Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation study (OECD 416), oral, rat:

NOAEL (reproductive toxicity): 17 000 ppm (corresponding to 1048.26 mg/kg bw/day (males) and 1158 mg/kg bw/day (females), worst case assumption over all generations and exposure periods)

NOAEL (general toxicity, P0/P1 females): 5000 ppm (corresponding to 330.51 mg/kg bw/day, worst case assumption over all generations and exposure periods)

NOAEL (general toxicity, P0/P1 males): 1500 ppm (corresponding to 91.93 mg/kg bw/day, worst case assumption over all generations and exposure periods)

NOAEL (general toxicity, F1/F2 pups): 5000 ppm (correlated to maternal toxicity)

Link to relevant study records
Reference
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
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
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
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
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




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



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
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.
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.
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: 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
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.
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.
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.
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.
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.
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
Critical effects observed:
no
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: 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.
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.
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.
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.
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.
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.
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.
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.




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.
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.
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.


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.
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.
Key result
Critical effects observed:
no
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

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 Indexb

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 Indexc

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 Indexd

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 Ratioe

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).

 

 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 048.26 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction

 

Toxicity to reproduction of the test substance was tested in a GLP-compliant Two-Generation Reproductive Toxicity study in rats (Crl:CD(SD)) according to OECD 416 (2011 m). 28 Male and 28 female rats (Crl:CD(SD) per generation were fed with a diet containing 500, 1500, 5000 and 17000 ppm test substance. Control animals received standard diet (Rodent Lab Diet®5002).

 

Parental animals (P0 and P1) were pre-treated over a period of 10 weeks followed by a mating period of up to 2 weeks, females also during gestation period (22 days) and lactation period (22 days). F1 and F2 offspring was weaned up to PND 21. At weaning, selected F1 offspring (one rat per sex per litter when possible) were randomly selected to serve as parents for the F2 generation. F1 and F2 litters were culled to 4 pups/sex/litter on postnatal day 4; all remaining pups were discarded without further evaluation.

 

Examinations in parental P0 and P1/F1 animals included mortality, clinical signs, body weights, food intake, food efficiency and test substance intake. In addition, analysis of reproductive function, including analysis of the oestrus cycle, sperm measures, reproductive performance, implantation sites and post-implantation losses, gestation length and pre-coital interval length were determined. The following reproductive indices were determined: mating, fertility index and post-implantation loss. At necropsy of parental animals (males after siring litters/females after weaning), gross pathology and histopathology (selected organs of control and high dose animals) were performed and organ weights determined.

 

In the F1 and F2 offspring abnormal behaviour and appearance, live and dead pups, number and sex of pups, pup weight, litter weight, stillbirths, live births, postnatal mortality and weight gain were determined. Furthermore, 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) were determined. Litter observations were performed on Day 0, 4, 7, 14 and 21 postpartum. In addition, the following indices were determined: live birth, viability and lactation index. 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 pathological examination on PND 21were performed on the following organs: brain, spleen, thymus and thyroid gland (randomly selected from F1 and F2 weanlings (one weanling/sex/litter). Presences of gross lesions were determined for all F1 and F2 weanlings. Individual absolute and relative organ weights (brain, spleen, and thymus) were determined. 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. Furthermore, a quantitative evaluation of primordial and growing follicles was conducted on 10 lactating F1 females (surviving to scheduled sacrifice) from control and high-dose groups.

 

In the P0 parental generation, death of one 17 000 ppm and two 500 ppm females was observed. The deaths were preceded by clinical symptoms, but since arterial thrombosis, pyelonephritis and dystocia were determined to be the respective cause of death, all findings were not considered treatment-related. All other clinical signs observed in P0 animals occurred with low frequency without dose-response and the reported observations were generally unremarkable and of a nature commonly seen in rats of this age and thus not considered treatment-related.

Adverse test substance-related effects in P0 animals consisted of the following: systemic toxicity evident as effects on adult body weight parameters and nutritional parameters (food consumption and/or food efficiency) in P0 males at 5000 ppm and above and P0 females at 17 000 ppm. Significant reduction in food consumption in males at 500 ppm was considered occasional and not treatment-related.

Gross pathology and histopathology did not reveal any treatment-related findings; observed lesions were within the normal ranges for this strain and age of rats.

Organ weight findings at high dose included increased mean relative weight of several organs (adrenal gland, kidneys, seminal vesicles, testes, epididymides, right cauda epididymis, and testes) in males and a decrease (4%) in the mean absolute brain weight in females. Effects on adrenal gland weights in males were furthermore detected at 5000 and 1500 ppm, the effect on brain weight was also visible in males at 5000 and 1500 ppm and in females of the 5000 ppm group. All observed organ weight findings were not considered treatment-related, since either microscopic correlates were missing and/or a dose-response was not visible; the findings were attributed to body weight changes seen at the same dose and/or the effects were not statistically significant.

No effects were observed on oestrus cycle and sperm parameters. Data on 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.

 

In the P1 parental generation, one female at 5000 ppm and one female of the control group were sacrificed due to clinical signs of dystocia, however this was only evident at necropsy in the control group animal. A further female in the 5000 ppm group was also sacrificed in extremis due to presence of a mammary gland tumor and a male of this dose group was killed accidentally. In addition, one male of the 500 ppm group died due to chronic progressive nephropathy. None of the observed deaths were considered treatment-related. Other clinical findings observed were all considered test substance unrelated since signs observed occurred at low frequency with no dose-response and were generally unremarkable and of a nature commonly seen in rats of this age. Adverse test substance-related effects in P1/F1 adult animals consisted of depressed body weights at 17,000 and 5000 ppm in males and at 17000 ppm in females. Occasional effects on body weights in females of the 1500 ppm ppm dose group were considered incidental and treatment unrelated. Occasionally observed instances of statistical significanct effects on body weights in females at 1500 ppm that did not appear to be dose-related were considered incidental. In addition, food consumption and/or food efficiency were reduced in P1 males at 17.000 ppm. Gross pathology did not reveal any treatment-related findings and observed lesions were within the normal ranges for this strain and age of rats.

Histopathological findings consisted of minimal thyroid follicular cell hypertrophy in P1/F1 adult females at 17,000 ppm. 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 also not demonstrate any test substance-related findings. Observed effects on organ weights in the highest dose group were decreased brain, liver, spleen, thyroid gland (absolute) and brain (relative) weights in males and decreases of absolute brain weights in females. In addition, in 5000 and 17,000 ppm dose groups, 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. All organ weight findings in P1/F1 adult rats were considered spurious or were related to significant effects on body weight and thus considered treatment-unrelated.

There were no significant differences in the total number of primordial and pre-antral follicles in selected females between the control and 17,000 ppm group. At 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 effects observed in P0 females at any dose level, this effect was not considered treatment-related. A statistically significant decrease in post-implantation loss was observed at 5000 ppm, but not at 17,000 ppm and thus, due to a missing dose-response, considered unrelated to the test substance. Other effects on oestrus cycle, sperm parameters or reproductive performance were not observed. Data on mating, fertility, pre-coital interval length, gestation length and implantation site counts were comparable across all dose groups.

 

There were no test substance-related clinical findings in F1 pups. Litter size, number of pups born/born alive and live birth, survival and lactation indices were comparable across all dose groups. Reduced F1 pup weights were observed at 17,000, 5000 and 500 ppm. At lower doses the magnitude of body weight reduction was minimal and transient and it was not observed at 1500 ppm. Thus, the effects observed on body weight in the lower dose groups are not considered as indicative for adverse effects of toxicological significance and hence, only the effects observed on body weight at the high dose level are considered as adverse.

A slight decrease in spleen weight parameters (absolute weight, relative weight/brain weight, relative weight/body weight) were observed in high-dose F1 males and females. Histopathology was not conducted on any F1 weanling spleens. The decrease in spleen weight parameters was interpreted to be evidence of retarded growth rather than splenic toxicity. There were no test substance-related gross pathology or histopathological findings (analyses were limited to thyroid glands in control and high dose F1 pups). Effects observed were consistent with normal background lesions in rats of this age and strain.

At the highest dose level a slight increase in the number of days to reach preputial separation compared to control males was observed. Analysis of covariance with body weight on the day of weaning as the covariate, revealed no statistically significant differences between the control group and the 17,000 ppm F1 males. Furthermore, the data were within the historical control range. Therefore, the delay in preputial separation was considered to be secondary to the lower body weight at weaning and non-adverse. There was no effect observed on the anogenital distance. At 1500 ppm a statistically significant increase in the number of male pups was observed. Since this effect was not observed at higher doses, it was considered unrelated to the test substance. In F1 females, no test substance-related effects on timing for achievement of vaginal patency was observed at any dietary level tested.

