw/day]
		0.25	0.5	2	4
No. of inseminated females	60	30	30	30	30
No. of death females	3	2	0	0	0
No. of abortions	0	0	0	2	11
No. of pregnant females	53	29	26	24	28
- with live foetuses	46	26	24	30	13
- with resorptions only	0	0	0	0	2
- with implantations only	4	1	2	2	2
No. of non-pregnant females	7	1	4	6	2
Pregnancy rate [%]	88	97	87	80	93

 

 

Table 2: Litter response and foetal parameters

 

	Control	dose groups [mg/kg bw/day]
		0.25	0.5	2	4
No. of all pregnant dams observed	50	27	26	22	17
No. of Corpora Lutea per dam	10.0	10.3	9.3	9.5	9.4
	0.3	0.4	0.5	0.6	0.7
No. of Implantation Sites per litter	8.0	7.6	7.4	7.0	6.3
	0.4	0.7	0.5	0.7	0.9
Pre-implantation loss per litter [%]	22.2	26.2	19.8	27.8	35.1
	3.2	5.7	4.2	4.9	7.5
No. of early resorptions per litter	0.340	0.519	0.423	0.182	0.294
	0.133	0.188	0.168	0.084	0.143
No. of late resorptions per litter	0.020	0	0.017	0	0.176
	0.020	0	0.053	0	0.128
Post-implantation loss per litter [%]	17.1	9.8	17.9	16.9	38.7
	3.9	4.2	5.8	6.3	10.0
No. of all pregnant dams with live foetuses	46	26	24	20	13
No. of Corpora Lutea per dam	10.4	10.3	9.8	10.1	10.5
	0.2	0.5	0.3	0.5	0.6
No. of Implantation Sites per litter	8.3	7.9	7.9	7.5	7.6
	0.4	0.7	0.4	0.6	0.9
Pre-implantation loss per litter [%]	21.0	23.7	18.5	26.9	28.6
	3.3	5.3	3.9	5.3	7.3
No. of early resorptions per litter	0.370	0.538	0.458	0.200	0.231
	0.144	0.194	0.180	0.092	0.166
No. of late resorptions per litter	0.022	0	0.083	0	0.231
	0.022	0	0.058	0	0.166
Post-implantation loss per litter [%]	9.9	6.3	11.0	8.6	19.8
	1.9	2.4	3.7	3.0	6.8
No. of live foetuses per litter	7.5	7.3	7.1	6.8	6.2
	0.4	0.6	0.5	0.6	0.9
No. of dead foetuses per litter	0.3	0	0.1	0.4	0.8
	0.1	0	0.1	0.2	0.4
No. of foetuses examined	359	191	174	143	90
Dead foetuses [%]	3.3	0	1.7	4.9	11.1
	0.9	0	0.9	2.7	5.3
Male foetuses [%]	45.8	46.1	46.2	52.9	51.3
	2.8	2.8	4.7	4.1	4.3
Fetal body weight [g]	39.7	39.8	40.5	37.4	38.3
	0.5	0.7	0.6	1.0	1.4

Table 3: Fetal observations (excerpt)

Group	1	5	6	2	3	4	1	5	6	2	3	4
Visceral anomalies
	Number of fetuses examined						Number of litters examined
	178	169	191	171	135	80	23	23	26	24	20	13
	Number of heads examined						Number of heads examined
	83	80	90	78	63	38	22	23	26	23	20	12
	Number of fetuses affected (mean % of fetuses affected)						Number of fetuses affected (mean % of fetuses affected)
Diaphragmatic hernia	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (15.4)
Skeletal anomalies
	Number of fetuses examined						Number of litters examined
	178	169	191	171	136	80	23	23	26	24	20	13
	Number of heads examined						Number of heads examined
	95	89	101	93	73	42	23	23	26	24	20	13
	Number of fetuses affected (mean % of fetuses affected)						Number of fetuses affected (mean % of fetuses affected)
Pubis (unilateral/bilateral): incomplete ossification	4 (3.1)	3 (1.2)	4 (2.5)	2 (1.0)	14 (8.2)	11 (11.0)	3 (13.0)	2 (8.7)	4 (15.4)	1 (4.2)	6 (30.0)	4 (30.8)
1^stmetacarpal: incomplete ossification or unossified. Forepaws: 4^thor/and 5^thmiddle phalanges: unossified.	18 (9.8)	19 (11.4)	20 (12.1)	17 (10.6)	39 (27.6)	31 (35.4)	6 (26.1)	12 (52.2)	13 (50.0)	11 (45.8)	13 (650.)	8 (61.5)

