6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March - 02 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

EC Number:

617-769-9

Cas Number:

858956-08-8

Molecular formula:

C8H8ClN3O2

IUPAC Name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately (P0) 7 wks; (P1) 7 wks
- Weight at study initiation: (P0) Males: 150.6 – 192.7 g; Females: 125.7 – 160.9 g; (P1) Males: 247 – 306 g; Females: 167 – 219 g
- Housing: Pretest/Premating: individually in stainless steel, wire-mesh cages suspended above cage boards; Mating/Cohabitation Period: rats were housed in the male’s cage as breeding pairs in stainless steel, wire-mesh cages suspended above cage boards; Gestation/Lactation period: females were housed individually (with their litters) in stainless steel, wire-mesh cages suspended above cage boards
- Use of restrainers for preventing ingestion: no
- Diet: PMI Nutrition International, Inc. Certified Rodent Lab Diet® 5002 (powdered), ad libitum
- Water: tap water (United water Delaware), ad libitum
- Acclimation period: approximately 5 days

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was added directly to the rodent diet and thoroughly mixed in the diet mixer until the test substance appeared to be homogeneously distributed throughout the diet.

DIET PREPARATION
- Rate of preparation of diet: at least biweekly
- Storage temperature of food: food was stored in the refrigerator

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation was observed or up to 2 weeks
- 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:

The sponsor reported purity of the test substance was 92.2%. Although the analysis for the analysed Batch-010 indicated that the purity was 90.9%, all formulations calculations were based on the sponsor reported purity (92.2%). However, the differences in reported purities for both compounds was considered minimal (1.3%) and these adjustments for purity were not expected to impact the study results in any way.

Diet samples containing the test substance at concentrations of 500,1500, 5000 and17,000 ppm were analysed for homogeneity/concentration verification analysis. Also a control diet sample was included for analysis. Stability of the test substance in diet at the concentrations of 300 and18000 ppm had been established at room temperature for 7 and14 days and refrigerated fo r14 and 21 days in a separate study (WR17053,SC1026,DuPont-21490). In addition, neat test substance was submitted for stability analysis near the beginning, middle (due to batch change) and end of the study. Concentrations of the test substance were determined by high-performance liquid chromatography (HPLC) with UV detection.The analytical results show that the test substance was homogeneously mixed in the diet at the targeted concentrations and that the test substance was stable in the diet under the study storage conditions for all dietary levels.The neat test substance was stable during the study period. Test substance was not detected in the control samples.

Duration of treatment / exposure:

P0: premating period (10 weeks), mating (up to 2 weeks), postmating period (males and females with no evidence of mating/no litter delivered: 5 - 6 weeks after the end of the gestation period), gestation (22 days), and lactation (22 days)
F1 (PND 21): premating period (10 weeks), mating (up to 2 weeks), postmating period (males and females with no evidence of mating/no litter delivered: 5 - 6 weeks after the end of the gestation period), gestation (22 days), and lactation (22 days)

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until aproximately 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: aproximately 10 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

corresponding to:
P0 male rats: 30.11 mg/kg bw/day
P0 female rats (premating period): 35.97 mg/kg bw/day
P0 female rats (gestation period): 33.03 mg/kg bw/day
P0 female rats (lactation period): 58.53 mg/kg bw/day

P1/F1 male rats: 42.32 mg/kg bw/day
P1/F1 female rats (premating period): 46.42 mg/kg bw/day
P1/F1 female rats (gestation period): 32.22 mg/kg bw/day
P1/F1 female rats (lactation period): 64.17 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

corresponding to:
P0 male rats: 91.93 mg/kg bw/day
P0 female rats (premating period): 110.02 mg/kg bw/day
P0 female rats (gestation period): 100.00 mg/kg bw/day
P0 female rats (lactation period): 174.72 mg/kg bw/day

P1/F1 male rats:126.33 mg/kg bw/day
P1/F1 female rats (premating period): 140.93 mg/kg bw/day
P1/F1 female rats (gestation period): 101.88 mg/kg bw/day
P1/F1 female rats (lactation period): 189.05 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

corresponding to:
P0 male rats: 299.09 mg/kg bw/day
P0 female rats (premating period): 367.00 mg/kg bw/day
P0 female rats (gestation period): 330.51 mg/kg bw/day
P0 female rats (lactation period): 599.47 mg/kg bw/day

P1/F1 male rats: 425.76 mg/kg bw/day
P1/F1 female rats (premating period): 465.20 mg/kg bw/day
P1/F1 female rats (gestation period): 335.75 mg/kg bw/day
P1/F1 female rats (lactation period): 651.05 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

17 000 ppm

Remarks:

corresponding to:
P0 male rats: 1048.26 mg/kg bw/day
P0 female rats (premating period): 1243.26 mg/kg bw/day
P0 female rats (gestation period): 1158.02 mg/kg bw/day
P0 female rats (lactation period): 2214.79 mg/kg bw/day

P1/F1 male rats: 1521.77 mg/kg bw/day
P1/F1 female rats (premating period): 1665.55 mg/kg bw/day
P1/F1 female rats (gestation period): 1192.19 mg/kg bw/day
P1/F1 female rats (lactation period): 2243.44 mg/kg bw/day