 

There were no test substance-related clinical findings observed in F2 pups. Litter size, number of pups born/born alive and live birth, survival and lactation indices were comparable across all dose groups. At 17 000 ppm a test substance-related reduction in mean pup weight was observed. Mean pup weights were 8% lower than controls on PND 21. A slight decrease in spleen weight parameters was observed in F2 males and females of the highest dose group (although it was less than that observed in the F1 weanlings). Histopathology was not conducted on any F2 weanling spleens. The decrease in spleen weight parameters was interpreted to be evidence of retarded growth rather than splenic toxicity.

There were no test-substance related gross pathology or histopathological findings (analyses were limited to thyroid glands in control and high dose F1 pups). Effects observed were consistent with normal background lesions in rats of this age and strain. No effects on sexual maturation or anogenital distance were observed. At 1500 ppm a statistically significant decrease in the number of male pups was observed. Since this effect was not observed at higher doses nor in the F1 generation (in the F1 generation a slight increase in males was observed), it was considered unrelated to the test substance.

 

Since there were no test substance-related effects observed on reproduction parameters in P0 and P1 parental animals, a NOAEL for reproduction was determined at 17,000 ppm for the P0 and P1 generation (corresponding to 1048.26 – 1521.77 mg/kg bw for P0 and P1 males, respectively and to 1158.02 – 1192.19 mg/kg bw/day in P0 and P1females, respectively (worst case assumption over all exposure periods)).

A LOAEL for general toxicity was determined at 5000 ppm (corresponding to 299.09-425.76 mg/kg bw (worst case assumption over all exposure periods)) and a NOAEL for general toxicity was determined at 1500 ppm (corresponding to 91.93-126.33 mg/kg bw (worst case assumption over all exposure periods)) for P0 and P1 adult males, based on reduced body weight and food parameters at 5000 ppm.

For adult P0/P1 females a LOAEL for general toxicity was determined at 17 000 ppm (corresponding to 1158.02 – 1192.19 mg/kg bw/day (worst case assumption over all exposure periods)) and a NOAEL for general toxicity was determined at 5000 ppm (corresponding to 330.51 – 335.75 mg/kg bw/day (worst case assumption over all exposure periods)), based on reduced body weight and food parameters at 17,000 ppm.

For F1/F2 pups a LOAEL of 17,000 ppm and a NOAEL of 5000 ppm (both correlated to maternal toxicity) were determined for general toxicity, based on reduced body weights and spleen weights. Taking into consideration that systemic toxicity was evident in maternal animals of the P0 and P1 generation at 5000 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 No. 1272/2008 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”.

 

A DNEL for workers was derived for longterm exposure, systemic effects to account for possible effects on body weight parameters and food efficiency which may be linked to maternal toxicity and consequently to secondary non-specific maternal developmental toxicity.

Effects on developmental toxicity

Description of key information

Developmental toxicity study (OECD 414), rats:

NOAEL (general toxicity, maternal animals): >= 1000 mg/kg bw/day (highest dose tested)

NOAEL (fetal developmental toxicity): >= 1000 mg/kg bw/day (highest dose tested)

 

Developmental toxicity study (OECD 414), rabbits:

NOAEL (general toxicity, maternal animals): 300 mg/kg bw/day

NOAEL (maternal developmental toxicity): 500 mg/kg bw/day (correlated to maternal toxicity)

NOAEL (fetal developmental toxicity): 1000 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September - 31 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: approximately 67 days
- Weight at study initiation: 222 - 270 g
- Housing: individually in stainless steel, wire-mesh cages suspended above cage boards
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet 5002 (pellets), 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

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations of the test substance in the vehicle were prepared daily. To achieve the respective test concentrations of active ingredient, the formulations were adjusted for sample purity (92.2%).

VEHICLE
- Other: Volume administered: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were taken 2 times near the beginning and the end of the study. Analyses addressed uniformity of mixing, concentration and stability. One vehicle sample and 3 samples (top,middle,and bottom samplings) of each concentrations were analysed to verify concentration and uniformity of mixing near study beginning and one vehicle sample and one sample of each concentration was analysed to verify concentration near end of the study. A fourth sample of each concentration was held for 5 h at room temperature and was then analysed for stability of the test substance in the formulation. Sample analyses were perfomed by Critical Path Services (CPS) and were shipped frozen. Data from the analysis of the formulation samples, including the 5-h stability sample, indicated that the test substance was uniformly mixed in the vehicle at the targeted concentrations and was stable in the vehicle under the conditions of the study. Test substance was not found in the control sample.

The COA included in the original finalised report listed a purity of 92.2% which did not fully account for all the inorganic impurities associated with this sample. Follow-up analysis of DPX-MAT28-009 was conducted after the final report had issued. The revised COA, included in this revised report, presents the purity as 90.5% and now accounts for the full impurity profile as determined in GLP analytical study. This reduction in determined purity of 1.7% had minimal impact on the reported doses determined by chemical analyses and on the values utilized for risk assessments. Therefore, the reported doses or dietary intake values have not been adjusted.

Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Days 6 -20
Frequency of treatment:
daily
Duration of test:
21 Days
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a pilot developmental toxicity study in rats with a similar compound (methyl ester of the test substance), the test substance was administered to time-mated female Crl:CD(SD) rats (8/group) once daily on GD 6 - 20 at dosages of 25, 300, or 1000 mg/kg/day. During the in-life portion of the study, maternal body weights, food consumption, and clinical signs were collected. On GD 21, all dams were euthanized and examined grossly. Gravid uterine weight was recorded to permit calculation of the adjusted maternal final body weight. The uterine contents were examined and described (number and status of implantation sites, and fetal assessment (viable, non-viable, location, sex, fetal weights, external alterations). There were no test substance-related maternal or developmental effects noted for any endpoint analyzed at any dose level. Based on these data, the dose levels selected for the current study were 30, 100, 300, and 1000 mg/kg/day.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (GD 6 - 20), once daily on GD 21

BODY WEIGHT: Yes
- Time schedule for examinations: once daily on GD 6 -21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Time schedule for examinations: GD 6, 8, 10, 12, 14, 16, 18, 20 and 21

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: livers were weighed and saved for histopathological examination

OTHER: Gross external and a visceral examinations were performed. In the absence of any demonstrated test substance-related effects on either the gross findings or liver weights, microscopic examination of the livers and gross lesions was not considered necessary.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of pregnant (%)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

- Remarks: In addition, all live fetuses with malformations visible at external examination were examined for soft tissue alterations; decapitation of these fetuses for head examination was performed at the discretion of the study director or designee. The frozen heads of decapitated fetuses (fetuses that were decapitated prior to visceral examination, approximately half of the fetuses) were examined by a serial sectioning technique. The skeletal bodies of all the fetuses and the skulls of half the fetuses (fetuses that were not designated for head examination) were examined for alterations.
Statistics:
The level of significance selected was p < 0.05.
For an detailed overiew on statistics, please refer to table 1 in the "Any other information on materials and methods incl. tables" section.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: stained skin/fur - brown was observed in a single animal

100 mg/kg bw/day: wet fur was observed in a single animal

control, 30, 100 and 300 mg/kg bw/day: hair loss was observed in single animals at different time points.

All observations that were recorded were unremarkable and occurred infrequently and were thus not considered adverse.

For details please refer to table 2 in the “Any other information on results incl. tables” section.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality 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
Description (incidence and severity):
For details please refer to table 3 in the “Any other information on results incl. tables” section.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day: liver discoloration was observed in one animal. This single event was considered incidental and non-adverse.
For details please refer to table 4 in the “Any other information on results incl. tables” section.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Dead fetuses:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Other effects:
not examined
Details on maternal toxic effects:
There were no test substance-related effects on reproductive outcome. The number of pregnant, the mean number of corpora lutea, implantation sites, resorptions (total, early and late), live/dead fetuses were comparable across all groups tested.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: single incidences of protruding tongue (malformation), anopthalmia (malformation) and small eye bulge (gross finding) were observed, but considered an incidental finding and not considered adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: single incidence of fused cervical arch (malformation) was observed, but considered an incidental finding and not considered adverse.
control, 30, 100, 300 and 1000 mg/kg bw/day: in all dose groups variations of the thoracic centrum and ribs were observed. In addition variations of the sternebrae were observed in all groups, exluding the 30 mg/kg bw/day dose group.
All observed variations were either incidental findings, not dose-related or occurrence did not differ significantly between dose groups and were thus not considered adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day: single incidence of discolored intestine (gross finding) was observed, but considered an incidental finding and thus not adverse.
30 mg/kg bw/day: single incidence of discolored liver (variation) was observed, but considered an incidental finding and thus not adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
There were no test substance-related effects on quantitative litter data. The number of live/dead fetuses, mean fetal weight and sex ratio were comparable across all groups tested. There were no test substance-related fetal malformations or variations observed at any dose level tested. The fetal alterations that were observed were unremarkable and occurred with low frequency across the dose levels tested.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 2: Summary of Maternal Clinical Observations