Groups: 1 and 5 (control), 6 (0.25 mg/kg bw/day), 2 (0.5 mg/kg bw/day), 3 (2.0 mg/kg bw/day), 4 (4.0 mg/kg bw/day)

Conclusions:

No deaths were considered to be treatment-related. Abortions occurred at 2 and 4 mg/kg bw/day with a high incidence at 4 mg/kg bw/day. All the abortions were between GD 21 and 28 and in all cases after a reduction of food intake for several days and a body weight loss observed in dams. Additionally, nearly all of the high dose group females that aborted had pale and/or accentuated lobular pattern of the liver.
Body weight changes and food consumption at 2 and 4 mg/kg bw/day were significantly reduced for many intervals during the dosing period, when compared to control values. The mean number of late resorptions was slightly increased at 4 mg/kg bw/day. Moreover, increased post-implantation loss and fetal death was observed at 4 mg/kg bw/day without reaching statistical significance. All other litter parameters did not show any significant treatment-related effects. Fetal body weights were considered unaffected by treatment. No malformations at external, visceral and skeletal examination were considered to reflect any abnormal development linked to treatment except two foetuses at 4 mg/kg bw/day with a diaphragmatic hernia. Incomplete ossification was noted for a very few bones; it was limited to 2 and 4 mg/kg bw/day and did not reflect any general ossification delay.
Based on the results of the conducted study, a NOAEL of 0.5 mg/kg bw/day was derived for maternal systemic and maternal developmental toxicity. In regard to fetal toxicity, a NOAEL of 2 mg/kg bw/day was established taking into account the overall effects on fetuses (reduced number of live fetuses (non-significant), diaphragmatic hernia (low incidence with 2/80 fetuses) and incomplete ossification (slight severity)). Due to clear maternal systemic toxicity at this dose levels, the effects observed on maternal developmental and fetal parameters are considered as secondary, non-specific consequence of maternal toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May - 04 Oct 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no data reported on dam thyroid weight and histopathological assessment, dam thyroid hormones and fetal anogenital distance

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

current version adopted in 2018

Deviations:

yes

Remarks:

no investigation of dam thyroid weight and histopathological assessment, dam thyroid hormones, fetal anogenital distance

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

guideline in place during study conduct: Draft 1998 (for updating guideline of 1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F., Japan, No. 4200

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl: CD (SD) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, St Aubin-les-Elbeuf, France
- Weight at study initiation: 237 - 301 g
- Housing: Pregnant females were individually housed in suspended stainless steel wire mesh cages.
- Diet: U.A.R. certified Rodent meal A04C 10 (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered water, ad libitum
- Acclimation period: 13 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution of 0.5% methylcellulose 400 (w/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Aqueous suspensions were prepared three times during the course of the animal study for each concentration: 0.3, 1 and 3 g/L. Based on the animal's most recent body weight, and at a volume of 10 mL/kg doses of 3, 10 and 30 mg/kg bw/day were gained. The suspensions were mixed continuously before and during dosing with an electromagnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.3, 1.0 and 3.0 g/L

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration were checked according to the analytical protocol. Homogeneity was checked on the formulations at 0.3, 1 and 3 g/L. The sampling was done at three levels, the surface, middle and bottom. At each level, 3 samples were taken. One sample was analysed, the last two were kept frozen. For this study the Study Director's target range requirements were 90-110%. As a consequence, when the analysis of first samples were outside his target ranges (but acceptable according to laboratory target ranges), the last two samples were analysed.
Mean values obtained from the homogeneity checks were used as measured concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: spermatozoa in a vaginal smear or sperm plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6-20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

21 days

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the range of dosages based on the results obtained in a previous range finding study in the rat (company study no. M-192047-01-2; report no. SA 99036). In this previous study, the doses of the test substance were 7.5, 25, 50 and 150 mg/kg bw/day. The following doses: 25, 50 or 150 mg/kg bw/day induced dose-related reduction in body weight changes and food consumption at the beginning of the dosing period. Thus, a dose of 30 mg/kg bw/day was expected to cause maternal toxicity in the present definitive developmental toxicity study.
- Rationale for animal assignment (if not random): The females were assigned to control and treated groups using a body weight procedure for each day of pairing. Body weight means were checked after the mating period to ensure similar means among all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon); once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 6, 8, 10, 12, 14, 16, 18 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: full feeder weights were measured on GD 1, 6, 8, 10, 12, 14, 16 and 18; empty feeder weights were measured on GD 6, 8, 10, 12, 14, 16, 18 and 21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 21
- Organs examined: macroscopic examination of visceral organs; livers were sampled and weighed

