Acetic acid, 2-[(5-chloro-8-quinolinyl)oxy]-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August to 23 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Cas Number:

88349-88-6

Molecular formula:

C11H8ClNO3

IUPAC Name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Test material form:

solid: particulate/powder

Remarks:

powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): X204558
- Molecular formula: C11H8ClNO3
- Molecular weight: 237.6
- Analytical purity: 98.3% +/- 0.03% wt/wt by liquid chromatography with identification by liquid chromatography-mass spectrometry (LCMS), gas chromatography mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR)y
- Lot/batch No.: Lot 2GHB0002, TSN303795

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

As recommended by the guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Males: on TD1 group means ranged from 299.3 to 301.5 g, Females: on TD1 group means ranged from 208.0 to 212.8 g
- Fasting period before study: None
- Housing: After assignment to study, animals were housed singly in solid bottom stainless steel cages, except during breeding and during the gestation and littering phases of the study. The solid bottom cages contained ground corn cob nesting material with some heat treated laboratory grade wood shavings for enrichment purposes. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above catch pans in order to better visualize copulation and plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material with some heat-treated laboratory grade wood shavings from approximately GD 0 until completion of lactation
- Diet: ad libitum LabDiet Certified Rodent Diet #5002
- Water: ad libitum municipal water
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark

IN-LIFE DATES: From: 16 August 2013 To: 23 September 2013

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The premix and diet were mixed throughout the study based on the 65 day stability (the test item was found to be stable in the feed at concentrations ranging from 0.0005 to 10% for 65 days in a previous study)
- Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. In addition, a Quadro Co-mil was used during preparation of the premixes to facilitate homogeneous distribution of the test material. Diets were prepared as a fixed percent of test material in rodent feed. The test material concentration was not adjusted for purity.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy occured or 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal lavage referred to as day 0 of pregnancy (gestational day 0 - GD0)
- If mating did not occir after 2 weeks the animals were separated without further opportunity for mating
- After successful mating each pregnant female was caged: singly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of all test diets from the first mix of the study was initiated prior to the start of dosing. The method used for analyzing the test materials in the diet was solvent extraction followed by analysis using LCMS. Representative samples from the test diets were evaluated concurrently with the concentration verification analyses to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed at least once during the study. Analyses of all test diets from the first mix of the study revealed mean test material concentrations ranging from 90.9 to 96.3% of targeted concentrations. Analyses of the low and high-dose diets indicated that the test material was homogeneously distributed based on relative standard deviations (RSD) of ≤ 6.4%.

Duration of treatment / exposure:

Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for at least 41 days.
The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days)

Frequency of treatment:

Daily in the diet

Details on study schedule:

- Due to previously-identified decreased palatability, four days were added to the beginning of the study where the high-dose diets were intentionally fed at a lower concentration of 700 ppm. During these test days, the 105 and 700 ppm dose groups were fed their target dose concentration. Females were dosed daily for at least two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through to postpartum day 4. Females were necropsied on post-partum days 5-7. The males were dosed for at least two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test days 42-43).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

plain diet control

Doses / concentrations 2

Dose / conc.:

105 ppm

Doses / concentrations 3

Dose / conc.:

700 ppm

Doses / concentrations 4

Dose / conc.:

2 100 ppm

No. of animals per sex per dose:

10 male and 10 female rats per group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dose of 2100 ppm X204558, representing a molecular weight equivalent dose of 3000 ppm X204559, was selected based upon previously demonstrated body weight loss and excessive decreases in feed consumption at a concentration of 3000 ppm X204558 and above. The mid- and low-doses of X204558 represent molecular weight equivalent doses of 1000 and 150 ppm X204559 as evaluated in the 90-day study. The high dose of X204558 and X204559 were expected to produce decreased feed consumption, some body weight depression and possible renal effects. The mid- and low-dose levels of X204558 were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low dose was expected to be a NOEL. In order to attenuate any potential palatability effects, as well as maintain consistency in dosing between these two groups, the high-dose levels of 700 on test days 1-3 was increased to 2100 ppm on day 4.
- Rationale for animal assignment: animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study

Positive control:

None - not required

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity.
In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted on all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plugpositive) females received clinical examinations on GD 0, 7, 14 and 20. Females were observed for signs of parturition beginning on or about GD 20 (see litter data). Females that delivered litters were subsequently evaluated on LD 0, 1, and 4.
Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and daily during the first week of the study. Male body weights were continued to be recorded weekly throughout the study. Females were weighed weekly during the remainder of the pre-mating phase of the study. During gestation, females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were weighed on LD 1 and 4.
Body weight gains were determined during the pre-mating phase and for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
Feed consumption was determined by weighing feed containers at the start and end of a measurement. Feed consumption was determined pre-exposure and daily during the first week of the study. Thereafter, feed crocks were measured weekly during the pre-mating phase. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption for males was not determined. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. For females delivering litters, feed consumption was measured on LD 1 and 4.
- Compound intake calculated was calculated using feed concentrations, body weights and feed consumption data: Yes

OTHER:
Kinetic Analysis of Blood – Pre-mating
In order to determine diurnal variations in systemic dose at steady state, blood samples from the first 4 surviving non-fasted rats/sex/dose via tail nick were collected at three time-points, 6:30 AM, 1:00 PM and 5:00 PM (approximately ± 30 minutes), near the end of the pre-breeding phase on TD 14.
Kinetic Analysis of Blood and Milk – Dams
Dams whose pups were used for blood collection were separated from their litters on LD 4 at approximately 12:30 PM for ~ 2 hours before milk collection began. During the separation time from the dam the individual litters were placed in tubs on warming blankets. At approximately 1:00 PM (approximately ± 30 minutes) blood was collected via jugular vein (~ 200 μL) from all dams selected for toxicokinetic sampling.
At approximately 3:00 PM (approximately ± 30 minutes) milk collection began by administration of two intraperitoneal doses of 1-2 IU oxytocin approximately 5-10 minutes apart to each of the dams. The dams were then placed under isoflurane anesthesia, and ~200 µL of milk was collected per animal.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

Females were observed for signs of parturition beginning on or about GD 20. Parturition was observed for signs of difficulty or unusual duration. The day of
parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery.

PARAMETERS EXAMINED
The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period.

GROSS EXAMINATION OF DEAD PUPS:
Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects. These pups were preserved in neutral, phosphate buffered 10% formalin.

Kinetic Analysis of Blood – F1 Pups
Systemic exposure of X204558 and X204559 to nursing pups was determined by collecting terminal blood samples from PND 4 pups. At approximately 12:30 P.M. pups were separated from the dams and then placed in tubs on warming blankets until blood was collected at approximately 1:00 PM (approximately ± 30 minutes). The blood samples were collected from two randomly selected culled pups/sex/litter from the first four available litters/dose on LD 4, and pooled by sex. If needed, samples may have been pooled from additional excess litter mates of the same sex to yield a sufficient blood volume. The pups were anesthetized using isoflurane and the blood collected by cardiac puncture. While under deep isoflurane anesthesia following blood collection, pups were euthanized by an intraperitoneal administration of sodium pentobarbital solution. Following euthanasia, the pups were examined for gross external and visceral alterations and preserved in phosphate-buffered 10% formalin.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males (fasted) were submitted for necropsy after at least four weeks of exposure.
- Maternal animals: Adult females (fasted) were terminated on LD 5-7, or at least 24 days after the end of the mating period for females not producing a litter.

GROSS NECROPSY
- Gross necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, reexamined and selected tissues were incised.

HISTOPATHOLOGY / ORGAN WEIGHTS
The uteri of all females were stained with an aqueous solution of 10% ammonium sulfide stain for approximately one minute and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin. Weights of the epididymides, kidneys, liver and testes were recorded, and organ:body weight ratios calculated.
Representative samples of the following tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides which were fixed in Bouin’s fixative: cervix, liver, prostate, coagulating glands, mammary gland, seminal vesicles, epididymides, ovaries, testes, gross lesions, oviducts, uterus, kidneys, pituitary and vagina.

Histologic examination of the tissues that were collected and preserved in neutral, phosphate-buffered 10% formalin was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (liver tissue from females) and relevant gross lesions.

The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. Microscopic evaluation included a
qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects.

Postmortem examinations (offspring):

All pups surviving to LD 4 that were not used for blood collection were euthanized by an intraperitoneal administration of sodium pentobarbital solution, examined for gross external and visceral alterations. The pups were saved in phosphate-buffered 10% formalin if alterations were noted during the examination. The pups with no alterations noted during the examination were discarded. Any pups found dead or which are euthanized in moribund condition were examined to the extent possible and preserved in phosphate-buffered 10% formalin.