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A one-generation reproduction study was conducted with methyl 6-amino-5-chloro-2-(cyclopropyl)-4-pyrimidinecarboxylate, of which the test substance is the major metabolite. In that study, dietary concentrations of 600, 5000, and 17,000 ppm were administered to P1 male and female CRL:CD(SD) rats (10 per sex per dietary level) during a premating period (4 weeks), mating (up to 2 weeks), gestation (3 weeks), and lactation (3 weeks). F1 weanlings (1 per sex per litter), randomly selected to comprise the F1 generation, were given the same dietary concentration as their respective P1 generation sires and dams until postnatal day 60. Organs of the reproductive tract, adrenals, pituitary and liver from P1 adults were weighed and examined microscopically. Previously identified target organs (pancreas, thyroid) were also examined microscopically for P1 and F1 adults and F1 weanlings. There were no effects on reproductive parameters at dietary concentrations up to 17,000 ppm. Test substance-related adverse effects on body weight and food consumption parameters occurred in P1 females at 17,000 ppm. F1 pup weights were decreased during the lactation period at 17,000 ppm and this continued, albeit with recovery, during the post-weaning period in F1 males and females. There were no adverse clinical pathology or neurobehavioral changes in P1 rats. Organ weights, gross observations and microscopic findings were not affected at any dietary concentration. Based on this data, dietary concentrations of 500, 1500, 5000, and 17,000 ppm were selected for the present study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (at the time of body weight determination)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during premating and postmating period; gestation day 0, 7, 14 and 21; lactational day 0, 7, 14 and 21; at scheduled sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

Vaginal lavage samples were collected daily from all P0 and P1 female rats in order to determine the stages of the estrous cycle. Vaginal lavage samples were collected beginning 3 weeks prior to the start of mating/cohabitation and continued until mating/copulation was confirmed or the mating/cohabitation period ended. The vaginal lavage sample, collected on the day copulation was confirmed, was not used for estrous cycle evaluation. Vaginal lavage samples were also collected from all P0 and P1 parental female rats at the time of sacrifice. Vaginal lavage samples were examined microscopically for determination of the stage of the estrous cycle (diestrus, estrus and proestrus).

Sperm parameters (parental animals):

Parameters examined in P0/P1 male parental generations: sperm motility, sperm morphology, right cauda epididymis weight, sperm count in right epididymides: determination of percentage of motile sperm cells/200 cells, determination of frequency of morphologically abnormal sperm, left testis and epididymis were flash frozen for analysis of sperm and spermatid counts, left testis and epididymis weight, sperm count per cauda epididymis and per gram cauda epididymis, and spermatid count per testis and per gram testis were determined.

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 F1 / F2 offspring: abnormal behaviour and appearance, postnatal mortality, live and dead pups, number and sex of pups, pup weight, litter weight, stillbirths, live births, postnatal mortality, weight gain, gross pathological examination on PND 21, presence of gross anomalies, vaginal patency and body weight on the day of achievement (F1 females), preputial separation and body weight on the day of achievement (F1 males) and anogenital distance (AGD) (F1). Litter observations were performed on Day 0, 4, 7, 14, and 21 postpartum.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (P0 and P1 adults) after siring litters.
- Maternal animals: All surviving animals (P0 and P1 adults) after the last litter of each generation was weaned (Day 21 postpartum).
- Nonnursing females: Approximately 5-6 weeks after the end of the mating period

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissue collected from P0 and P1 adult rats for analysis: testis, epididymis, prostate, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, vagina, liver, kidneys, spleen, brain, pituitary gland, adrenal glands, thyroid gland and gross observations. Tissues collected at necropsy were processed and evaluated microscopically for all 28 rats per sex in the control and high-dose (17,000 ppm) P0 and P1 adult rats. Target organs (P1 adult female thyroid gland) from the female P1 adult intermediate groups were subsequently processed and evaluated in order to determine an NOAEL. In addition, the same tissues examined microscopically from the control and high-dose adults were examined from all mated P1 adult rats that failed to produce a litter.

Organ weight of P0 and P1 adult rats was determined for the following organs and tissues: testis, epididymis, right cauda epididymis, prostate, seminal vesicles with coagulating glands, ovaries with oviducts, uterus with cervix, liver, kidneys, spleen, brain, pituitary gland, adrenal glands, thyroid gland. Uteri and presence of number of implantation sites were determined in all cohabitated females.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and culled pups of the F2 offspring were sacrificed at PND 4 days of age.
- Weanlings of the F1 and F2 generation were sacrificed at the day of weaning (Day 21 postpartum), except F1 rats selected for continued evaluation.

GROSS NECROPSY
- Gross necropsy on PND 21consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Analysis were performed on the following organs: brain, spleen, thymus and thyroid gland (randomly selected from F1 and F2 weanlings (one weanling/sex/litter). Presence of gross lesions were determined for all F1 and F2 weanlings.

HISTOPATHOLOGY / ORGAN WEIGTHS
Individual organ weights (brain, spleen, and thymus) were determined on PND 21. Group mean values and organ weight ratios (% body weight and % brain weight) were calculated. Tissues were not collected from pups (nursing offspring) that died (found dead, sacrificed in extremis, or accidentally killed) during the lactation period and microscopic examination of pup tissue was not performed.

Target organs identified in the P1 and F1 adults (F1 adult female thyroid gland) were processed and evaluated microscopically from selected (1 weanling/sex/litter) F1 and F2 control and high-dose female weanlings.

A quantitative evaluation of primordial and growing follicles was conducted on 10 lactating F1 females (surviving to scheduled sacrifice) from control and high-dose 17,000 ppm groups. 6 ovarian cross sections were taken from the central area of the ovary. Primordial and growing follicles (up to but not including antral follicles) were enumerated for up to 12 ovarian sections per animal.

Statistics:

For an overiew on statistics, please refer to table 1 in the "Any other information on materials and methods incl. tables" section.