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

All maternal and litter parameters were analyzed using a combination of parametric ANOVA and nonparametric techniques, followed by adjustment for multiple comparisons and multiple endpoints via bootstrap resampling methods (Westfall and Young, 1993). Fetal parameters were analyzed using Generalized Estimating Equations (GEE) linear and logistic regression models, which assumed exchangeable intralitter correlations and used a robust variance estimator for regression parameters (Zeger and Liang, 1986). The linear regression model was used for analyzing fetal body weight and fetal death status. The logistic model was used for analyzing fetal sex. Although fetal linear regression model, because the distribution of the variable was too sparse for computations required for logistic regression. The GEE methods are robust to misspecifications of the response variance as well as assumptions about the intralitter correlation structure, as long as robust variance estimators are used. Individual t-tests for pairwise comparisons to control were evaluated when the overall treatment effect was statistically significant (p<0.05 via a Wald chi-square test), and a Wald chi-square test for linear trend was also conducted regardless of the significance of the overall treatment effect.

Indices:

Percentage of pre-implantation loss: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] * 100

Percentage of post-implantation loss: [(no. of implantations - no. of live foetuses) / no. of implantations] * 100

Percentage of live fetuses: [no. of live fetuses / (no. of live fetuses + no. of dead fetuses)] * 100

Percentage of dead fetuses: [no. of dead fetuses / (no. of live fetuses + no. of dead fetuses)] * 100

Percentage of male fetuses: (no. of male fetuses / total no. of live fetuses) * 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the treated groups, the general conditions of females were similar to those of controls. A few observations concerned fur (hairloss, soiled), incisors (broken) and eyes (ocular discharge) but were unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No animals died during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, body weight loss (-0.6 g) was noted during GD 6-8, while the control group gained weight (6.0 g). Further, the mean body weights at 30 mg/kg bw/day were lower than controls from GD 8 to 21. At 30 mg/kg bw/day, the mean body weight change from GD 6 to 8 was significantly lower than the control value. In the 10 mg/kg bw/day group, the body weight gain from GD 6 to 8 was reduced (-43%) when compared to the control group, but not statistically significant. Nevertheless, from GD 8 to 21, no changes in body weight gain were recorded at 10 mg/kg bw/day when compared to control. At 3 mg/kg bw/day, maternal body weight changes were considered unaffected by treatment (please refer to Table 1).

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):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Maternal liver weight was significantly increased at 10 and 30 mg/kg bw/day when compared to control value.
Gravid uterine weight was comparable among the groups (106.1 ± 4.4 , 108.3 ± 2.3, 109.8 ± 2.6 and 106.0 ± 3.0 for control, 3, 10 and 30 mg/kg bw/day, respectively) (please refer to Table 1).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Accentuated lobular pattern of the liver lobes was observed in 5/25 pregnant females at 30 mg/kg bw/day. Other findings were not considered to be treatment-related as no apparent dose-related effect was noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy rate: There were no differences between treated and control groups. All pregnant females had live fetuses at cesarean section. In the control group and the 30 mg/kg bw/day group, the pregnancy rate was 100%. At 3 and 10 mg/kg bw/day, the pregnancy rate was 92% with 2/25 non-pregnant females per group (non-treatment-related, non-adverse).

At 30 mg/kg bw/day, maternal food consumption was significantly reduced during intervals GD 6-8, 8-10 and 10-12 when compared to controls (treatment-related, adverse). At 3 and 10 mg/kg bw/day, the food consumption was considered unaffected by treatment (please refer to Table 2).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no effects observed at this dose

Key result

Dose descriptor:

NOAEL

Remarks:

overall toxicity

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed up to 10 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

overall toxicity

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

other: significant increase in absolute liver weights and accentuated lobular pattern of the liver lobes, reduced body weight and body weight gain; reduced food consumption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

No malformed fetuses were noted at skeletal examination. A few anomalies were observed in different groups but no dose-related increase was noted except at 30 mg/kg bw/day for the short 13th ribs (with no, a short or a discontinuous costal cartilage) in 9/191 fetuses (control group: 4/189 foetuses). At 30 mg/kg bw/day, a slight ossification delay was noted for the 7th cervical centrum (13/191 fetuses; control group: 6/189 fetuses), 1st metatarsal (55/191 fetuses; control group: 11/189 fetuses) and for the dumbell thoracic centrum (18/191 fetuses; control group: 6/189 fetuses). In all cases, the associated cartilages were present and normal. At 3 and 10 mg/kg bw/day, the degree of ossification of bones was not considered to be affected by treatment (please refer to Table 5).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, enlarged thymus was noted in 16/176 fetuses. The number of fetuses with enlarged thymus in the 3 and 10 mg/kg bw/day groups were comparable to the control group.
Otherwise, variations were distributed among all groups with no dose relationship (please refer to Table 5).