Dose group (mg/kg bw/day)

 

0

 

30

 

100

 

300

1000

Number of animals

 

25

 

25

 

25

 

25

25

Hair loss

Number of Observations

Number of animals

 

                       

 

 

4

1

 

 

17

3

 

 

16

3

 

 

3

3

 

 

Days from - to

16

19

12

21

15

21

19

21

 

Scheduled sacrifice

Number of Observations

Number of Animals

25

25

25

25

25

25

25

25

25

25

Days from - to

21

21

21

21

21

21

 21

21

 21

21

Stained skin/fur - brown – chin Number of Observations

Number of Animals

 

 

 

 

 

1

1

Days from - to

 

 

 

 

19

19

Wet fur

– chin/perinasal Number of Observations

Number of Animals

 

 

 

1

1

 

 

Days from - to

 

 

18

18

 

 

 

Table 3: Mean Final Body Weights, Absolute and Relative Organ Weights for Maternal Rats

Dose group (mg/kg bw/day)

0

30

100

300

1000

Final body weight

 

392.6

 

394.6

 

397.8

 

393.5

 

386.6

 

19.9(25)

21.8(25)

23.3(25)

24.2(25)

25.7(25)

Liver

Mean

15.40

15.04

15.49

15.71

14.72

Standard deviation (n)

1.70(24)

1.31(25)

1.66(25)

2.00(25)

1.38(24)

Liver/Final body weight x 100

 

3.922

 

3.816

 

3.891

 

3.994

 

3.813

Standard deviation (n)

0.350(24)

0.315(25)

0.294(25)

0.439(25)

0.345(24)

 

Table 4: Summary of Maternal Gross Observations

Dose group (mg/kg bw/day)

0

30

100

300

1000

Number of animals

25

25

25

25

25

Liver

No visible lesions

Discoloration

 

              

 

25

0

 

24a

0

 

23

1

 

23b

0

 

25

0

 

Whole Body

No visible lesions

 

25

 

 

24a

 

 

24

 

 

23b

 

 

25

 

 

a Maternal gross observations were inadvertently not recorded for Animal #204.

b Maternal gross observations were inadvertently not recorded for Animal #401 and #409.

 

Table 5: Reproductive Outcome

Dose group (mg/kg bw/day)

0

30

100

300

1000

Number of animals in group

25

25

25

25

25

Not Pregnant (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Died/Killed

0

0

0

0

0

Survived to scheduled kill

0

0

0

0

0

Pregnant (%)

25(100.0)

25(100.0)

25(100.0)

25(100.0)

25(100.0)

Died/Killed/Aborted

0

0

0

0

0

with total resorption

0

0

0

0

0

with live fetus at scheduled kill

25

25

25

25

25

Corpora Lutea (Mean)

13.5

13.3

13.6

13.7

13.7

Standard Deviation (n)

3.1(25)

2.6(24)

3.0(25)

2.0(25)

2.2(25)

Implants (Mean)

11.7

12.2

11.9

12.3

12.3

Standard Deviation (n)

1.3(25)

2.2(25)

1.9(25)

1.7(25)

2.1(25)

Total Resorptions (Mean)

0.08

0.08

0.20

0.56

0.40

Standard Deviation (n)

0.28(25)

0.28(25)

0.50(25)

2.02(25)

0.65(25)

Early Resorptions (Mean)

0.08

0.08

0.20

0.56

0.40

Standard Deviation (n)

0.28(25)

0.28(25)

0.50(25)

2.02(25)

0.65(25)

Late Resorptions (Mean)

0.00~

0.00

0.00

0.00

0.00

Standard Deviation (n)

0.00(25)

0.00(25)

0.00(25)

0.00(25)

0.00(25)

Dead Fetuses (Mean)

0.0~

0.0

0.0

0.0

0.0

Standard Deviation (n)

0.0(25)

0.0(25)

0.0(25)

0.0(25)

0.0(25)

Live Fetuses (Mean)

11.6

12.2

11.7

11.7

11.9

Standard Deviation (n)

1.3(25)

2.2(25)

2.0(25)

2.7(25)

2.1(25)

Male Fetuses (Mean)

5.5

5.6

5.6

6.2

6.0

Standard Deviation (n)

1.8(25)

2.0(25)

2.3(25)

1.9(25)

2.1(25)

Female Fetuses (Mean)

6.1

6.6

6.2

5.6

5.8

Standard Deviation (n)

1.7(25)

2.1(25)

2.0(25)

2.0(25)

2.1(25)

Fetal Weight (Mean)

5.70

5.53

5.64

5.50

5.53

Standard Deviation (n)

0.24(25)

0.30(25)

0.25(25)

0.45(25)

0.27(25)

Male Weight (Mean)

5.89

5.70

5.77

5.67

5.66

Standard Deviation (n)

0.28(25)

0.30(25)

0.26(25)

0.41(25)

0.27(25)

Female Weight (Mean)

5.55

5.38

5.52

5.28

5.44

Standard Deviation (n)

0.26(25)

0.31(25)

0.28(25)

0.48(24)

0.32(25)

Sex Ratio (Mean)

0.47

0.47

0.47

0.54

0.51

Standard Deviation (n)

0.14(25)

0.15(25)

0.17(25)

0.16(25)

0.15(25)

~ next to control mean indicates no analyses were performed. Statistical analyses are only conducted on the total mean fetal weight; the means for males and females are presented for information only. Remarks: The incidence of pregnancy, maternal mortality, the total resorptions, and early deliveries/abortions were statistically analyzed using the Cochran-Armitage test. Live fetuses, dead fetuses, resorptions, corpora lutea, and implantations were analyzed by One-Way Analysis of Variance and Dunnett’s test. Fetal weight and sex ratio were analyzed using Analysis of Covariance and Dunnett-Hsu.

 

Table 6: Incidence of Fetal Malformations and Variations

Dose group (mg/kg bw/day)

0

30

100

300

1000

Total number of foetuses examined

290

304

293

293

297

External Defects: Number of Fetuses Examined

 

290

304

293

293

297

External Defects: Number of Litters Examined

25

25

25

25

25

Head

Tongue, Protruding (Malformation)

 

 

 

1 (0.3)

1 (4.0)

 

Head

Eye bulge, Small (Gross finding)

 

 

 

1 (0.3)

1 (4.0)

 

Head Defects: Number of fetuses examined

140

145

140

142

143

Head Defects: Number of litters examined

25

25

25

25

25

Head

Eye, Anophthalmia (Malformation)

 

 

 

1 (0.7)

1 (4.0)

 

Visceral Defects: Number of fetuses examined

140

145

140

142

143

Visceral Defects: Number of litters examined

25

25

25

25

25

Abdomen

Intestines, discolored (Gross finding)

 

 

1 (0.7)

1 (4.0)

 

 

Abdomen

Liver, discolored (Variation)

 

1 (0.7)

1 (4.0)

 

 

 

Skeletal - Head Defects: Number of fetuses examined

150

159

153

151

154

Skeletal - Head Defects: Number of litters examined

25

25

25

25

25

Skull

Frontal, Incomplete ossification (Variation)

 

 

5 (3.3)

1 (4.0)

 

 

Skull

Zygomatic, Incomplete ossification (Variation)

 

1 (0.7)

1 (4.0)

 

 

 

Skull

Interparietal, Incomplete ossification (Variation)

 

 

 

1 (0.7)

1 (4.0)

 

Skull

Supraoccipital, Incomplete ossification (Variation)

3 (2.0)

2 (8.0)

2 (1.3)

2 (8.0)

1 (0.7)

1 (4.0)

3 (2.0)

2 (8.0)

 

Skull

Parietal, Incomplete ossification (Variation)

 

1 (0.6)

1 (4.0)

1 (0.7)

1 (4.0)

2 (1.3)

2 (8.0)

 

Skeletal - Body Defects: Number of fetuses examined

290

304

293

293

297

Skeletal - Body Defects: Number of litters examined

25

25

25

25

25

Vertebrae

Cervical arch, Fused (Malformation)

 

 

 

1 (0.3)

1 (4.0)

 

Vertebrae

Thoracic centrum, Unossified (Variation)

 

 

 

1 (0.3)

1 (4.0)

 

Vertebrae

Thoracic centrum, Bipartite ossification (Variation)

6 (2.1)

6 (14.0)

1 (0.3)

1 (4.0)

2 (0.7)

2 (8.0)

1 (0.3)

1 (4.0)

8 (2.7)