For litter parameters, the proportion of affected pups per litter or the litter mean was used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05. Additional statistical tests were used and other parameters analyzed, if deemed necessary.

Reproductive indices:

Mating index (%) = Number of mated* / Number paired x 100
Fertility index (%) = Number pregnant females**/Number of mated* x 100
Post-Implantation loss (%) *** = Number of Implantation sites - Number of pups born females with live pups / Number of Implantation sites x 100

* Mated = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
** Pregnant = uterine implantation sites.
*** Determined for each litter.

Offspring viability indices:

Live birth index (%)* = Number of pups born a live / Total number of pups born x 100
Viability index (%)*, ** = Number of pups alive on PND 4 pre-culling / Number of pups born alive x 100
Lactation index (%)*, ** = Number of pups alive at weaning (PND 21) / Number of pups alive on PND 4 pre-culling x100

* Determined for each litter.
** Excluding litters sacrificed due to death of dam during lactation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

17,000 ppm: one female rat was found dead on gestation day 19. Prior to death, this animal showed clinical signs including increased breathing, dehydration, and lack of feces present on cage board.
All other clinical signs observed in males and females occurred with low frequency with no dose-response across all study groups. In addition, the reported observations were generally unremarkable and of a nature commonly seen in rats of this age.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

17,000 ppm: one female rat was found dead on gestation day 19. Prior to death, this animal showed clinical signs including increased breathing, dehydration, and lack of feces present on cage board. The cause of death was pulmonary arterial thrombosis.

5000 ppm: one animal was found dead on TD 4, the cause of death was pyelonephritis. The animal was replaced with an animal on TD 5. Another animal was sacrificed in extremis on TD 99 following clinical observations indicative of dystocia. The aforementioned deaths were not considered to be test substance-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related and/or statistically significant reductions in mean body weights and weight gains in males and females. In males, beginning after approximately 5 weeks after start of the study, weekly mean body weights were 6 to 9% lower than that of controls. Cumulative weight gains calculated from TDs 0 to 70 or 119 were 17% or 13% lower than that of controls, respectively, as a result of reduced body weight gains observed during the testing period.
In females, weekly mean body weights were 6 to 7% lower than that of controls for the last 3 weeks of premating, and resulted in reduced cumulative weight gains (TDs 0 to 70) that were 16% lower than that of controls. During the gestation period, mean weekly body weights were 8% lower than that of controls after the first week of gestation. Cumulative body weight gains during gestation were 12% lower than that of controls and were attributed by a 26% reduction in body weight gains during the first week of gestation as compared to the control group. During the lactation period, mean weekly body weights were reduced 5 to 9% as compared to controls. Overall (LD 0-21) body weight gains were increased (not statistically significant) as compared to controls (gain of 13.2 g vs. 0.5 g in the control group).

5000 ppm: test substance-related and/or statistically significant reductions in mean body weights and weight gains in males. Mean body weights were 6 to 7% lower than that of controls from TDs 42 to 105. Cumulative weight gains calculated from TDs 0 to 70 were 14% lower than that of controls as a result of reduced body weight gains observed during the testing period.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related statistically significant reductions in food consumption in males and females. In males, food consumption was reduced at test day 56 to 63 (premating period), at test days 35 to 70 (premating period), at test day 0 to 7 ( premating period). In females, food consumption was reduced at test day 42 to 49 (premating period), at test day 7 to 14 (gestation period), at test day 7 to 14 (lactation period)

5000 ppm: test substance-related statistically significant reductions in food consumtion in males. In males, food consumption was reduced at test day 63 to 70 (premating period)

500 ppm: one instance of statistically significant reductions in food consumption in males. This occasional instance did not appear to be dose-related and was thus considered incidental.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: there were test substance-related statistically significant reductions in food efficiency in males and females. In males, cumulative food efficiency was 15% lower than that of controls on test days 0 to 70 as a result of statistically significant reductions in body weight parameters observed throughout the testing period. This affect was mostly attributed to body weight effects observed throughout premating and a lack of food consumption effects for this same range.
In females, cumulative premating (days 0 to 70) food efficiency was 12% lower than that of controls, as a result of statistically significantly reduced body weight gain for the same interval and a lack of food consumption effects. Furthermore, food efficiency was 20% lower than that of controls during the first week of gestation.

5000 ppm: test substance-related statistically significant reductions in food efficiency in males. Cumulative food efficiency was 10% lower than that of controls on test days 0 to 70. This effect was mostly attributed to body weight effects observed throughout premating and a lack of food consumption effects for the same period.

1500 ppm: test substance-related statistically significant reductions in food efficiency in males. Cumulative food efficiency was 8% lower than that of controls on test days 0 to 70 primarily as a result of a 36% reduction (compared to control) on test days 56 to 63. This effect is mostly attributed to a transient weight gain reduction (37% lower than controls) for the same interval.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

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, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All microscopic findings were consistent with normal background lesions in rats of this age and strain.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of reproductive failure in the P0 adult pairs was 5/28, 2/28, 4/28, 3/28, and 4/28 in the 0, 500, 1500, 5000, and 17,000 ppm groups, respectively and was not considered to be related to the test substance.