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Fetal evaluation: In the control group, 25 litters were present with 369 live fetuses. At 3 and 10 mg/kg bw/day, 23 litters were present with 348 and 346 live fetuses, respectively. In the 30 mg/kg bw/day group, 25 litters were present with 367 live fetuses ((please refer to Table 4). External observations were performed on all live fetuses. Internal observations were performed on 180, 168, 169 and 176 live fetuses of the control, 3, 10 and 30 mg/kg bw/day group, respectively. Skeletal observations were performed on 189, 180, 177 and 191 live fetuses of the control, 3, 10 and 30 mg/kg bw/day group, respectively.

External observations: No abnormal external fetal observations were noted in any dose-group. The number of runt foetuses (bw < 4.0 g) was not increased in a dose-related manner (2/369 fetuses in the control group and 5/348, 1/346 and 1/367 fetuses at 3, 10 and 30 mg/kg bw/day, respectively).

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Remarks on result:

other: occurence in the presence of maternal overall toxicity

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

visceral/soft tissue: immune system

other: anomalies: ossification delay (7th cervical centrum, 1st metatarsal and dumbell thoracic centrum)

Description (incidence and severity):

enlarged thymus: 3.8, 1.2, 3.6 and 9.3% of affected fetuses in control, 3, 10 and 30 mg/kg bw/day, respectively; 20.8, 8.7, 17.4 and 44% on litter basis in control, 3, 10 and 30 mg/kg bw/day, respectively

13th rib shortened: 2.1, 0.6, 1.7 and 5.0% of affected fetuses in control, 3, 10 and 30 mg/kg bw/day, respectively; 12.0, 4.3, 8.7 and 24% on litter basis in control, 3, 10 and 30 mg/kg bw/day, respectively

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Maternal data

OBSERVATIONS	DOSE (mg/kg bw/day)
	Control	3	10	30
Number of mated females	25	25	25	25
Scheduled sacrifice (day 21) :
Number of pregnant females (with live fetuses)	25	23	23	25
Number of non pregnant females	0	2	2	0
Pregnancy rate (%) #	100	92	92	100
Body weight (g),^B	n = 25	n = 23	n = 23	n = 25
GD0	267.0 ± 2.6	267.6 ± 3.1	267.1 ± 2.7	267.7 ± 3.6
GD6	301.0 ± 3.2	299.4 ± 3.3	301.1 ± 3.7	300.6 ± 4.1
GD8	307.0 ± 3.5	305.0 ± 3.8	304.5 ± 4.5	300.0 ± 4.4
GD10	317.7 ± 3.5	315.1 ± 4.1	315.9 ± 4.3	309.6 ± 4.4
GD12	328.8 ± 3.8	324.2 ± 4.1	328.0 ± 4.4	320.6 ± 4.8
GD14	337.7 ± 3.9	334.0 ± 4.3	338.8 ± 4.9	329.3 ± 5.0
GD16	355.2 ± 4.5	353.5 ± 4.5	357.2 ± 5.3	347.2 ± 5.5
GD18	383.4 ± 5.0	382.7 ± 4.4	387.2 ± 5.7	377.9 ± 6.0
GD 21	438.8 ± 6.6	439.9 ± 5.1	443.7 ± 7.0	430.5 ± 7.3
Maternal Body Weight Change (g)	n = 25	n = 23	n = 23	n = 25
GD0 – GD6	34.1 ± 1.6	31.8 ± 1.6	34.0 ± 2.1	32.9 ± 1.1
GD6 – GD8	6.0^¶¶± 1.0^ƛƛ	5.6 ± 0.9	3.4 ± 1.7	-0.6^¤¤± 0.7^φ φ
GD8 – GD10	10.7 ± 0.6	10.1 ± 0.7	11.3 ± 1.3	9.7 ± 0.8
GD10 – GD14	20.0 ± 0.8	18.9 ± 0.9	23.0 ± 1.1	19.6 ± 1.1
GD14 – GD18	45.7 ± 2.0	48.7 ± 1.1	48.4 ± 1.6	48.6 ± 1.6
GD18 – GD21	55.4 ± 2.4	57.1 ± 1.5	56.5 ± 1.9	52.6 ± 1.9
Mean body weight change (corrected) (g)	65.8 ± 3.6	64.0 ± 3.6	66.8 ± 4.8	56.8 ± 3.2
Organ weights (g)
Gravid uterine weight	106.1 ± 4.4	108.3 ± 2.3	109.8 ± 3.6	106.0 ± 3.0
Liver weight	14.2^±±± 0.3^§§	14.8 ± 0.4	16.4***± 0.4^φ φ	18.8*** ± 0.4^φφφ

#: (Number of pregnant females/number of mated females) x 100.

