Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest concentration

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest concentration

Key result

Reproductive effects observed:

no

Conclusions:

Rat NOEL (Reproductive parameters): 1.0 mg/kg/day

Executive summary:

Male and female Sprague-Dawley rats ingested 0, 0.01, 0.065 or 1.0 mg/kg/day of the test substance for 8 weeks. Male and female rats ingesting the same concentration of the test substance on a mg/kg basis were mated twice to produce the f1a and f1b litters. Selected f1b rats ingested the same concentration of test material as their parents for 11 weeks at which point they were mated twice to produce the f2a and f2b litters.

Ingestion of 1.0 mg/kg/day of the test substance did result in a slight decrease in flat f1a, f1b and f2a weanling body weights and f1 adult body weights. However, concentrations as high as 1.0 mg/kg/day did not affect the ability of the parental animals to mate, produce a litter of normal size and number and raise a litter. Thus the no-observable-effect level (NOEL) for reproductive parameters in Sprague-Dawley rats is 1.0 mg/kg/day for the test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female Sprague-Dawley rats ingested 0, 0.01. 0.065 or 1.0 mg/kg/day of the test substance for 8 weeks. Male and female rats ingesting the same concentration of the test substance on a mg/kg basis were mated twice to produce the f1a and f1b litters. Selected f1b rats ingested the same concentration of test material as their parents for 11 weeks at which point they were mated twice to produce the f2a and f2b litters.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyphenoxy]propanoic acid

Cas Number:

95977-27-8

Molecular formula:

C15H11ClF3NO4

IUPAC Name:

(2S)-2-[4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyphenoxy]propanoic acid

Specific details on test material used for the study:

DOWCO 453
Lot # AGR 187381
Purity: 99.4%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This strain of rat was selected in order to assess the reproductive effects of the test substance in an outbred strain which has an extensive data base in the literature.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NewYork
- Age at study initiation: 6 wks
- Housing: Housed in wire-mesh bottomed, stainless steel cages
- Diet: Purina's Certified Rodent Chow #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature: Approximately 22°C
- Humidity: 40-60%
- Photoperiod: 12hrs dark /12 hrs light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- A 0.1% premix of the test substance in Purina Laboratory Chow was shown to be stable for up to 1 year. The premix diets were prepared bi-weekly throughout the study. Test diets were prepared from the working premix to provide the selected dose levels of the test substance on a mg/kg/day basis. The concentration of the test substance in the diets was adjusted weekly during the pre-mating period based on group mean body weight and food consumption to maintain the appropriate dose levels.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: two 7-day cohabitation periods
- After the first 7-day mating, the males were rotated and allowed to mate with a different female from the respective treatment group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of control and test diets were analyzed 4 times per generation to determine the concentration of the test substance in the diet. Reference diet samples (working premix and one/sex/dose level) were retained weekly. weekly samples of Certified ground chow used to prepare the diet mixes were retained also.

Duration of treatment / exposure:

Test animals were maintained on diets containing sufficient test substance to provide dose levels of 0, 0.01, 0.065 and 1.0 mg/kg/day during the first 8 weeks and 11 weeks of the treatment period of f0 and f1 generations, respectively. During mating, gestation, and the first week of lactation, females from each treatment group were provided with the appropriate diet containing the respective concentration of the test substance given during the week immediately prior to mating. During the second and third weeks of lactation, females received 1/2 and 1/3, respectively, of the dietary concentration given during breeding and gestation in order to avoid overdosing of the neonates. To avoid potential overdosing of females during the mating period, males received the same concentration in the diet as their female cage mate. Following completion of the breeding period, males from each treatment group continued to receive the appropriate fixed concentration of the test substance equivalent to the dietary concentration administered to males during the week prior to mating. Until all f1 litters had been weaned, weanlings chosen for the f1 generation received the same concentration of the test substance in the diet as was given to the f0 rats during their first week of treatment.