6 (24.0)

Ribs

Rib, Short (Variation)

2 (0.7)

2 (8.0)

1 (0.3)

1 (4.0)

5 (1.7)

2 (8.0)

1 (0.3)

1 (4.0)

2 (0.7)

1 (4.0)

Ribs

Rib, Cervical rib (Variation)

3 (1.0)

2 (8.0)

3 (1.0)

1 (4.0)

4 (1.4)

2 (8.0)

1 (0.3)

1 (4.0)

 

Ribs

Rib, Full supernumerary rib (Variation)

3 (1.0)

1 (4.0)

 

 

 

 

Ribs

Rib, Thickened (Variation)

3 (1.0)

1 (4.0)

 

 

 

 

Ribs

Rib, Wavy (Variation)

4 (1.4)

1 (4.0)

 

1 (0.3)

1 (4.0)

 

 

Ribs

Rib, Short supernumerary (Variation)

2 (0.7)

2 (8.0)

2 (0.7)

2 (8.0)

2 (0.7)

2 (8.0)

 

2 (0.7)

1 (4.0)

Ribs

Rib, Extra ossification site (Variation)

19 (6.6)

10 (40.0)

13 (4.3)

6 (24.0)

10 (3.4)

9 (36.0)

 

9 (3.1)

4 (16.0)

5 (1.7)

5 (20.0)

Sternebrae

Unossified (Variation)

 

 

 

4 (1.4)

2 (8.0)

 

Sternebrae

Misaligned (Variation)

 

 

2 (0.7)

2 (8.0)

 

1 (0.3)

1 (4.0)

Sternebrae

Fused (Variation)

1 (0.3)

1 (4.0)

 

 

1 (0.3)

1 (4.0)

 

Upper line denotes number of affected fetuses

Lower line denotes number of affected litters

Figures in parenthesis denote percentage incidence

Calculated values do not include animals which either, were not pregnant, did not survive to the scheduled kill, had a total litter loss, aborted or are marked for exclusion

Conclusions:
CLP: not classified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August - 18 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Kalamazoo, Michigan, USA
- Age at study initiation: approximately 5.5 months (at the time of mating by the supplier)
- Weight at study initiation: 2981 - 4355 g
- Housing: individually in clean, stainless steel cages suspended above ground corncob bedding (Pel O’Cobs®; The Andersons, Cob Products Division, Maumee, Ohio, USA).
- Diet: PMI Nutrition International, LLC, Certified Rabbit LabDiet® 5322, ad libitum
- Water: reverse osmosis purified (on site) drinking water, ad libitum
- Acclimation period: approximately 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 - 19.2
- Humidity (%): 55.0 - 67.6
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance formulations were prepared daily, and maintained at room temperature for no more than 5 h from the completion of preparation to the completion of dosing. The test substance formulations were stirred for at least 30 min prior to dosing and continuously throughout use. The appropriate amount of the test substance for each formulation was weighed into a tared, glass mortar. A small amount of the vehicle was added to each mortar, and the test substance and vehicle were ground with a pestle until a uniform mixture was obtained. The resulting mixture was quantitatively transferred into a calibrated glass container. Approximately 70% of the total volume of the vehicle was added to each container and the suspension was mixed using a magnetic stirrer. Vehicle was then added to each container to bring the formulations to the calibration mark. The time at which the formulations were complete was recorded.

VEHICLE
- Amount of vehicle: 0.5% methylcellulase (w/v)
- Lot/batch no.: VX0285 (Spectrum Quality Products, Inc., New Brunswick, New Jersey, USA)
- Other: The appropriate amount of methylcellulase powder was dissolved in deionized water that had been heated to approximately 70°C. After cooling, additional deionized water was added to achieve the required final volume, and the solution was mixed until uniform using a magnetic stirrer. A sufficient volume of the vehicle was prepared approximately weekly. Aliquots were prepared for daily dispensation and stored refrigerated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration, duplicate samples for homogeneity determination were collected on 7 August 2007 from the top, middle and bottom of the 25 and 250 mg/mL dosing formulations. Formulations were mixed using a magnetic stirrer for at least 30 min prior to sampling. Duplicate samples for concentration analyses were collected from the middle strata of each dosing formulation and vehicle prepared on the first (7 August 2007), tenth (16 August 2007) and last (30 August 2007) day of test substance administration. Concentration analyses were performed using High Performance Liquid Chromatography. In this study, the formulations were prepared at theoretical test article concentrations ranging from 25 to 250 mg/mL, and the QC sample concentrations were 25.1, 100 and 251 mg/mL. All reported results met the acceptance criteria (concentration level had to be 85% to 115% of the QC target).
Stability of the test substance was established in a previous study (WIL-189192, 2008) for 4.25 h at room temperature at a concentration range of 25 to 250 mg/mL. The formulations analyzed met the WIL SOP requirement for homogeneity, i.e., the RSD for the mean concentration was 10% or less at a concentration within the acceptable limits (85% to 115% of target concentration).

The COA included in the original finalized report listed a purity of 92.2% which did not fully account for all the inorganic impurities associated with this sample. Follow-up analysis of the test substance presented the purity as 90.5%. This reduction in determined purity of 1.7% had minimal impact on the reported doses determined by chemical analyses and on the values utilized for risk assessments. Therefore, the reported doses or dietary intake values have not been adjusted.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Days 7 - 28
Frequency of treatment:
daily
Duration of test:
22 Days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on a previously conducted dose range-finding prenatal developmental toxicity study with the test substance in rabbits. In this study, the test substance was administered orally by gavage to 4 groups of 8 time-mated female New Zealand White rabbits once daily from gestation days 7 through 28. Dosage levels were 100, 300, 500 and 1000 mg/kg bw/day. Controls received the vehicle (0.5% methylcellulose) on a comparable regimen.
One female in the 1000 mg/kg/day group aborted on gestation day 23 following 10 days reduced food consumption. Furthermore, 1 female in the control group delivered on gestation day 29 following 11 non consecutive days of reduced food consumption. All other animals survived to the scheduled necropsy on gestation day 29; no test substance-related clinical findings were noted and no remarkable macroscopic findings were noted at any dosage level. Mean maternal body weights, body weight gains, net body weights, net body weight gains and food consumption in all test substance-treated group were generally similar to those in the control group. The mean litter proportion of post-implantation loss (primarily early resorptions) in the 1000 mg/kg bw/day group (19.2% per litter) was higher than in the control group value (4.2% per litter), and corresponded to a lower mean litter proportion of viable fetuses in this group (80.8% per litter compared to 95.8% in the control group); however, these differences from the control group were due primarily to 1 doe with only 2 viable fetuses. Mean fetal body weight (combined sexes) in the 1000 mg/kg/day group was also lower (10.7%) than the control group value, but was due primarily to 1 litter. Intrauterine growth and survival, as well as gravid uterine weights, were unaffected by test substance administration at dosage levels of 100, 300 and 500 mg/kg bw/day. The only external fetal malformation noted in this study was open eyelid for 1 fetus and 1 late resorption in the 300 and 500 mg/kg bw/day groups, respectively. No external developmental variations were observed in fetuses in this study.
Potential effects on intrauterine growth and survival, indicated by a higher mean litter proportion of post-implantation loss and a corresponding lower mean litter proportion of viable fetuses, as well as a reduction in mean fetal body weight, were noted at 1000 mg/kg/day when compared to the control group. The increase in post-implantation loss was due primarily to 1 doe with only 2 viable fetuses. Collectively, these differences from the control group were not of sufficient magnitude to preclude selection of 1000 mg/kg bw/day as the highest dosage level for use in a definitive prenatal developmental toxicity study. Thus, in the absence of any other evidence of maternal or fetal toxicity, dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for the definitive prenatal developmental toxicity study in pregnant New Zealand White rabbits.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked were moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0 (by the supplier), 4 and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for GD 7-10, 10-14, 14-17, 17-20, 20-24, 24-29 and 7-29. In addition, gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 7-29 body weight change exclusive of the weight of the uterus and contents) were calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: GD 4 -29
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: kidneys, liver, stomach and all gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination.

OTHER: The cranial, thoracic, abdominal and pelvic cavities were opened and the organs examined for females found dead, aborted or delivered. Maternal tissues were retained in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location of implantation sites, corpora lutea and viable fetuses were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Percent difference from control was presented for body weights and organ weights. Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, fetal body weights (separately by sex and combined) and organ weights were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group. Data obtained from non-gravid animals were excluded from statistical analyses.