B: Statistical analysis was not conducted on this endpoint

C: Weight change during gestation minus gravid uterus.

#: Bartlett's test for homogeneity of variances was significant (p<0.05) or could not be done because there was zero variance in one or more groups, therefore non-parametric statistical procedures were employed.

±: p<0.05; ANOVA Test for overall treatment effect

±±: p<0.001; ANOVA Test for overall treatment effect

§§: p<0.01; Linear Regression Test for linear trend

ƛƛ: p<0.001; Linear trend t-test, adjusted for multiple testing via bootstrap resampling methods (Westfall and Young, 1993). Adjustments computed separately for parameters related to maternal body weights and organ weights, food consumption, and litter outcomes.

**:p<0.001; Dunnett's Test for pairwise comparisons to control

φ φ: p<0.01; Individual t-test for pairwise comparison to control, adjusted for multiple testing via bootstrap resampling methods (Westfall and Young, 1993). Adjustments computed separately for parameters related to maternal body weights and organ weights, food consumption, and litter outcomes

φ φ φ: p<0.001; Individual t-test for pairwise comparison to control, adjusted for multiple testing via bootstrap resampling methods (Westfall and Young, 1993). Adjustments computed separately for parameters related to maternal body weights and organ weights, food consumption, and litter outcomes

¶¶: p<0.001; Kruskal-Wallis Test for overall treatment effect.

Ұ Ұ: p<0.001; Jonckheere's Test for trend

¤¤: p<0.001; Mann-Whitney U Test for pairwise comparisons to control

 

Table 2: Maternal food consumption                           

OBSERVATIONS	DOSE (mg/kg/day)
	Control	3	10	30
	25	25	25	25
Maternal food comsumption (g/day)
GD1 – GD6	27.2 ± 0.5	27.2 ± 0.7	27.8 ± 0.7	27.9 ± 0.6
GD6 – GD8	27.6^+± 0.8 §§	27.6 ± 0.8	26.9 ± 1.0	24.4* ± 0.6
GD8 – GD10	28.5^++± 0.8 §§§	28.2 ± 1.0	28.0 ± 0.7	24.9 ** + 0.8φ
GD10 – GD12	28.5^+± 0.7 §§	28.1 ± 0.7	28.5 ± 0.8	25.6*±0.7
GD12 – GD14 #	28.6 ± 0.5	28.4 ± 0.8	29.0 + 0.9	27.3 ± 0.6
GD14 – GD16	27.8 ± 0.7	28.3 ± 0.7	29.4 ± 0.8	27.1 ± 0.6
GD16 – GD18	30.5 ± 0.7	30.4 ± 0.9	31.2 ± 0.8	30.3 ± 0.7
GD18 – GD21	29.3 ± 0.5	29.7 ± 0.6	30.2 ± 0.9	29.2 ± 0.7

#: *Bartlett's test for homogeneity of variances was significant (p<0.05) or could not be done because there was zero variance in one or more groups, therefore non-parametric statistical procedures were employed

⁺: p<0.05; ANOVA Test for overall treatment effect

⁺⁺: p<0.01; ANOVA Test for overall treatment effect

*: p<0.05; Dunnett's Test for pairwise comparisons to control

**: p<0.01; Dunnett's Test for pairwise comparisons to control

Φ: Individual t-test for pairwise comparison to control, adjusted for multiple testing via bootstrap

Table 3. Litter response (Caesarean section data)

	Control	dose groups [mg/kg bw/day]
		3	10	30
No. of dams/litters examined	25	23	23	25
No. of Corpora Lutea per dam	16.9	17.2	17.0	17.0
	± 0.3	± 0.5	± 0.4	± 0.4
No. of Implantation Sites per litter	15.6	16.1	16.1	15.5
	± 0.7	± 0.3	± 0.4	± 0.4
Pre-implantation loss per litter [%]	7.6	5.4	4.9	8.4
	± 3.6	± 1.6	± 1.3	± 1.8
No. of early resorptions per litter	0.7	1	1	0.8
	± 0.2	± 0.3	± 0.2	± 0.2
No. of late resorptions per litter	0.1	0	0	0.1
	± 0.1			± 0.1
Post-implantation loss per litter [%]	6.6	6	6.5	5.2
	± 2.1	± 1.6	± 1.4	± 1
No. of live fetuses per litter	14.8	15.1	15	14.7
	± 0.7	± 0.3	± 0.4	± 0.4
No. of dead fetuses per litter	0	0	0	0
No. of female fetuses per litter	7.4	7.6	7.3	7.9
	± 0.5	± 0.5	± 0.4	± 0.5
No. of male fetuses per litter	7.4	7.5	7.7	6.8
	± 0.4	± 0.4	± 0.5	± 0.5