For details please refer to table 2 and 3 in the “Any other information on results incl. tables” section

Details on results (P0)

The data for mating, fertility, pre-coital interval length, gestation length, implantation site counts and post-implantation losses were comparable across all groups tested for each respective generation.
For details please refer to table 3 in the “Any other information on results incl. tables” section.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect on reproduction observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

3

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

4

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

5

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: corresponding to 299.09 and 367.00 mg/kg bw/day in males and females, respectively

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

5000 ppm: clinical signs evidenced due to dystocia (prior to sacrifice). However, no conclusive pathological evidence of dystocia was observed for this animal.

control: clinical observations indicative of dystocia (prior to sacrifice)

There were no test substance-related increases in any clinical observations reported during either the premating, gestation, or lactation phases in males and females. The signs observed occurred with low frequency with no dose-response across all study groups. In addition, the reported observations were generally unremarkable and of a nature commonly seen in rats of this age. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

5000 ppm: one female rat was sacrificed in extremis on test day (TD) 104 due to presence of a mammary gland tumor (adenocarcinoma). One male was accidentally killed on TD 0 and replaced with another animal on the same day.

500 ppm: one male was found dead on TD 54. The cause of death was chronic progressive nephropathy.

control: one female rat was sacrificed in extremis on TD 106 due to clinical observations indicative of dystocia.

The observed deaths in males and females were not determined to be test substance-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related and/or statistically significant reductions in mean body weights and weight gains in males and females. In males, beginning at TD 0 to the end of the premating period (TD 70) and terminal sacrifice (TD 105), weekly mean body weights were 12 to 15% lower than that of controls. Cumulative weight gains calculated from test days 0 to 70 or 105 were 13% or 15% lower than that of controls, respectively.
In females, during the first 3 weeks (including TD 0) of the premating period, mean body weights were up to 14% lower than that of controls. Mean final weights at the end of the premating period were 7% lower than that of controls and mean cumulative weight gain during this premating period was within 5% of controls.

5000 ppm: test substance-related and/or statistically significant reductions in mean body weights and weight gains in males and females. In males mean body weights were 7 to 9% lower than that of controls from TD 63 to 105. Cumulative weight gains calculated from TDs 0 to 70 or 105 were 7% or 9% lower than that of controls, respectively, as a result of reduced body weight gains observed during the testing period. Body weights observed in P1 males on the day of weaning (LD 21) were slightly reduced (6%) compared to controls. In females, body weights were slightly reduced by (6%), compared to controls, and only at the day of weaning (LD 21).

1500 ppm: occasional observed instances of statistical significanct effects on body weights in females that did not appear to be dose-related were considered incidental.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related statistically significant reductions in food consumption was observed in males. Cumulative food consumption was 5% lower than that of controls, respectively, on TD 0 to 70. This effect was mostly attributed to body weight affects observed throughout the study.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related statistically significant reductions in food efficiency was observed in males. Cumulative food efficiency was 9% lower than that of controls, respectively, on TD 0 to 70. This effect was mostly attributed to body weight affects observed throughout the study.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

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, non-treatment-related

Description (incidence and severity):

17,000 ppm: statistically significant decreases in the absolute weights of the brain (4%), liver (17%), spleen (16%), and thyroid gland (17%), as compared to control values were observed in males. Furthermore, statistically significant decreases in relative organ weights (% of brain weight) of liver and spleen were observed in males. Since the mean final body weight of the 5000 and 17,000 ppm groups were also decreased, the organ weight decreases were all attributed to the decrease in body weight and were not considered to be organ specific effects. There was no test substance-related microscopic effect on any of these 4 male organs. In high-dose females a statistically significant decrease (5%) in the mean absolute brain was observed. This decrease was accompanied by a similar decrease (4%) in mean final body weights. Since there was no difference in relative brain weights and no test substance-related microscopic findings in the brain, the lower mean absolute brain weight was interpreted to be a reflection of the lower body weights and not an organ specific effect.

5000 and 17,000 ppm: statistically significant increases in several mean relative (% body weight) organ weight values (adrenal glands, brain, epididymides, kidneys, pituitary gland, right cauda epididymides, seminal vesicles, and testes) were observed in males. Since the decrease in mean body weight was proportionally greater than the decrease in mean absolute organ weights, the relative values were statistically increased. These increases in mean relative organ weights were all attributed to the decreases in mean final body weight at 5000 and 17,000 ppm. There was no test substance-related microscopic pathology in any of the male organs. No effects on organ weights were noted in females.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross observations were consistent with normal background lesions in rats of this age and strain. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

17,000ppm: a small increase in thyroid follicular cell hypertrophy was observed in females. The thyroid cell hypertrophy was characterized by an increase in the size of the follicular lining cell and a change in shape from flat or cuboidal cell to columnar and was observed in 2/28, 0/28, 0/28, 0/28, and 8/28 females given 0, 500, 1500, 5000 and 17,000 ppm, respectively. All were graded as minimal (grade 1 of 4). The increased incidence in the high-dose group was considered test substance-related. However, as there were no test substance-related microscopic findings in the P0 adult males and females or the P1 adult males the biological significance of this finding remains unclear. Examination of the thyroid gland in the F1 and F2 weanlings did not demonstrate any test substance-related findings. For details please refer to table 4 in the “Any other information on results incl. tables” section.

All other microscopic observations in this study were consistent with normal background lesions in rats of this age and strain.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Ovarian follicle counts
There were no significant differences in the total number of primordial and pre-antral follicles between the control and 17,000 ppm females.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

5000 ppm: the mean cycle length was statistically significantly increased. Since this effect was primarily due to the unusually high value observed for one animal of this dose group, biological significance of this finding is questionable. Further, the mean cycle length was unaffected in the high dose group and there were no similar effects observed in P0 females at any level thus the effect on oestrus cycle length observed in the mid-dose group at 5000 ppm was not considered treatment-related.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

5000 ppm: statistically significant decrease in post-implantation loss observed in F1 litters that occurred at 5000 ppm, but not at 17,000 ppm, was considered unrelated to the test substance and therefore not toxicologically significant.