Indices:
Group mean litter basis:
Postimplantation loss/litter = Number of dead fetuses, Resorptions (Early/Late)/Group / Number of gravid female/group

Proportional litter basis:
Summation per group (%) = Σ Postimplantation loss/litter (%) / Number of litters/group

Where:
Postimplantation loss/ litter (%) = Number of dead fetuses, Resorptions (Early/Late)/litter / Number of implantation sites/litter

Fetal developmental findings:

Summation per group (%) = Σ Viable fetuses affected/litter (%) / Number of litters per group

Where:
Viable fetuses affected/litter (%) = [Number of viable fetuses affected/litter / Number of viable fetuses/litter] x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased incidences of soft stool (beginning on GD 18 until end of the study) and hair loss were observed. Death of an animal on GD 13 was preceded by the following clinical findings: increased respiration at the 1 h post-dose observation on gestation day 7, rales and decreased defecation at the daily examinations during gestation days 9-13 and body weight loss. Two females of the highest dose group aborted and showed the following clinical findings: body weight loss, reduced food consumption, decreased defecation several days prior to abortion and red material in the cage pan or anogenital area on the day of abortion.

500 mg/kg bw/day: increased incidences of soft stool (beginning on GD 18 until end of the study) were observed.

300 mg/kg bw/day: one female that aborted showed the following clinical findings: body weight loss, decreased defecation and soft stool (the abortion was not considered test substance-related, due to absence of dose response)

Other clinical findings, including decreased defecation, rales and increased respiration, occurred infrequently, in a manner that was not dose-related, and were not considered test substance-related.

For details please refer to table 1 in the “Any other information on results incl. tables” section.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: two females were found dead. One animal was found dead at GD 13. Dead was preceded by clinical observations, but necropsy could not reveal the cause of death, but it was considered treatment-related. The other animal died on GD 27 due to an intubation error (non-treatment-related)

100 mg/kg bw/day: on gestation day 22 one female was found dead. Clinical observations were not noted prior to death. The death was not considered test substance-related in the absence of a dose-response.

For details please refer to table 2 in the “Any other information on results incl. tables” section.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: lower mean body weight gains and/or losses were observed during the treatment period (calculations were made for the following gestation periods: 14-17, 17-20, 20-24 and 24-29; gestation days 7-29). Only the mean body weight loss observed in this group during gestation days 14-17 achieved statistical significance (p<0.01). Females that aborted had test substance-related body weight losses of 636 g to 643 g (approximately 19% to 17%) from the first day of dosing (gestation day 7) through the day of abortion.

500 mg/kg bw/day: mean body weight loss was observed during gestation days 24-29 (not statistically significant, but considered treatment-related, because of the observed dose-response)

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

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
500 and 1000 mg/kg bw/day: mean food consumption was lower during geststaion day 14 -17, 17-20 and 20-24. Statistical significance was only achieved (p<0.05) at 1000 mg/kg bw/day (GD 14-17). Females that aborted showed reduced food consumption, generally 17 g/day from gestation days 13 and 17 for the same respective females through the day of abortion.

300 mg/kg bw/day: increases in food consumption during gestation days 11-12 and 12 -13 (statistically significant, but not considered test substance-related).

For details please refer to table 4 in the “Any other information on results incl. tables” section.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Scheduled necropsy on GD 29:
control, 100 and 1000 mg/kg bw/day: at scheduled necropsy on gestation day 29, 1 female in the control and one female in the 100 mg/kg/day groups and 2 females in the 1000 mg/kg/day group were determined to be non-gravid.

Unscheduled necropsy due to death or abortion:
1000 mg/kg bw/day: the dead female (GD 13) had red material around the nasal area, accessory spleen and white precipitate in the abdominal cavity adjacent to the right kidney; 9 normally developing implantations were noted in utero. A further animal that was found dead (GD 27) had red fluid in thoracic cavity and an esophageal perforation indicating an intubation error. This female was also noted with 9 dead fetuses in utero with no apparent external malformations. Two females aborted. The female that aborted on GD 20, had 1 early resportion and red material around the urogenital area, dark red contents in the vagina and 9 early resorptions in utero. The female that aborted on GD 26 had 1 late resorption and had red material around the urogenital area, cystic oviducts, thick white contents in the stomach and 9 late resorptions (mummified) in utero.

300 mg/kg bw/day: one female aborted 2 live and 7 dead pups with no apparent external malformations on gestation day 28. This female was internally normal (non-treatment-related, incidental finding, since no abortions observed at 500 mg/kg bw/day, absence of a dose-response).

100 mg/kg bw/day: one female that was found dead on gestation day 22, had an accessory spleen, cystic oviducts and 7 normally developing implantations and 1 early resorption in utero (The death was not considered test substance-related in the absence of a dose-response).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: two females aborted on GD 20 and GD 26. Both females had test substance-related body weight losses of 636 g to 643 g (approximately 19% to 17%) from the first day of dosing (gestation day 7) through the day of abortion. These maternal effects corresponded with reduced food consumption, generally 17 g/day from gestation days 13 and 17 for the same respective females through the day of abortion. The abortions were considered to be test substance-related but likely secondary to the decreased food consumption. Increased risk of abortion has been associated with decreased food consumption and greater than 10% body weight loss in rabbits.

300 mg/kg bw/day: one female aborted on GD 28 (non-treatment-related, incidental finding, since no abortions observed at 500 mg/kg bw/day, absence of a dose-response).

For details please refer to table 2 in the “Any other information on results incl. tables” section.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Control, 100, 300, 500, 1000: mean number of pre-implantation losses were 0.7, 1.3, 0.8, 0.6 and 1.0, respectively and were comparable among dose groups.

Control, 100, 300, 500, 1000: mean number of ost-implantation loss were 0.4, 0.6, 0.3, 0.5 and 0.7, respectively and were comparable among dose groups.

For details please refer to table 7 and 8 in the “Any other information on results incl. tables” section.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Control, 100, 300, 500, 1000: mean number of early resorptions were 0.2, 0.5, 0.2, 0.3 and 0.1, respectively and were comparable among dose groups.

Control, 100, 300, 500, 1000: mean number of late resorptions were 0.2, 0.2, 0.1, 0.2 and 0.6, respectively and were comparable among dose groups.

For details please refer to table 2, 7 and 8 in the “Any other information on results incl. tables” section.
Dead fetuses:
no effects observed
Description (incidence and severity):
For details please refer to table 7 and 8 in the “Any other information on results incl. tables” section.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
control, 100 and 1000 mg/kg bw/day: at scheduled necropsy on gestation day 29, 1 female in the control and 100 mg/kg/day groups and 2 females in the 1000 mg/kg/day group were determined to be non-gravid.

For details please refer to table 2 and 7 in the “Any other information on results incl. tables” section.

Other effects:
no effects observed
Description (incidence and severity):
No effects were observed on
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effect observed
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effect observed
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
number of abortions
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
500 and 1000 mg/kg bw/day: mean fetal weights (36.7 g and 38.1 g, respectively) were lower than that of controls (39.7 g). The difference was not statistically significant and no dose-response was visible, thus this finding was not considered treatment-related.

For details please refer to table 7 and 8 in the “Any other information on results incl. tables” section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
For details please refer to table 7 and 8 in the “Any other information on results incl. tables” section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
For details please refer to table 7 in the “Any other information on results incl. tables” section.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
For details please refer to table 8 in the “Any other information on results incl. tables” section.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
control, 100, 300, 500 and 1000 mg/kg bw/day: external malformations were noted in 4(3), 1(1), 1(1), 1(1) and 0(0) fetuses (litters), respectively. None of the findings differed statistical significantly from the control group. In addition, these external malformations occurred in single fetuses or litters or in a manner that was not dose-related and therefore, were not considered test substance-related.

For details please refer to table 9 and 10 in the “Any other information on results incl. tables” section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
control, 100, 300, 500 and 1000 mg/kg bw/day: skeletal malformations occurred in 1(1), 0(0), 4(3), 1(1) and 3(2) fetuses (litters), respectively.

300, 500 and 1000 mg/kg bw/day: Vertebral anomaly with or without associated rib anomaly was noted in one fetus of each dose group, respectively. Since the difference from the concurrent control group was not statistically significant, the finding was limited to three litters and occurs commonly in the historical control data, it was not considered to be test substance-related.

1000 mg/kg bw/day: two fetuses had only 6 cervical vertebrae present. The mean litter proportion of extra site of ossification (4.2% per litter) was higher (not statistically significant) than in the concurrent control group (0.0% per litter). This value exceeded the maximum mean value of the historical control data (2.3% per litter). Because this finding was primarily observed in five fetuses in one litter, it was not considered test substance-related.

300 mg/kg bw/day: centra anomaly (cervical centra nos. 3 and 4 fused) was noted in one fetus, the fetuses had costal cartilage anomaly consisting of the 7th cervical rib fused to the right costal cartilage no. 1 distally and associated with sternum in normal position.

control: one fetus had a forked rib.