Table 4. Examination of the fetuses

	Control	dose groups [mg/kg bw/day]
		3	10	30
No. of fetuses	369	348	346	367
Male fetuses [%]	50.1	49.7	51.2	46.3
	± 2.2	± 2.8	± 2.6	± 3
Fetal body weight [g]	5.38	5.33	5.40	5.35
	± 0.05	± 0.06	± 0.05	± 0.06
Male fetal body weight [g]	5.52	5.48	5.47	5.51
	± 0.05	± 0.06	± 0.06	± 0.06
Female fetal body weight [g]	5.24	5.19	5.33	5.22
	± 0.05	± 0.06	± 0.04	± 0.07
No. of fetuses with malformations	0	1	5	2
External malformations	No malformations observed.
Internal malformations	No. of fetuses
Eosophagus: malpositioned (laterally); ventricular sytem defect in cranial region; all lung lobes and kidney (unilateral): misshapen; adrenal glands (bilateral): enlarged	0	1	0	0
Right ventricle: enlarged and wall partially thin with muscular defect; ventricular septum: thin; ascending aorta and aortic arch: dilated; aortic valves: absent	0	0	1	0
Skeletal malformations	No. of fetuses
Cervical rib:(unilateral) on 7th cervical vertebra: short; 3rd and 4th cervical centrum: cartilage bipartite and fused (bilateral)	0	0	0	1
1st and 2nd costal cartilages (unilateral): fused and/or 2nd costal cartilage(s) (unilateral/bilateral): not attached to the sternum and/or 1st and 2nd sternebrae: fused	0	0	4	1

Table 5: Observations in fetuses

 

	Control	3	10	30	Control	3	10	30
Skeletal anomalies
Number of fetuses examined	189	180	177	191
Number of litters examined					25	23	23	25
	Number of fetuses affected (mean% of fetuses affected)				Number of litters affected (mean% of litters affected)
7th cervical centrum: unossified / normal cartilage.	6 (3.2)	4 (2.2)	14 (7.9)	13 (6.8)	5 (20)	3 (13)	7 (30.3)	9 (36)
13 th rib (unilateral/bilateral) with no, a short or a discontinuous costal cartilage	4 (2.1)	1 (0.6)	3 (1.7)	9 (5.0)	3 (12.0)	1 (4.3)	2 8.7)	6 (24)
1^stmetatarsal (bilateral): unossified / normal cartilage	11 (5.6)	25 (14.0)	24 (13.8)	55 (29.1)	6 (24.0)	11 (47.8)	11 (47.8)	19 (76.0)
Visceral anomalies
Number of fetuses examined	180	168	169	176
Number of litters examined					24	23	23	25
	Number of fetuses affected (mean% of fetuses affected)				Number of litters affected (mean% of litters affected)
Thymus (unilateral/bilateral): enlarged	7 (3.8)	2 (1.2)	6 (3.6)	16 (9.1)	5 (20.8)	2 (8.7)	4 (17.4)	11 (44.0)

 

 Further information on maternal body weight, weight gain, food consumption and organ weights are included as attachment.

Conclusions:

No deaths occurred in the study. Adverse maternal effects were noted at 30 mg/kg bw/day through body weight loss from GD 6 to 8 and reduced food consumption from GD 6 to GD 12. The general conditions of females at 3 and 10 mg/kg bw/day were similar to those of controls. Macroscopic examination of females revealed increased incidence of accentuated lobular pattern at 30 mg/kg bw/day and increased mean liver weight at 10 and 30 mg/kg bw/day. Neither gestational parameters nor mean fetal body weights were affected by treatment. At 30 mg/kg bw/day, a few fetal findings showed increased incidences for enlarged thymus and slight ossification delay for a few bones.
In conclusion, the test substance had no specific effect on intrauterine development at doses up to 10 mg/kg bw/day. At the next higher dose, signs evident for maternal toxicity and few developmental anomalies were observed in fetuses which were considered as secondary non-specific consequences of maternal toxicity effects.


CLP: not classified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Species:

rabbit

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex IX-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity

 

Two studies are available addressing prenatal developmental toxicity of the test substance. The studies were conducted in rats and rabbits in accordance with OECD 414 under GLP conditions. For dose selection, dose range finding studies were performed in both species.