Statistics:

Least squares regression analysis was performed on dependent variables plasma AUC24h and milk concentration. The independent variables were dose and quadratic term (dose squared). Parental body weights, gestation, lactation body weight gains, litter mean body weights, feed consumption, organ weights (absolute and relative) were evaluated by Bartlett's equality of variances. Based upon the outcome either a parametric or non-parametric analysis of variance (ANOVA) was performed. If significant; Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If significant; Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Outlierswere identified by the sequential method of Grubbs and only excluded from analysis for documented, scientifically sound reasons. Mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test with Bonferroni's correction. Evaluation of neonatal sex ratio on PND1 was performed by binomial distribution. Gender was determined for pups found dead on PND0 and these data were included in sex ratio calculations. Survival indices, post-implantation loss, and other incidence data among neonates were analyzed using litter as the experimental unit by the censored Wilcoxon test as modified by Haseman and Hoel with Bonferroni’s correction. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control to keep the error rate at 0.05.

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100

Offspring viability indices:

Offspring viability indices were calculated for all dose level groups as follows:
Gestation index = (No. females delivering a viable litter/No. females delivering a litter) x 100
Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Adult females (2100 ppm) had periportal hepatocytes vacuolization consistent with fatty change considered non-adverse due to minimal nature and absence of any other degenerative, inflammatory or necrotic liver changes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetics:
Pre-Breeding: Plasma AUC24hr values in treated adult males and females were generally dose proportional through the high dose, though there appeared to be a slight trend toward supralinearity. No X204559 was detected in any of the blood samples from the X204559-treated animals. However, X204558 was present at quantifiable levels in all samples from all animals, except controls. Estimated systemic exposures (plasma AUC24h) at TD 14, for the high dose X204558-treated animals, were approximately double those of the X204559-treated animals.
Dams and Milk: Blood levels in the dams on LD 4 were generally dose proportional through the high dose. No X204559 was detected in any of the blood or plasma samples on LD 4. However, X204558 was present at quantifiable levels in all samples from all treated animals. LD 4 plasma concentrations for the high dose X204558-treated dams were slightly lower than those of the X204559-treated dams. However, milk concentrations within the high dose X204558-treated group were slightly higher than those of the X204559-treated group, suggesting overall exposure to X204558 was very similar between the two groups. This was slightly different from the trends observed on TD 14. Differences in plasma X204558 levels may have been due to higher body fat content and higher plasma lipid levels associated with lactation for the LD 4 animals. Since X204559 is highly lipophilic, bioavailability may have been increased in the LD 4 animals due to improved solubility in blood. No X204559 was detected in any of the milk samples. No significant deviation from linearity was identified in the milk concentrations of X204558, though there was a clear trend toward supralinearity at the highest dose.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until scheduled termination. No treatment-related effects on behavior or demeanor were observed in male or female rats at any dose level during any phase of the study. There were no notable observations made during the cage-side observations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the premating, gestation, and lactation phases of the study there were no treatment-related effects on male or female body weight or body weight gain at all dose levels tested. There were no treatment-related effects on gestation or lactation body weight or body weight gain at any dose level tested. During the premating, gestation, and lactation phases of the study there were no treatment-related effects on feed consumption at all dose levels tested.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Time-weighted average doses were 0, 7.38, 48.2, or 123 mg/kg/day for males, and 0, 7.42, 48.9, or 125 mg/kg/day for females during pre-breeding. During the gestation and lactation phases, respective time-weighted average doses were 0, 6.98, 49.1, or 143 mg/kg/day and 0, 11.1, 75.5, or 227 mg/kg/day were obtained for females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on mating, conception, fertility, time to mating or gestation length, percent post-implantation loss or sex ratio.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no statistically significant differences or treatment-related changes in the terminal body weights or any of the organ weights for males and females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment related gross pathological observations. All gross pathological observations were interpreted as spontaneous alterations, unassociated with exposure.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related changes were confined to the liver of high-dose females. Adult females (5 of 10) given 2100 ppm had very slight periportal hepatocytes vacuolization consistent with fatty change. This was only a very minimal change characterized by the presence of very fine round cytoplasmic vacuoles consistent with fine lipid droplets within the periportal hepatocytes, particularly in the right medial lobe of the liver. In some cases, slightly larger round cytoplasmic lipid vacuoles were also noted scattered within individual periportal and midzonal hepatocytes. Due to the minimal nature of this change and absence of any other degenerative, inflammatory or necrotic changes in the liver, it was interpreted to be a non-adverse change. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

2 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No adverse effects seen at any dose level.