Details on study schedule:

After 8 weeks of administration of the test material, f0 animals were mated using one male to one female of the respective treatment group to produce the f1a litters. After the last f1a litter was weaned, the f0 animals were allowed to mate a second time to produce the f1b litters. At the time of weaning, thirty animals/sex/dose level were randomly chosen from the f1b litters as parents for the next generation and placed on the respective test diets. After 11 weeks of ingesting the test material, these f1 animals were allowed to mate for the f2 generation. The first breeding of these f1 adults resulted in the f2a litters. After the last f2a litter was weaned, the f1 adults were allowed to mate a second time to produce the f2b litters.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.01 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

0.065 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 mg/kg bw/day

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The highest dose selected in this study (1 mg/kg/day) was identical to the original three-generation reproduction study in Fischer 344 rats. The lower two levels, 0.01 and 0.065 mg/kg/day, are similar to middle and low levels employed in the two-year chronic toxicity and oncogenicity study in Fischer 344 rats.
- Fasting period before blood sampling for clinical biochemistry: Yes (overnight)

Examinations

Parental animals: Observations and examinations:

PHYSICAL OBSERVATIONS
- Each animal on study was observed for signs of toxicity and changes in demeanor at least once a day.

BODY WEIGHT AND FOOD CONSUMPTION
- Body weights and food consumption for all f0 and f1 adult animals were recorded weekly prior to mating. Since the male and female rats were still gaining weight prior to mating, the males were weighed weekly after the mating period for the f1a and f2a litters and the females were weighed once after weaning the f1a and
f2a litters.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in all offspring:
the date of parturition, the number of live and dead pups on the day of parturition (day zero), the number of live pups on day 1, 4, 7, 14 and 21 after delivery, the weight of the litter and lactating female on days 1, 4 (post-culling), 7, 14 and 21 of lactation, and individual body weights for each male and female pup on day 21. Any visible physical abnormalities in the neonates were recorded during the lactation period.

Postmortem examinations (parental animals):

After weaning the last litter from the f1b and f2b generations. all surviving adult f0 and f1 males and females were fasted overnight, weighed and anesthetized with methoxyflurane. The trachea was clamped and they were sacrificed by decapitation. The adults were subjected to a complete gross pathologic examination. The eyes were examined in situ by gently pressing a glass slide against the cornea and observing the eyes under fluorescent light illumination. Tissues were saved from these animals and preserved in neutral phosphate buffered 10% formalin with the exception of the testes and epididymides which were fixed in Bouin's solution. No histologic examination of these tissues was made. Adult rats that were moribund or died spontaneously were necropsied in a similar manner; however, a detailed eye examination was not performed and their testes and epididymides were fixed in formalin with the other tissues.
The following tissues are collected during the gross necropsy examination:
Liver, pancreas, peripheral nerve, adrenal, small intestine, mesenteric lymph node, epididymis, prostate, oviduct, urinary bladder, salivary gland, mediastinal lymph node, thyroid gland, larynx, eye, lacrimal gland, oral cavity, heart, brain, spinal cord, kidney, cecum, mesenteric tissue, seminal vesicle, uterus, cervix, lungs, thymus, aorta, parathyroid gland, skin, tongue, zymbal gland, spleen, pituitary, bone marrow, stomach, large intestine, testicle, coagulating gland, ovary, vagina, skeletal muscle, mediastinal tissue, esophagus, trachea, mammary gland, nasal turbinates, bone

Postmortem examinations (offspring):

Weanling Rats: The f1a and f2a weanlings were discarded at weaning on day 21 of lactation; no gross or histopathological examination was conducted. For f1b and f2b weanlings, 10 randomly selected animals/sex/dose level were sacrificed and subjected to a complete gross pathologic examination as described for the adults. Tissues were saved as indicated, for adults and no histologic examination was performed.

Statistics:

Body weights were evaluated by Bartlett's test for equality of variances (α=0.01). Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, a Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed.
Statistical outliers (p <0.02) of food consumption data were identified by the method described by Grubbs (1969) and excluded from analysis. Descriptive statistics of food consumption data were subsequently determined. The fertility index was analyzed by the Fisher exact probability. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates were analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Several f1a pups from dams ingesting 0.065 mg/kg/day of the test substance were hypothermic or jaundice-appearing at some point during the lactation period. However, all of these pups survived and appeared normal at weaning. In addition, pups from dams ingesting higher concentrations of the test material did not exhibit symptoms of hypothermia or jaundice.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The spontaneous deaths of the female rats occurred primarily during delivery and did not appear to be treatment related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weights of 14 and 21-day old f1a neonates from dams ingesting 1.0 mg/kg body weight/day of the test substance were statistically significantly decreased from control animals.
The f1a pups from dams ingesting 0.065 mg/kg/day of the test substance weighed significantly less than control animals on days 14 and 21 postpartum. Similarly, the female pups from dams ingesting 0.01 mg/kg/day weighed significantly less than controls on day 21 post-partum.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