None of the observed malformations was statistically significant. In addition, each malformation occurred in a single animal or litter or in a manner that was not dose-related and therefore, were not considered to be related to test substance administration. With exception of ossification, other skeletal developmental variations occurred similarly in the control group, were not observed in a dose-related manner and/or occurred in single fetuses and were thus not considered treatment-related.

For details please refer to table 9 and 10 in the “Any other information on results incl. tables” section. For Historical control data please refer to the attached PDF in the “Overall remarks, attachments” section.




Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
control, 100 and 500 mg/kg bw/day: one animal of each dose group had visceral malformations (persistent truncus arteriosus (control); retroesophageal aortic arch (100 mg/kg bw/day); hydrocephaly (500 mg/kg bw/day)). Evaluated on a percent per litter basis, none of the differences from the concurrent control group were statistically significant. Furthermore, these visceral malformations occurred in single animals or litters or in a manner that was not dose-related and therefore, were not considered to be test substance-related. Soft tissue developmental variations were noted in all groups, including the control group, but were not considered treatment-related, since they occurred similar in the control group, were not observed in a dose-related manner and/or the mean litter proportions were not statistically significantly different from the concurrent control group.

For details please refer to table 9 and 10 in the “Any other information on results incl. tables” section.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: 1 fetus had an irregularly shaped cyst attached to the right accessory lobe of the lung.

500 mg/kg bw/day: 3 fetuses had gas-filled stomachs. 1 fetus had cystic oviducts ranging in size from 1-3 mm.

300 mg/kg bw/day: 1 fetus had renal papilla(e) not fully developed.
These findings were not classified as either a malformation or developmental variation.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no advere effects observed
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Table 1: Summary of clinical findings

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Normal

-no significant clinical observations

543/22

526/22

539/22

521/22

480/22

Disposition

-found dead

0/ 0

1/ 1

0/ 0

0/ 0

2/ 2

-aborted; sent to necropsy

0/ 0

0/ 0

1/ 1

0/ 0

2/ 2

-scheduled euthanasia; gestation day 29

14/14

13/13

15/15

15/15

12/12

Behavior/cns

-head tilt

21/ 1

0/ 0

0/ 0

0/ 0

0/ 0

-impaired use of right forelimb

0/ 0

0/ 0

0/ 0

3/ 1

0/ 0

Body/integument

-hair loss urogenital area

2/ 1

0/ 0

5/ 2

5/ 2

23/ 3

-hair loss ventral abdominal area

3/ 2

0/ 0

0/ 0

1/ 1

21/ 5

-hair loss left hindlimb

5/ 1

3/ 2

1/ 1

5/ 2

21/ 4

-hair loss right hindlimb

2/ 2

6/ 1

9/ 2

6/ 2

28/ 3

-hair loss left lateral abdominal area

3/2

2/1

1/1

0/0

1/1

-dried brown material base of tail

4/2

0/0

2/1

0/0

0/0

-hair loss left forelimb

14/3

21/4

0/0

4/1

8/2

-hair loss left inguinal area

0/0

0/0

1/1

7/3

23/4

-hair loss right forelimb

13/2

14/3

0/0

0/0

4/1

-hair loss right lateral abdominal area

0/0

6/1

2/1

0/0

1/1

-Hair loss right lateral thoracic area

0/0

5/1

0/0

0/0

2/1

-Hair loss right inguinal area

2/1

0/0

4/1

4/2

19/4

-Hair loss ventral thoracic area

0/0

0/0

0/0

1/1

2/2

-Hair loss left axillary area

0/0

1/1

0/0

4/2

6/1

-Wet red material anogenital area

0/0

0/0

0/0

0/0

1/1

-Hair loss anogenital area

0/0

0/0

0/0

0/0

1/1

Cardio-pulmonary

-rales

4/2

18/5

6/2

5/1

5/1

Eyes/ears/nose

-scabbing right ear

0/0

0/0

0/0

0/0

4/1

-Scabbing left ear

0/0

0/0

0/0

0/0

4/1

Excreta

-soft stool

0/0

1/1

8/3

16/9

13/7

-Decreased defecation

15/5

32/9

41/11

58/16

36/8

-Wet red material in cage pan

0/0

0/0

0/0

0/ 0

3/2

-Feces small

2/1

0/0

2/1

4/ 2

2/1

 

Table 2: Summary of maternal survival and pregnancy status

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Number of females

22

%

22

%

22

%

22

%

22

 

Females that aborted or delivered

0

0.0

0

0.0

1

4.5

0

0.0

2

9.1

Females that died

0

0.0

1

4.5

0

0.0

0

0.0

2

9.1

Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

1

100.0

0

0.0

0

0.0

2

100.0

Females that were euthanized

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Females examined at scheduled necropsy

22

100.0

21

95.5

21

95.5

22

100.0

18

81.8

Non gravid

1

4.5

1

4.8

0

0.0

0

0.0

2

11.1

Gravid with resorptions only

21

95.5

20

95.2

21

100.0

22

100.0

16

88.9

Gravid with viable fetuses

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

 

Table 3: Summary of body weight changes during gestation (g)

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Day 7-10

 

 

 

 

 

Mean

Standard deviation

Standard error

n

38.0

53.4

11.7

21

39.0

66.6

14.5

21

70.0

59.6

12.7

22

36.0

45.8

9.8

22

36.0

92.2

20.6

20

Day 10 -14

 

 

 

 

 

Mean

Standard deviation

Standard error

n

66.

56.5

12.3

21

70.

39.0

8.5

21

67.0

72.9

15.6

22

66.0

42.1

9.0

22

52.0

67.6

15.5

19

Day 14-17

 

 

 

 

 

Mean

Standard deviation

Standard error

n

70.0

63.4

13.8

21

68.0

41.0

8.9

21

 

34.0

93.5

19.9

22

36.0

85.8

18.3

22

-9.0**

79.0

18.1

19

Day 17-20

 

 

 

 

 

Mean

Standard deviation

Standard error

n

44.0

50.3

11.0

21

45.0

34.9

7.6

21

41.0

70.7

15.1

22

42.0

54.7

11.7

22

13.0

89.9

20.6

19

Day 20-24

 

 

 

 

 

Mean

Standard deviation

Standard error

n

39.0

102.8

22.4

21

21.0

87.8

19.6

20

63.0

83.0

17.7

22

49.0

98.0

20.9

22

13.0

88.3

20.8

18

Day 24-29

 

 

 

 

 

Mean

Standard deviation

Standard error

n

15.

111.9

24.4

21

-20.

124.5

27.8

20

0.0

119.5

26.1

21

-56.0

174.0

37.1

22

-35.0

185.1

46.3

16

Day 7-29

 

 

 

 

 

Mean

Standard deviation

Standard error

n

218.0

201.1

45.0

20

218.0

201.1

45.0

20

292.0

161.6

35.3

21

173.0

190.9

40.7

22

166.0

234.7

58.7

16

Gestation Day 7 Body weight

 

 

 

 

 

Mean

Standard deviation

Standard error

n

3640.

243.9

53.2

21

3644.

256.5

57.4

20

3612.

189.4

41.3

21

3595.

207.0

44.1

22

3634.

279.9

70.0

16

Terminal Body weight

 

 

 

 

 

Mean

Standard deviation

Standard error

n

3913.0

298.1

65.1

21

3862.0

363.5

81.3

20

3903.0

243.5

53.1

21

3768.0

244.5

52.1

22

3800.0

373.1

93.3

16

Gravid uterine weight

 

 

 

 

 

Mean

Standard deviation

Standard error

n

508.3

87.00

18.98

21

474.4

79.83

17.85

20

510.3

84.12

18.36

21

481.0

86.42

18.42

22

462.9

61.38

15.35

16

Net Body weight

 

 

 

 

 

Mean

Standard deviation

Standard error

n

3404.4

258.60

56.43

21

3387.7

332.56

74.36

20

3393.1

219.85

47.98

21

3286.7

208.27

44.40

22

3337.2

335.72

83.93

16

Net Body weight change

(Gestation Day 7-29)

 

 

 

 

 

Mean

Standard deviation

Standard error

n

-235.2

165.86

36.19

21

-256.4

182.39

40.78

20

-218.6

170.62

37.23

21

-307.8

165.43

35.27

22

-297.2

206.06

51.51

16

** = Significantly different from the control group at 0.01 using Dunnett’s test

Mean differences calculated from individual differences

Nongravid weight(s) not included in calculation of mean

 

Table 4: Summary of food consumption during gestation (g/animal/day)

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Day 7-10

 

 

 

 

 

Mean

Standard deviation

Standard error

n

163.0

21.5

4.7

21

170.0

27.6

6.0

21

181.0

25.3

5.4

22

169.0

28.5

6.1

22

158.0

42.5

9.5

20

Day 10 -14

 

 

 

 

 

Mean

Standard deviation

Standard error

n

151.0

27.9

6.1

21

163.0

29.4

6.4

21

172.0

28.9

6.2

22

156.0

24.4

5.2

22

152.0

28.7

6.6

19

Day 14-17

 

 

 

 

 

Mean

Standard deviation

Standard error

n

156.0

43.9

9.6

21

165.0

34.9

7.6

21

157.0

74.9

16.4

21

120.0

54.8

11.7

22

112.0*

45.0

10.3

19

Day 17-20

 

 

 

 

 

Mean

Standard deviation

Standard error

n

158.0

46.0

10.0

21

155.0

37.8

8.2

21

143.0

61.2

13.0

22

133.0

50.7

11.1

21

129.0

53.2

12.2

19

Day 20-24

 

 

 

 

 

Mean

Standard deviation

Standard error

n

136.