2

Key result

Dose descriptor:

NOEL

Remarks:

Reproductive effects

Effect level:

2 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No adverse effects seen at any dose level.

Remarks on result:

not measured/tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetics: As in the adult animals, no X204559 was present at quantifiable levels in pup blood or plasma. Plasma levels of X204558 were greater than dose proportional at the 2100 ppm dose group in both male and female pups. Blood levels of X204558 in pups were 25 ± 6%, on average, of those of the dams in the low dose X204558 group, increasing to 51 ± 15% in the high dose X204558 group. The relative milk concentrations also increased with increasing dose but not enough to account for the dose-dependent increase in pup blood concentrations. A likely explanation for this phenomenon is that saturation of metabolism and/or elimination may occur in pups from X204558 treated dams. Pup plasma levels of X204558 from X204558-administered dams were roughly 2- fold higher than pup plasma X204558 levels from X204559-administered dams at the equimolar concentration. Pup plasma levels of X204558 were 19 ± 5% of dam plasma levels in the X204559 treatment group. The reason for lower relative levels of X204558 in pups from X204559-treated dams is unknown. Milk concentrations are also slightly lower in X204559-treated dams, but not enough to account for the difference.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no treatment-related effects on litter size or sex ratio at any dose level tested. There were no treatment-related effects on pup body weights at any dose level tested.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No adverse effects seen at any dose level.

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Reproduction Indices and Pup Survival

	Dose ppm
	0	105	700	2100
No. males	10	10	10	10
No. females	10	10	10	10
Male mating index %^A	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Female mating index %^B	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Male conception index %^C	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Female conception index %^D	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Male fertility index %^E	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Female fertility index %^F	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Gestation index %^G	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10)
Gestation survival index %^H	100.0 (144/144)	100.0 (154/154)	100.0 (135/135)	99.3 (147/148)
Day 1 survival index %^I	96.5 (139/144)	100.0 (154/154)	99.3 (134/135)	99.3 (146/147)
Day 4 survival index %^I	95.8 (138/144)	100.0 (154/154)	99.3 (134/135)	99.3 (146/147)
Sex ratio on Day 1 Male:Female	52:48	47:53	51:49	53:47
Time to mating (days)	2.4 ± 1.1	3.0 ± 1.0	2.7 ± 1.1	3.0 ± 0.9
Gestation length (days)	21.6 ± 0.5	21.8 ± 0.4	21.5 ± 0.5	21.5 ± 0.5
% Postimplantation loss^K	5.07 ± 6.35	2.05 ± 4.73	2.82 ± 3.65	2.19 ± 3.58

^A# MALES WITH EVIDENCE OF MATING/TOTAL # MALES COHOUSED WITH FEMALES X 100%.

^B# FEMALES WITH EVIDENCE OF MATING/# FEMALES COHOUSED WITH MALES X 100%.

^C# MALES WHICH SIRED A LITTER/# MALES MATED X 100%.

^D# FEMALES WITH EVIDENCE OF PREGNANCY/# FEMALES MATED X 100%.

^E# MALES WHICH SIRED A LITTER/# MALES COHOUSED WITH FEMALES X 100%.

^F# FEMALES WITH EVIDENCE OF PREGNANCY/# FEMALES COHOUSED WITH MALES X 100%.

^G# FEMALES WHICH DELIVERED A LIVE LITTER/# FEMALES WITH EVIDENCE OF PREGNANCY X 100%.

^HPERCENTAGE OF NEWBORN PUPS THAT WERE ALIVE AT BIRTH.

^I[# OF LIVE PUPS ON DAY 1 OR 4/# OF LIVE PUPS ON DAY 0] X 100.

^KMEAN PERCENT/LITTER (CALCULATED AS [(NO. IMPLANTS – NO. LIVE BORN)/NO. IMPLANTS] X 100.

THERE WERE NO STATISTICAL DIFFERENCES FROM CONTROL AT ALPHA=0.05.