Rats

In a preliminary study, 8 pregnant Sprague-Dawley rats per group received the test substance at dose levels of 7.5, 25, 50 or 150 mg/kg bw/day by gavage from gestation days (GD) 6 to 20 (M-192047-01-2, 1999). No treatment-related deaths occurred during the study period and there were no adverse effects on gestational parameters. The dose of 150 mg/kg bw/day led to adverse clinical signs including curling, clonic convulsions and reduced motor activity. Further, decreased body weight gain and food consumption was observed. At 50 and 150 mg/kg bw/day the corrected body weight change was lower than the control values, reaching statistically significance at 150 mg/kg bw/day. At the same dose, food consumption was reduced during the dosing period, showing statistical differences during each interval except from GD 16 to 18. At 25 and 50 mg/kg bw/day, the mean food consumption was significantly lower than the control values from GD 6 to 8 and GD 8 to 10 and from GD 12 to 14 at 50 mg/kg bw/day only. At dose levels ≥ 25 mg/kg bw/day, a significant increase in absolute liver weights associated with accentuated lobular pattern and/or abnormal colour were observed.

The mean number of corpora lutea, uterine implantation sites and the mean pre-implantation loss percentage in the treated groups did not show any significant differences from the control group. There were no adverse effects on gestational parameters including number of early and late resorptions, dead and live fetuses and post-implantation loss percentages.

In regard to fetal toxicity, a decreased mean fetal body weight was observed at 150 mg/kg bw/day . Further, the number of runt fetuses (10/44 foetuses in three litters) was increased. Based on the outcome of this study, dose levels of 3 - 30 mg/kg bw/day were selected for the main study.

 

In the main study, 25 female Sprague-Dawley rats per group were mated on a one-to-one basis with stock males of the same strain. As a result, 23 to 25 pregnant Sprague-Dawley rats per group were exposed to the test substance at dose levels of 3, 10 or 30 mg/kg bw/day by gavage from GD 6 to 20 (M-196111-01-2, 2000). No deaths occurred during the study period. The pregnancy rate was 92 to 100% and there was no difference between the treated and control groups. Clinical signs included hairloss and soiled fur, broken incisors and ocular discharge, and were found to be not treatment-related. Starting at 10 mg/kg bw/day, reduced body weight gain and food consumption was observed in dams reaching statistical significance at 30 mg/kg bw/day. Briefly, at 30 mg/kg bw/day, body weight loss (-0.6 g) was noted during GD 6-8, while the control group gained weight (6.0 g). Further, the mean body weights at 30 mg/kg bw/day were lower than controls from GD 8 to 21. At 30 mg/kg bw/day, the mean body weight change from GD 6 to 8 was significantly lower than the control value. In the 10 mg/kg bw/day group, the body weight gain from GD 6 to 8 was reduced (-43%) when compared to the control group, but not statistically significant. Nevertheless, from GD 8 to 21, no changes in body weight gain were recorded at 10 mg/kg bw/day when compared to control. At 3 mg/kg bw/day, maternal body weight changes were considered unaffected by treatment. Correlated to the reduced body weight gain, maternal food consumption was significantly reduced during intervals GD 6-8, 8-10 and 10-12 when compared to controls at 30 mg/kg bw/day. At 3 and 10 mg/kg bw/day, the food consumption was considered unaffected by treatment. Increased mean liver weights were noted in dams in the 10 and 30 mg/kg bw/day groups. Necropsy findings revealed an increased incidence of accentuated lobular pattern at 30 mg/kg bw/day.

In regard to maternal developmental toxicity, no alterations in the elevated gestational parameters including abortions, pre- and post-implantation losses and resorptions were observed. 

Fetal body weights were unaffected by treatment and the number of live offspring, sex ratio and litter size and weights were comparable among the groups. No malformed fetuses were noted at skeletal examination. A few anomalies were observed in different groups but no dose-related increase was noted except at 30 mg/kg bw/day for the short 13th ribs (with no, a short or a discontinuous costal cartilage) which was detected in 4/189 and 9/191 fetuses in the control and high dose group, respectively (2.1, 0.6, 1.7 and 5.0% of affected fetuses in control, 3, 10 and 30 mg/kg bw/day, respectively; 12.0, 4.3, 8.7 and 24% on litter basis in control, 3, 10 and 30 mg/kg bw/day, respectively). Further, at 30 mg/kg bw/day, a slight ossification delay was noted for the 7th cervical centrum (6/189 and 13/191 fetuses in the control and high dose group, respectively), 1st metatarsal (11/189 and 55/191 fetuses in the control and high dose group, respectively) and for the dumbell thoracic centrum (6/189 and 18/191 fetuses in the control and high dose group, respectively). In all cases, the associated cartilages were present and normal. At 3 and 10 mg/kg bw/day, the degree of ossification of bones was not considered to be affected by treatment. At visceral examination, enlarged thymus was noted in 16/176 fetuses at 30 mg/kg bw/day compared to 7/180 fetuses in the control group (3.8, 1.2, 3.6 and 9.3% of affected fetuses in control, 3, 10 and 30 mg/kg bw/day, respectively; 20.8, 8.7, 17.4 and 44% on litter basis in control, 3, 10 and 30 mg/kg bw/day, respectively). The number of fetuses with enlarged thymus in the 3 and 10 mg/kg bw/day groups were comparable to the control group.