The incidence of reproductive failure in the P1 adult pairs was 4/28, 9/28, 3/28, 3/28, and 3/28 in the 0, 500, 1500, 5000 and 17,000 ppm groups, respectively and was not considered to be related to the test substance.

For details please refer to table 5 and 6 in the “Any other information on results incl. tables” section.

Details on results (P1)

The data for mating, fertility, pre-coital interval length, gestation length and implantation site counts were comparable across all groups tested for each respective generation.
For details please refer to table 6 in the “Any other information on results incl. tables” section.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect on reproduction observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

3

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

4

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

5

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related clinical observations in either F1 offspring at any dietary level tested. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pups
17,000 ppm: 11 male pups and 4 female pups died during the lactation period.
5,000 ppm: 2 male pups died during the lactation period.
1500 ppm: 4 male pups and 8 female pups died during the lactation period.
500 ppm: 5 male pups and 3 female pups died during the lactation period.
control: 2 male pups and 5 female pups died during the lactation period.

Most pups died within the first 4 days of lactation. The gross observations were non-specific and typical of control rat pups that die early in the nursing period. Gross observations, when present, were usually indicative of a failure to breath at birth (e.g., lungs not expanded), failure to suckle following birth (e.g., no milk spot in stomach), or postmortem changes (e.g., cannibalized, amputation, autolysis). All gross observations were interpreted as incidental and unrelated to the test substance. For details please refer to table 7 in the “Any other information on results incl. tables” section.

Survival indices were comparable across all groups. For details please refer to table 8 in the “Any other information on results incl. tables” section.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: statistically significant test substance-related reductions in mean pup weight. Weights were slightly lower (7%) than controls on PND 0. This continued through PNDs 4 to 14 with a more pronounced difference between PNDs 14 and 21. Mean pup weights were 7%, 7%, 8%, and 15% lower than controls on PNDs 4, 7, 14, and 21, respectively. Weight gains within this dose group were comparable to controls from PND 0 through 14; however, as reflected in the mean body weight data, weight gains were significantly lower than controls from approximately PND 14 to 21 (approximately 27% lower).

5000 ppm: mean pup weights were statistically significant lower (7%) compared to controls at PND 21. Body weight gains were comparable to controls from PND 0 though 14 with a minimal (12%) decrease between PND 14 and 21. This lower mean body weight at 5000 ppm is not considered adverse because of the minimal magnitude and the transient nature of the reduction.

500 ppm: statistically significant decrease in pup weight. This effect was not considered related to the test substance, since it was not observed at 1500 ppm. Therefore, this finding was considered not toxicologically significant (non-adverse, because of the minimal magnitude and the transient nature of the reduction).

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

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, non-treatment-related

Description (incidence and severity):

17,000 ppm: a slight increase in the number of days to reach preputial separation compared to control males was observed (44.0 days for 17,000 ppm males compared to 42.9 days for control males). Since delays in sexual maturation can be associated with lower body weight, the statistical evaluation was performed using analysis of covariance with body weight on the day of weaning as the covariate. Using this evaluation, there were no statistically significant differences between the control group and the 17,000 ppm F1 males. In addition, the value observed in the high dose F1 males was within DuPont Haskell’s historical control range (20 studies; 1990-present, mean 42.9; range 39.9-45.0). Therefore, the delay in preputial separation was considered to be secondary to the lower body weight at weaning and consequently regarded as non-adverse.

Vaginal patency
There were no test substance-related effects on timing for achievement of vaginal patency in F1 offspring at any dietary level tested.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

17.000 ppm: slight decrease in spleen weight parameters (absolute weight, relative weight/brain weight, relative weight/body weight) in males and females. In males, mean absolute, relative (% brain weight) and relative (% body weight) spleen weights were decreased 25%, 23%, and 13%, respectively, as compared to control values. In females, mean absolute, relative (% brain weight) and relative (% body weight) spleen weights were decreased 31%, 29%, and 19%, respectively, as compared to control values. Histopathology was not conducted on any F1 weanling spleens (male or female). The decrease in spleen weight parameters was interpreted to be evidence of retarded growth rather than splenic toxicity. For details please refer to table 9 in the “Any other information on results incl. tables” section.

For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross observations in weanlings were consistent with normal background lesions in rats of this age and strain. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related microscopic findings observed. Microscopic examination of weanlings was limited to the thyroid gland of the control and 17,000 ppm weanlings. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

1500 ppm: statistically significant effect on sex ratio (more males). Since this effect was not observed at t 5000 or 17,000 ppm, it was considered unrelated to the test substance and, therefore, not toxicologically significant. For details please refer to table 8 in the “Any other information on results incl. tables” section.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter size, number of pups born/born alive and survival indices (Live-Birth Index, Viability Index and Lactation Index) were comparable across all dose groups.

Body weight and weight changes
Reductions in mean body weights and weight gains are considered test substance related and reflect the relatively high levels of test substance intake during this period of rapid growth when offspring is still nursing and also consuming the test diets. Dietary intake could not be quantified for offspring; however, because of their relative size, their exposure likely approached or exceeded that of maternal rats. Offspring weights were comparable to controls through lactation day 14 when the pups are nursing and unable to consume test diets directly. The lower mean body weight at 5000 ppm is not considered adverse because of the minimal magnitude and the transient nature of the reduction. On lactation day 21, selected pups are randomly assigned to serve as the parental generation for production of F2 litters. Thus, lactation day 21 is equal to test day 0 for the F1 generation adults. Despite the fact that the lactation day 21 offspring weights were lower, the test day 0 weights from a subset of these offspring on the same day were generally comparable to controls, as were the body weight gains for this group throughout premating for 5000 ppm F1 rats. This observation underscores the minimal magnitude of this change. Lastly, this slight effect was even less pronounced among F2 offspring; at 5000 ppm, F2 offspring weights were comparable to controls during lactation.