43.4

9.5

21

127.0

53.7

12.0

20

132.0

49.4

10.5

22

129.0

45.3

9.7

22

111.0

42.9

10.1

18

Day 24-29

 

 

 

 

 

Mean

Standard deviation

Standard error

n

78.0

29.4

6.4

21

70.0

39.6

8.9

20

81.0

35.9

7.8

21

68.0

38.3

8.2

22

77.0

42.7

10.7

16

Day 7-29

 

 

 

 

 

Mean

Standard deviation

Standard error

n

135.0

24.4

5.3

21

135.0

28.0

6.3

20

139.0

27.4

6.0

21

124.

22.3

4.8

22

126.0

21.3

5.3

16

* = Significantly different from the control group at 0.05 using Dunnett’s test

 

Table 5: Summary of organ weights (g)

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Liver

 

 

 

 

 

Mean

% Difference

Standard deviation

Standard error

n

82.25

 

11.862

2.589

21

80.35

-2.3

12.970

2.900

20

84.72

3.0

9.636

2.103

21

81.17

-1.3

14.076

3.001

22

82.46

0.3

14.415

3.604

16

Kidneys

 

 

 

 

 

Mean

% Difference

Standard deviation

Standard error

n

15.72

 

1.329

0.290

21

15.62

-0.6

1.647

0.368

20

16.20

3.1

1.820

0.397

21

15.68

-0.3

1.451

0.309

22

16.22

3.2

1.980

0.495

16

 

Table 6: Summary of maternal macroscopic findings

Dose group (mg/kg bw/day)

Control

100

300

500

1000

Number examined

22

22

22

22

22

No significant changes observed

10

6

9

10

10

Nongravid - ammonium sulfide negative

1

1

0

0

2

Skin: matting, red

0

0

0

0

3

Spleen: accessory

5

4

5

5

2

Vagina: contents, dark red

0

0

0

0

2

Aborted gestation day 20

0

0

0

0

1

Oviducts: cyst(s)

4

11

8

7

6

Died gestation day 22

0

1

0

0

0

Stomach: contents, thick white

0

0

0

0

1

Aborted gestation day 26

0

0

0

0

1

Thoracic cavity: contents, red fluid

0

0

0

0

1

Esophagus: perforation

0

0

0

0

1

Cause of death: intubation error

0

0

0

0

1

Died gestation day 27

0

0

0

0

1

Lungs: area(s), dark red

0

1

2

0

0

Liver: pale

1

0

0

3

0

Kidneys: pale

0

0

0

1

0

Skin: hair loss

0

0

0

0

1

Lungs: mottled

0

0

1

0

0

Skin: matting, brown

1

0

0

0

0

Thoracic cavity: contents, clear fluid

1

0

0

0

0

Thymus: edematous

1

0

0

0

0

Liver: area(s), white

2

0

0

0

0

Aborted gestation day 28

0

0

1

0

0

Uterus: contents, brown

0

0

0

0

1

 

Table 7: Summary of fetal data at scheduled necropsy

 

 

 

 

 

 

 

 

 

 

Dose

mg/kg bw/day

Sex

Viable fetuses

Dead fetuses

Resorptions

Implantation loss

Implantation sites

Corpora lutea

Pre-implantation loss

Fetal weights in (g)

Number of gravid females

Control

 

male

female

 

 

Early

Late

 

 

 

 

 

 

 

Total

81

114

195

0

4

4

8

203

217

14

NA

21

 

Mean

3.9

5.4

9.3

0.0

0.2

0.2

0.4

9.7

10.3

0.7

39.7

 

 

Standard deviation

 

1.39

1.57

1.87

0.00

0.51

0.51

0.67

2.06

2.20

0.73

3.95

 

 

Standard error

0.30

0.34

0.41

0.00

0.11

0.11

0.15

0.45

0.48

0.16

0.86

 

100

Total

l 79

88

167

0

9

3

12

179

204

25

NA

20

 

Mean

4.0

4.4

8.4

0.0

0.5

0.2

0.6

9.0

10.2

1.3

41.1

 

 

Standard deviation

 

1.28

2.16

2.16

0.00

0.76

0.37

0.82

2.09

1.91

1.21

5.53

 

 

Standard error

0.29

0.48

0.48

0.00

0.17

0.08

0.18

0.47

0.43

0.27

1.24

 

300

Total

107

86

193

0

4

3

7

200

217

17

NA

21

 

Mean

5.1

4.1

9.2

0.0

0.2

0.1

0.3

9.5

10.3

0.8

40.4

 

 

Standard deviation

 

1.41

1.41

1.66

0.00

0.68

0.48

0.80

1.72

1.68

0.98

5.26

 

 

Standard error

0.31

0.31

0.36

0.00

0.15

0.10

0.17

0.38

0.37

0.21

1.15

 

500

Total

97

112

209

0

6

4

10

219

232

13

NA

22

 

Mean

4.4

5.1

9.5

0.0

0.3

0.2

0.5

10.0

10.5

0.6

36.7

 

 

Standard deviation

 

1.56

1.80

1.82

0.00

0.46

0.50

0.67

1.73

1.57

0.96

5.00

 

 

Standard error

0.33

0.38

0.39

0.00

0.10

0.11

0.14

0.37

0.33

0.20

1.07

 

1000

Total

61

77

138

0

2

9

11

149

165

16

NA

16

 

Mean

3.8

4.8

8.6

0.0

0.1

0.6

0.7

9.3

10.3

1.0

38.1

 

 

Standard deviation

 

1.64

1.97

1.50

0.00

0.34

1.15

1.20

1.49

2.18

1.26

5.44

 

 

Standard error

 

0.41

0.49

0.38

0.00

0.09

0.29

0.30

0.37

0.55

0.32

1.36

 

Na = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, Fetal weights compared using Dunnett’s test

 

Table 8: Summary of fetal data at scheduled necropsy (% per litter)

Dose group

(mg/kg bw/day)

Control

100

300

500

1000

Corpora lutea

 

 

 

 

 

Mean

Standard deviation

Standard error

N

10.3

2.20

0.48

21

10.2

1.91

0.43

20

10.3

1.68

0.37

21

10.5

1.57

0.33

22

10.3

2.18

0.55

16

Implanation sites

 

 

 

 

 

Mean

Standard deviation

Standard error

N

9.7

2.06

0.45

21

9.0

2.09

0.47

20

9.5

1.72

0.38

21

10.0

1.73

0.37

22

9.3

1.49

0.37

16

Viable fetuses (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

96.4

6.34

1.38

21

93.3

9.57

2.14

20

96.8

7.62

1.66

21

95.4

6.63

1.41

22

93.1

11.52

2.88

16

Dead fetuses (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

0.0

0.00

0.00

21

0.0

0.00

0.00

20

0.0

0.00

0.00

21

0.0

0.00

0.00

22

0.0

0.00

0.00

16

Early resorptions (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

2.1

5.68

1.24

21

5.1

9.09

2.03

20

2.0

6.97

1.52

21

2.9

4.90

1.04

22

1.5

4.04

1.01

16

Late resorptions (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

1.5

3.81

0.83

21

1.6

4.00

0.89

20

1.2

3.82

0.83

21

1.7

4.56

0.97

22

5.5

10.62

2.65

16

Total resorptions (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

 

3.6

6.34

1.38

21

 

6.7

9.57

2.14

20

 

3.2

7.62

1.66

21

 

4.6

6.63

1.41

22

 

6.9

11.52

2.88

16

Pre-implantation loss (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

 

6.2

7.11

1.55

21

 

12.2

12.40

2.77

20

 

7.7

8.83

1.93

21

 

5.5

8.90

1.90

22

 

8.4

9.83

2.46

16

Post-implantation loss (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

 

3.6

6.34

1.38

21

 