Litter size summary (n = 10)

Dose ppm	Litter size (n = 10), mean ± SD
	Born live	Born dead	Lactation day 1	Lactation day 4
0	14.4 ± 2.2	0.0 ± 0.0	13.9 ± 2.0	13.9 ± 2.0
105	15.4 ± 1.6	0.0 ± 0.0	15.4 ± 1.6	15.4 ± 1.6
700	13.5 ± 1.9	0.0 ± 0.0	13.4 ± 1.8	13.4 ± 1.8
2100	14.7 ± 1.9	0.1 ± 0.3	14.6 ± 1.9	14.6 ± 1.9

THERE WERE NO STATISTICAL DIFFERENCES FROM CONTROL AT ALPHA=0.05.

Applicant's summary and conclusion

Conclusions:

The NOAEL for general toxicity and the NOEL for reproductive effects was the highest concentration tested, 2100 ppm (equivalent to 123 mg/kg/day for males and 125 mg/kg/day for females during pre-breeding and 143 mg/kg/day for females during the gestation and 227 mg/kg/day for females during lactation).

Executive summary:

A study was conducted to evaluate the potential effects of X204558 (cloquintocet acid) on reproductive function, prenatal/neonatal survival and growth of the offspring (according to OECD 421).

Groups of 10 male and 10 female Crl:CD(SD) rats were fed diets supplying 0, 105, 700, or 2100 ppm X204558. Time-weighted average doses were 0, 7.38, 48.2, or 123 mg/kg bw/day for males, and 0, 6.98-11.1, 48.9-75.5, or 125-227 mg/kg bw/day for females given 0, 105, 700, or 2100 ppm during the prebreeding, gestation and lactation phases of the study. Due to previously-identified decreased palatability, four days were added to the beginning of the study where the high-dose diets were intentionally fed at lower concentrations of 700 ppm. During these test days, the 105 and 700 ppm dose groups were fed their target dose concentration. An additional treatment group was fed diet containing 3000 ppm cloquintocet mexyl (X204559), to allow for a comparison of systemic exposure and any observed effects in animals administered an equimolar concentration of X204558; cloquintocet mexyl is hydrolysed to cloquintocet acid. As cloquintocet mexyl will be the subject of a separate submission, the results are not reported in detail here.

Females were dosed daily for at least two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through to postpartum day 4. Females were necropsied on post-partum days 5-7. The males were dosed for at least two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test days 42-43). Effects on reproductive function as well as general toxicity were evaluated. In addition, post-mortem examinations included a complete necropsy of the adults with collection of organ weights and histopathologic examination of tissues with emphasis on organs of the reproductive system. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. Levels of X204558 and X204559 were measured in whole blood or plasma in males and females on test day 14, in adult female whole blood or plasma and milk on lactation day 4, and in pup whole blood or plasma on post-natal day 4 to determine systemic exposure.

During the ramp-up phase of the study, decreased palatability of the test material resulted in decreases in body weight and/or body weight gain in males and females in the 2100 and 700 ppm groups. Attributed to the decreased palatability of the test diets, there were treatment-related decreases in feed consumption of male and female rats given 700 or 2100 ppm. These decreases occurred initially with test diet administration and were most prominent on test days 1-2. Following the ramp-up phase, feed consumption, returned to normal levels for the remainder of the study. During the premating, gestation, and lactation phases of the study, there were no treatment-related effects in clinical observations, body weight and body weight gain, feed consumption, reproductive function, prenatal/early neonatal growth and survival of the offspring, organ weights, or gross pathology in either sex at all dose levels tested. Females given 2100 ppm had very slight periportal hepatocyte vacuolization consistent with fatty change. Due to the minimal nature of this change and absence of any other degenerative, inflammatory or necrotic changes in the liver, it was interpreted to be a non-adverse effect.

Overall exposure to X204558 in adult rats was very similar between the X204558- and X204559-treated groups. X204558 was shown to partition into the milk; therefore, pups were exposed to X204558 via the milk. The resulting pup blood levels of X204558 were approximately 51% of the dam blood levels after treatment with 2100 ppm X204558. Blood levels of pups from dams treated with X204558 were roughly 2- fold higher than those observed in pups of dams treated with the equimolar dose of X204559 (19% of dam blood levels). The data also indicated X204558 exhibits nonlinear kinetics in offspring of dams exposed to 2100 ppm X204558.

Based on these results, the NOAEL for general toxicity was 2100 ppm. The NOEL for reproductive effects was 2100 ppm, the highest concentrations tested. The overall toxicity profile was very similar between the X204558- and X204559-treated groups.