Effect levels (F1)

1

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related clinical observations in either F2 offspring at any dietary level tested. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Weanlings
5000 ppm: one male and one female weanling were found dead. They were partially cannibalized shortly before their scheduled weanling euthanasia. Both weanlings underwent gross pathological examination, but the cause of death could not be determined.

Pups
17,000 ppm: 5 male pups and 3 female pups died during the lactation period.
5,000 ppm: 9 male and 10 female pups died during the lactation period.
1500 ppm: 3 male pups pups died during the lactation period.
500 ppm: 3 male pups and 1 female pups died during the lactation period. One 500 ppm F1 female pup had multiple congenital deformities that were considered incidental.
control: 6 male pups and 5 female pups died during the lactation period.

Most pups died within the first 4 days of lactation. The gross observations were non-specific and typical of control rat pups that die early in the nursing period. Gross observations, when present, were usually indicative of a failure to breath at birth (e.g., lungs not expanded), failure to suckle following birth (e.g., no milk spot in stomach), or postmortem changes (e.g., cannibalized, amputation, autolysis). All gross observations were interpreted as incidental and unrelated to test substance dietary exposure. For details please refer to table 7 in the “Any other information on results incl. tables” section.

Survival indices were comparable across all groups. For details please refer to table 10 in the “Any other information on results incl. tables” section.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

17,000 ppm: test substance-related reduction in mean pup weight. Mean pup weights were 8% lower than controls on PND 21. For details please refer to the attached PDF (Summary Tables) in the “Overall remarks, attachments” section.

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:

no effects observed

Description (incidence and severity):

For details please refer to table in the “Any other information on results incl. tables” section.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

For details please refer to table in the “Any other information on results incl. tables” section.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

17.000 ppm: slight decrease in spleen weight parameters in males and females (although it was less than that observed in the F1 weanlings). In males, the mean absolute, relative (% brain weight) and relative (% body weight) spleen weights were decreased 16%, 15%, and 7%, respectively, as compared to control values. The 16% and 15% decreases were statistically significant. In females, the mean absolute, relative (% brain weight) and relative (% body weight) spleen weights were decreased 15%, 14%, and 8%, respectively, as compared to control values. These decreases were not statistically significant. Histopathology was not conducted on F2 weanling spleens. The decrease in spleen weight was interpreted to be evidence of retarded growth rather than splenic toxicity.

For details please refer to table 11 in the “Any other information on results incl. tables” section.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross observations in weanlings were consistent with normal background lesions in rats of this age and strain. For details please refer to table in the “Any other information on results incl. tables” section.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related microscopic findings observed. Microscopic examinations of weanlings was limited to the thyroid gland of the control and 17,000 ppm weanlings. For details please refer to table in the “Any other information on results incl. tables” section.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

1500 ppm: statistically significant effect on sex ratio (less males). Since this effect was not observed at 5000 or 17,000 ppm, it was considered unrelated to the test substance and, therefore, not toxicologically significant. For details please refer to table 10 in the “Any other information on results incl. tables” section.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Litter size, number of pups born/born alive and survival indices (Live-Birth Index, Viability Index and Lactation Index) were comparable across all dose groups.

Effect levels (F2)

1

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

17 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

2

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: Please refer to the "Doses/Concentrations" section for corresponding dose values given as mg/kg bw/day.

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

17 000 ppm

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Overall reproductive toxicity:

For F1/F2 pups a LOAEL of 17,000 ppm and a NOAEL of 5000 ppm were determined, based on reduced body weights and spleen weights, which was considered to be evidence of retarded growth rather than splenic toxicity, at the highest dose level. Taking into consideration that systemic toxicity was evident in maternal animals of the P0 and P1 generation at 17000 ppm, the effects observed in the F1 and F2 offspring are rather considered as a secondary, non-specific consequence of maternal toxicity. According to Annex I, 3.7.2.4.3 of EC regulation 1272/2009 (CLP), the data are not considered as sufficient and conclusive for classification as “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity”.

Table 2: Causes of Reproductive Failure in P0 Male and Female Rats

Dose group	Sex	Gross and/or microscopic findings
500 ppm	male	testes: degeneration/atrophy, seminiferous tubules
5000 ppm	female	ovaries: absence of recent corpora lutea (decreased ovulation)
5000 ppm	female	ovaries: absence of recent corpora lutea (decreased ovulation)
5000 ppm	female	ovaries: absence of recent corpora lutea (decreased ovulation)
17,000 ppm	female	death during pregnancy
17,000 ppm	female	failure to mate
17,000 ppm	male

 

Table 3: Summary of Reproductive Outcome: P0 Generation

Dose group (ppm)	control	500	1500	5000	17,000
Number of females paired	28	28	28	28	28
No. of females with evidence of copulationa	28	28	27	26	8
No. of females pregnantb	23	26	24	25	24
No. of females that littered	21	26	24	23	24
Mating Index (%) c	100	100	100	100	96.4
Fertility Index (%) d	82.1	92.9	85.7	89.3	88.9
Precoital Interval (Days)	2.3e 1.5(28)	2.6 1.9(28)	2.5 1.8(27)	2.7 1.7(28)	3.0 2.3(26)
Gestation Length (Days)	22.3 0.5(21)	22.3 0.7(26)	22.2 0.5(23)	22.2 0.4(23)	22.3 1.1(23)
Number of Implantation Sites	13.4 4.6(23)	13.2 4.1(24)	15.0 1.7(22)	13.8 4.1(24)	14.3 2.8(24)
Post-Implantation Loss (%) f	3.6 6.5(21)	2.9 5.6(24)	4.4 6.8(22)	3.6 8.2(23)	5.4 8.4(24)

a Evidence of copulation = intravaginal copulatory plug or sperm in vaginal lavage.