the reproduction indices were comparable between experimental groups

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The spontaneous deaths of the female rats occurred primarily during delivery and did not appear to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of 14 and 21-day old f1b neonates from dams ingesting 1.0 mg/kg body weight/day of the test substance were statistically significantly decreased from control animals. The pups from the f1b litters ingesting 1.0 mg/kg/day which were raised to adulthood always weighed less than control animals. This was due in part to an age difference. Two litters born from dams ingesting 1.0 mg/kg/day were 7 days younger than litters from other dose levels.

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross lesions observed at necropsy of the f1b litters were considered to be spontaneous in nature and not treatment related.

Reproductive function / performance (P1)

Reproductive performance:

no effects observed

Description (incidence and severity):

the reproduction indices were comparable between experimental groups

Details on results (P1)

The size of the f1b litters were comparable between experimental groups

Effect levels (P1)

Key result

Dose descriptor:

NOEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

As with the f0 animals, several of the spontaneous deaths of the f1 rats occurred during delivery and did not appear to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight decrease in day 21 weights of the f2a neonates was observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of male f1 rats and female f1 rats was comparable between experimental groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathologic observations of the f1 adults and the f2b weanlings were considered to be spontaneous in nature and not treatment related.

Other effects:

no effects observed

Description (incidence and severity):

No effects were observed between controls and treated animals in the reproduction indices, and the size of the f2a litters

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest concentration

Results: F2 generation

General toxicity (F2)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

As with the f0 animals, several of the spontaneous deaths of the f1 rats occurred during delivery and did not appear to be treatment related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weights of the f1 dams during the lactation period for the f2b litters were comparable between experimental groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of male f1 rats and female f1 rats was comparable between experimental groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathologic observations of the f1 adults and the f2b weanlings were considered to be spontaneous in nature and not treatment related.

Details on results (F2)

The reproduction indices and the size of the f2b litters were comparable between experimental groups.

Effect levels (F2)

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest concentration

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Body weight data (significant observations)

Day (post-partum)	0 mg/kg/day	0.01 mg/kg/day	0.065 mg/kg/day	1.0 mg/kg/day
Body weights of f1a litter
14	33.4±2.1	31.8±2.8	31.3±3.4*	31.5±2.9*
21 (male)	55.7±4.0	52.9±4.1	51.5±4.9*	50.6±5.3*
21 (male)	53.5±4.0	50.2±3.6*	49.4±4.9*	48.7±5.0*
Body weights of f1a litter
14	33.7±2.8	32.6±3.2	32.5±2.6	31.7±2.9*
21 (male)	56.1±5.1	53.5±6.4	52.9±5.1	51.9±5.3*
21 (male)	53.8±4.2	51.0±5.7	50.6±4.5	49.8±5.1*
Body Weights of f1 Dams During the Lactation Period for f2a Litters
14 (male)	357±17	337±29*	349±32	342±26
Body Weights of f2a Litters
21 (male)	55.3±4.3	53.6±5.3	55.1±5.3	51.8±4.1*

Applicant's summary and conclusion

Conclusions:

Rat NOEL (Reproductive parameters): 1.0 mg/kg/day

Executive summary:

Male and female Sprague-Dawley rats ingested 0, 0.01, 0.065 or 1.0 mg/kg/day of the test substance for 8 weeks. Male and female rats ingesting the same concentration of the test substance on a mg/kg basis were mated twice to produce the f1a and f1b litters. Selected f1b rats ingested the same concentration of test material as their parents for 11 weeks at which point they were mated twice to produce the f2a and f2b litters.

Ingestion of 1.0 mg/kg/day of the test substance did result in a slight decrease in flat f1a, f1b and f2a weanling body weights and f1 adult body weights. However, concentrations as high as 1.0 mg/kg/day did not affect the ability of the parental animals to mate, produce a litter of normal size and number and raise a litter. Thus the no-observable-effect level (NOEL) for reproductive parameters in Sprague-Dawley rats is 1.0 mg/kg/day for the test substance.