Under the conditions of this study, a NOAEL of 10 mg/kg bw/day was defined for systemic toxicity in maternal animals based on the effects observed at 30 mg/kg bw/day on body weight gain, food consumption and the liver. It should be noted that the liver was also identified as target organ following repeated exposures. No maternal abnormalities pointing to maternal developmental toxicity were observed. In regard to fetal toxicity, no adverse effects have been observed up to 10 mg/kg bw/day. At 30 mg/kg bw/day, visceral and skeletal anomalies were observed leading to a NOAEL of 10 mg/kg bw/day for fetal developmental toxicity. Due to clear maternal systemic toxicity at this dose level, the effects observed on fetal parameters are considered as secondary, non-specific consequence of maternal toxicity.

 

Rabbit

In a preliminary range-finding study, 4 to 6 pregnant New Zealand White rabbits were treated with 0.25, 0.75, 2 or 5 mg/kg bw/day of the test substance from GD 6 to 28 by gavage (M-192052-01-2, 1999). There were no treatment-related deaths during the study period. In the 2 and 5 mg/kg bw/day group, body weight loss and reduced food intake were observed in the dams reaching statistical significance at 5 mg/kg bw/day. At this dose, all females had few feces during GD9 – 21. Otherwise, no adverse clinical signs were found in any dose group. Abortion was observed in one female of the 5 mg/kg bw/day group on GD 26 and in one female of the 0.75 mg/kg bw/day group on GD 20. Analysis of litter and fetal parameters revealed no significant treatment effects. At external foetal examination, no treatment-related findings were observed. Based on the outcome of this study, dose levels of 0.25 - 4 mg/kg bw/day were selected for the main study.

In the main study, 30 pregnant New Zealand White rabbits per group were treated from GD 6 to 28 by gavage with doses of 0.25, 0.5, 2 and 4 mg/kg bw/day of test substance (M-192124-02-2, 2000). The pregnancy was a result of artificial insemination with semen from bucks of the same strain and the same supplier. During the study period, no treatment-related deaths were observed. The pregnancy rate was between 80 and 97% across all groups. At 2 and 4 mg/kg bw/day, body weight changes and food consumption were found to be statistically significantly reduced for many intervals during the dosing period compared to the controls. There were no treatment-related clinical signs during the study period and no adverse findings at necropsy in any dose group. In the 4 mg/kg bw/day group, 11/28 pregnant females aborted between GD 21 and 27. Nearly all of these females (10/11) had pale and/or accentuated lobular patterns of the liver. In the 2 mg/kg bw/day group, 2/24 pregnant females aborted on GD 25 and 28. All females aborted after reduced food consumption and body weight loss, indicating maternal toxicity as cause of the abortions.

In the 4 mg/kg bw/day group, the mean number of late resorptions was statistically significantly increased for females with live fetuses. Moreover, increased post-implantation loss and fetal death were observed at 4 mg/kg bw/day without reaching statistical significance. All other litter parameters did not show any significant treatment-related effects. Diaphragmatic hernia was observed in two out of 80 fetuses in two litter, and incomplete ossification of pubis, metacarpal and/or phalanges were reported at 4 mg/kg bw/day. All other litter parameters including fetal body weights were not affected by treatment.

Based on the results of the conducted study, a NOAEL of 0.5 mg/kg bw/day was derived for maternal systemic and maternal developmental toxicity. In regard to fetal toxicity, a NOAEL of 2 mg/kg bw/day was established taking into account the overall effects on fetuses (reduced number of live fetuses (non-significant), diaphragmatic hernia (low incidence with 2/80 fetuses at 4 mg/kg bw/day, no similar finding in the control or other dose groups) and incomplete ossification (slight severity)). Due to clear maternal systemic toxicity at this dose levels, the effects observed on maternal developmental and fetal parameters are considered as secondary, non-specific consequence of maternal toxicity.

Mode of Action Analysis / Human Relevance Framework

No data available.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification on reproductive and developmental toxicity according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.