b Pregnant = uterine implantation sites or delivery.

c No. of females mated ÷ No. of females paired X 100

d No. of females pregnant ÷ No. of females mated X 100

e Data summarized as: Mean Standard Deviation (n)

f (No. of implantation sites – No. of Pups Born) ÷ No. of implantation sites X 100.

Note: Mated = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites,or delivery of a litter.

 

Table 4: Incidence of thyroid follicular cell hypertrophy in female P1 rats

Dose group (ppm)	control	500	1500	5000	17,000
Number of rats per sex	28	28	28	28	28
Thyroid: hypertrophy	2	0	0	0	8

 

Table 5: Causes of Reproductive Failure in P1 Male and Female Rats

Dose group	Sex	Gross and/or microscopic findings
control	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	male	failure to mate
500 ppm	female	ovaries: absence of recent corpora lutea (decreased ovulation)
5000 ppm	female	failure to mate
17,000 ppm	female	failure to mate
17,000 ppm	female	ovaries: absence of recent corpora lutea (decreased ovulation)

 

Table 6: Summary of Reproductive Outcome: P1 Generation

Dose group (ppm)	control	500	1500	5000	17,000
Number of females paired	28	28	28	28	28
No. of females with evidence of copulationa	26	22	28	26	25
No. of females pregnantb	24	19	25	25	25
No. of females that littered	23	19	25	24	25
Mating Index (%) c	92.9	78.6	100	96.4	96.4
Fertility Index (%) d	92.3	86.4	89.3	92.6	92.6
Precoital Interval (Days)	3.1e 2.3(26)	3.6 3.4(22)	3.1 2.6(28)	3.5 1.8(26)	3.8 3.4(24)
Gestation Length (Days)	22.2 0.7(23	22.0 0.4(19)	22.2 0.5(25)	22.0 0.3(23)	22.0 0.4(22)
Number of Implantation Sites	14.3 4.3(23)	14.9 1.8(19)	14.8 2.5(25)	15.0 2.8(24)	15.4 2.0(25)
Post-Implantation Loss (%) f	10.1 10.0(23)	7.7 10.2(19)	5.5 7.3(25)	2.3@ 3.8(23)	4.1 6.4(25)

a Evidence of copulation = intravaginal copulatory plug or sperm in vaginal lavage.

b Pregnant = uterine implantation sites or delivery.

c No. of females mated ÷ No. of females paired X 100

d No. of females pregnant ÷ No. of females mated X 100

e Data summarized as: Mean Standard Deviation (n)

f (No. of implantation sites – No. of Pups Born) ÷ No. of implantation sites X 100.

Note: Mated = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites,or delivery of a litter.

 

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

^ Cochran-Armitage test for trend

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

# Trend test (Jonckheere-Terpstra) significant

~ next to control mean indicates no analyses were performed

 

Table 7: Incidence of deaths in F1 and F2pups

Dose group (ppm)	control	500	1500	5000	17,000
F1 male pups	2	5	4	2	11
F1 female pups	5	3	8	0	4
F2 male pups	6	3	3	9	5
F2 female pups	5	1	0	10	3

 

Table 8: Summary of pup survival: F1 Generation

Dose group (ppm)	control	500	1500	5000	17,000
Number of viable litters	21	25	24	23	23
PND 4	21	25	24	23	22
PND 7	21	25	24	23	22
PND 14	21	25	24	23	22
PND 21	21	25	24	23	22
Number of pups born	13.9a	13.0	14.4	13.7	13.6
	3.7(21)	4.0(26)	2.0(24)	3.6(23)	3.1(24)
born alive	13.6	12.7	14.0	13.7	13.0
	3.8(21)	4.2(26)	2.1(24)	3.7(23)	3.4(24)
Number of pups alive on PND 0	13.6	12.7	14.0	13.7	13.0
	3.8(21)	4.2(26)	2.1(24)	3.7(23)	3.4(24)
PND 4 pre-cull	13.4	13.1	13.8	13.6	13.0
	3.8(21)	3.4(25)	2.0(24)	3.7(23)	2.0(22)
PND 4 post-cull	7.6	7.7	8.0	7.7	8.0
	1.4(21)	1.2(25)	0.0(24)	1.1(23)	0.0(21)
PND 7	7.5	7.7	8.0	7.7	8.0
	1.4(21)	1.2(25)	0.2(24)	1.1(23)	0.0(22)
PND 14	7.5	7.7	8.0	7.7	8.0
	1.4(21)	1.2(25)	0.2(24)	1.1(23)	0.0(22)
PND 21	7.5	7.7	7.9	7.7	7.8
	1.4(21)	1.2(25)	0.3(24)	1.1(23)	1.1(22)
Live Born Index^b	97.4	94.3	97.1	99.2	92.8
	5.6(21)	19.5(26)	5.0(24)	2.7(23)	20.4(24)
Viability Index^c	98.9	99.4	98.3	99.7	94.9
	2.8(21)	2.0(25)	3.5(24)	1.5(23)	10.8(22)
Lactation Index^d	98.9	99.5	98.5	100.0	97.2
	3.6(21)	2.4(25)	4.1(24)	0.0(23)	13.2(22)
Sex Ratio^e	44.9	50.8	56.7!	49.4	50.0
	11.6(21)	19.0(26)	14.8(24)	13.8(23)	19.7(24)

a Data summarized as: Mean

Standard Deviation (n)

b Number of pups born alive ÷ Number of pups born X 100

c Number of pups alive on day 4 (Preculling) ÷ Number of pups born alive X 100

d Lactation Index: Mean percent survival from Day 4 Postculling to Day 21.

e Number of male fetuses born ÷ total number fetuses born X 100

 

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

! Analysis of Covariance and Dunnett-Hsu

~ next to control mean indicates no analyses were performed

 

Table 9: F1 Weanlings: Mean Absolute and Relative Spleen Weights in Male and Female Rats

Dose group (ppm)	control	500	1500	5000	17,000
Male - spleen
spleen mean final body weight (g)	59.5	56.8	56.3	53.1*	51.0*
spleen absolute weight (g)	0.266	0.254	0.238	0.232	0.199*
spleen weight/brain weight x 100	17.480	16.838	15.735	15.466	13.379*
spleen weight/body weight x 100	0.448	0.448	0.421	0.433	0.391*
Female - spleen
spleen mean final body weight (g)	56.0	54.8	55.0	52.2	47.7*
absolute weight (g)	0.264	0.243	0.247	0.232	0.182*
spleen weight/brain weight x 100	17.913	16.609	16.811	15.890	12.646*
spleen weight/body weight x 100	0.471	0.443	0.450	0.444	0.382**

* statistically significant by parametric comparison to control (Dunnett/Tamhane-Dunnett Test).

** statistically significant by nonparametric comparison to control (Dunn’s Test).

 

Table 10: Summary of pup survival: F2 Generation

Dose group (ppm)	control	500	1500	5000	17,000
Number of viable litters	22	19	25	24	25
PND 4	21	19	25	24	25
PND 7	21	19	25	24	25
PND 14	21	19	25	24	25
PND 21	21	19	25	24	25
Number of pups born	13.1a	13.7	13.9	14.8	14.7
	4.1(23)	2.2(19)	2.5(25)	2.9(24)	1.9(25)
born alive	12.8	13.6	13.8	14.7	14.4
	4.1(23)	2.2(19)	2.5(25)	2.8(24)	2.1(25)
Number of pupsalive on PND 0	12.8	13.6	13.8	14.7	14.4
	4.1(23)	2.2(19)	2.5(25)	2.8(24)	2.1(25)
PND 4 pre-cull	13.1	13.6	13.6	14.6	14.3
	2.9(21)	2.2(19)	2.5(25)	2.8(24)	2.2(25)
PND 4 post-cull	7.9	8.0	8.0	7.9	8.0
	0.5(21)	0.0(19)	0.2(25)	0.4(24)	0.0(25)
PND 7	7.8	8.0	8.0	7.9	8.0
	0.6(21)	0.0(19)	0.2(25)	0.4(24)	0.2(25)
PND 14	7.8	8.0	7.9	7.9	8.0
	0.6(21)	0.0(19)	0.3(25)	0.4(24)	0.2(25)
PND 21	7.8	7.9	7.9	7.8	8.0
	0.6(21)	0.3(19)	0.3(25)	0.5(24)	0.2(25)
Live Born Indexb	93.8	99.3	99.5	99.0	98.2
	21.4(23)	2.2(19)	2.6(25)	3.9(24)	6.4(25)
Viability Indexc	98.9	99.6	98.5	99.8	98.8
	3.7(21)	1.6(19)	3.1(25)	1.2(24)	3.5(25)
Lactation Indexd	98.8	98.7	99.5	99.0	99.5
	5.5(21)	3.8(19)	2.4(25)	3.4(24)	2.4(25)
Sex Ratioe	50.6	47.9	43.8!	45.6	50.4
	16.4(23)	14.1(19)	12.9(25)	11.0(24)	10.8(25)

a Data summarized as: Mean

Standard Deviation (n)

b Number of pups born alive ÷ Number of pups born X 100

c Number of pups alive on day 4 (Preculling) ÷ Number of pups born alive X 100

d Lactation Index: Mean percent survival from Day 4 Postculling to Day 21.

e Number of male fetuses born ÷ total number fetuses born X 100

 

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

! Analysis of Covariance and Dunnett-Hsu

~ next to control mean indicates no analyses were performed

 

Table 11: F2 Weanlings: Mean Absolute and Relative Spleen Weights in Male and Female Rats

Dose group (ppm)	control	500	1500	5000	17,000
Male - spleen
spleen mean final body weight (g)	61.9	60.2	60.7	59.0	56.1*
absolute weight (g)	0.289	0.253	0.275	0.256	0.243*
spleen weight/brain weight x 100	18.929	16.590	18.101	16.756	16.172*
spleen weight/body weight x 100	0.467	0.420	0.450	0.432	0.432
Female - spleen
mean final body weight (g)	57.8	58.0	57.6	55.5	52.3**
spleen absolute weight (g)	0.267	0.258	0.272	0.261	0.226
spleen weight/brain weight x 100	18.056	17.180	18.429	17.370	15.566
spleen weight/body weight x 100	0.459	0.443	0.469	0.469	0.424

* statistically significant by parametric comparison to control (Dunnett/Tamhane-Dunnett Test).

** statistically significant by nonparametric comparison to control (Dunn’s Test).