6.7

9.57

2.14

20

 

3.2

7.62

1.66

21

 

4.6

6.63

1.41

22

 

6.9

11.52

2.88

16

Males (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

41.5

12.51

2.73

21

49.3

16.99

3.80

20

55.5

13.20

2.88

21

46.8

14.33

3.06

22

45.0

21.33

5.33

16

Females (%)

 

 

 

 

 

Mean

Standard deviation

Standard error

N

58.5

12.51

2.73

21

50.7

16.99

3.80

20

44.5

13.20

2.88

21

53.2

14.33

3.06

22

55.0

21.33

5.33

16

Male fetal weights (g)

 

 

 

 

 

Mean

% Differences

Standard deviation

Standard error

N

40.0

 

4.06

0.89

 

41.4

 

3.5

5.78

1.29

40.8

 

2.0

5.64

1.23

37.3

 

-6.8

5.22

1.11

38.0

 

-5.0

5.62

1.45

Fale fetal weights (g)

 

 

 

 

 

Mean

% Differences

Standard deviation

Standard error

N

39.5

 

4.20

0.92

21

40.8

 

3.3

5.78

1.29

20

40.1

 

1.5

5.01

1.09

21

36.2

 

-8.4

4.99

1.06

22

37.8

 

-4.3

5.58

1.39

16

Combined (g)

 

 

 

 

 

Mean

% Differences

Standard deviation

Standard error

N

39.7

 

3.95

0.86

21

41.1

 

3.5

5.53

1.24

20

40.4

 

1.8

5.26

1.15

21

36.7

 

-7.6

5.00

1.07

22

38.1

 

-4.0

5.44

1.36

16

 

Table 9: Summary of fetuses and litters with malformations (absolute number)

 

Fetuses

Litters

Dose group

(mg/kg bw/day)

Control

100

300

500

1000

Control

100

300

500

1000

Number examined externally

195

167

193

209

138

21

20

21

22

16

Microcephaly

0

0

0

1

0

0

0

0

1

0

Proboscis-like nose

0

0

0

1

0

0

0

0

1

0

Mandibular micrognathia

0

0

0

1

0

0

0

0

1

0

Maxillary micrognathia

0

0

0

1

0

0

0

0

1

0

Microphthalmia and/or anophthalmia

0

0

1

1

0

0

0

1

1

0

Astomia

0

0

0

1

0

0

0

0

1

0

Macroglossia

1

1

0

0

0

1

1

0

0

0

Carpal and/or tarsal flexure

3

0

0

0

0

2

0

0

0

0

Paw hyperflexion

2

0

0

0

0

1

0

0

0

0

Number examined viscerally

195

167

193

209

138

21

20

21

22

16

Hydrocephaly

0

0

0

1

0

0

0

0

1

0

Retroesophageal aortic arch

0

1

0

0

0

0

1

0

0

0

Persistent truncus arteriosus

1

0

0

0

0

1

0

0

0

0

Number examined skeletally

195

167

193

209

138

21

20

21

22

16

Vertebral anomaly with or without associated rib anomaly

0

0

1

1

1

0

0

1

1

1

Costal cartilage anomaly

0

0

2

0

0

0

0

1

0

0

Centra anomaly

0

0

1

0

0

0

0

1

0

0

Rib anomaly

1

0

0

0

0

1

0

0

0

0

Only 6 cervical vertebrae present

0

0

0

0

2

0

0

0

0

1

Total number with malformations

 

 

 

 

 

 

 

 

 

 

External

4

1

1

1

0

3

1

1

1

0

Soft tissue:

1

1

0

1

0

1

1

0

1

0

Skeletal

1

0

4

1

3

1

0

3

1

2

Combined

6

2

5

2

3

5

2

3

2

2

 

Table 10: Summary of fetuses and litters with variations (absolute number)

 

Fetuses

Litters

Dose group

(mg/kg bw/day)

Control

100

300

500

1000

Control

100

300

500

1000

Number examined externally

195

167

193

209

138

21

20

21

22

16

Number with findings

0

0

0

0

0

0

0

0

0

0

Number examined viscerally

195

167

193

209

138

21

20

21

22

16

Heart- extra papillary muscle

8

7

12

12

11

5

5

7

8

9

Spleen- pale

0

0

2

5

0

0

0

1

1

0

Spleen- small

2

0

4

5

2

2

0

1

2

2

Retrocaval ureter

4

5

3

4

4

3

2

3

4

3

Accessory spleen

26

21

17

15

11

12

7

10

9

7

Gallbladder- absent or small

9

4

17

2

5

8

4

8

2

4

Major blood vessel variation

15

8

7

11

8

9

7

5

4

4

Hemorrhagic ring around the iris

0

0

0

1

0

0

0

0

1

0

Number examined skeletally

195

167

193

209

138

21

20

21

22

16

13th full rib(s)

47

60

70

88

52

16

18

15

21

14

Hyoid arch(es) bent

19

13

16

11

6

12

9

10

6

5

13th rudimentary rib(s)

34

35

29

32

33

18

17

11

16

13

Sternebra(e) #5 and/or #6 unossified

18

18

22

32

12

9

11

9

12

8

Sternebra(e) malaligned(slight or moderate)

4

4

6

6

1

4

3

4

5

1

Extra site of ossification anterior to sternebra #1

2

4

5

1

2

2

4

4

1

1

Sternebrae with thread-like attachment

0

0

3

0

0

0

0

2

0

0

27 presacral vertebrae

12

18

9

29

16

7

11

5

12

4

7th cervical rib(s)

5

0

10

5

3

3

0

7

4

2

Hyoid body and/or arch(es) unossified

0

0

0

4

0

0

0

0

4

0

Reduced ossification of the skull

1

0

0

1

1

1

0

0

1

1

Accessory skull bone(s)

0

1

1

1

0

0

1

1

1

0

Extra site of ossification ventral to cervical #2

0

1

0

0

6

0

1

0

0

2

Vertebral centra not fully ossified

0

0

0

0

1

0

0

0

0

1

Pubis unossified

0

0

0

1

0

0

0

0

1

0

25 presacral vertebrae

1

0

1

0

0

1

0

1

0

0

7th sternebra

0

1

0

0

0

0

1

0

0

0

Sternebra(e) #1,#2,#3 and/or #4 unossified

0

1

0

0

0

0

1

0

0

0

 

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Experimental exposure time per week (hours/week):
168
Species:
rabbit
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity was tested in a GLP compliant study in rats (Crl:CD(SD)) according to OECD 414 (2011 n). In this study 25 timed-mated rats per dose level were treated daily by gavage with 30, 100, 300 and 1000 mg test substance /kg bw/day suspended in 0.5% methylcellulose from gestation day 6 to day 20. The animals of the control group received vehicle only (0.5% methylcellulose). To achieve targeted concentrations of active ingredient, the formulations were adjusted for sample purity (92.2%). This purity was revised by a later GLP analytical study and reported to be 90.5%. This reduction in determined purity of 1.7% was considered to have only minimal impact on the reported doses. Therefore, the reported doses have not been adjusted. In addition, sample analysis indicated that the test substance was uniformly mixed in the vehicle at the targeted concentrations and was stable under the conditions of the study.

 

Examinations of maternal animals included clinical observations, mortality, body weight, food consumption and gross pathological examinations. In addition, livers were weighed and saved for histopathological examination. At day 21 of gestation fetuses were delivered by caesarian section. The following parameters were determined at caesarian section: number of corpora lutea, number of implantations, uterus weight, number of resorptions, number of pregnant, number of live/dead fetuses, sex of live fetuses, individual weights of fetuses and external (all fetuses), visceral (one half of the fetuses) and skeletal (all fetuses) malformations and variations.

There were no test substance-related deaths and clinical signs. Stained or wet skin/fur and hair loss occurred infrequently in single animals of different dose groups and were thus not considered treatment-related or adverse. There were no effects observed on body weight and food consumption. Gross pathology only revealed liver discoloration in one animal dosed at 100 mg/kg bw/day. This single event was considered incidental and non-adverse. There were no effects observed on liver weights. In the absence of any test substance-related effect on gross findings, microscopic examination of the livers was not considered necessary. The number of pregnant, the mean number of corpora lutea, implantation sites, resorptions (total, early and late), live/dead fetuses were comparable across all groups tested. Also the number of live offspring, fetal body weight and sex ratio were comparable among all groups tested.

Single incidences of external malformation, protruding tongue (malformation), anopthalmia (malformation) and small eye bulge (gross finding) were observed in the 300 mg/kg bw/day dose group, but considered incidental due to missing dose-response. Skeletal findings included a single incidence of fused cervical arch (malformation) in the 300 mg/kg bw/day dose group, variations of the thoracic centrum and ribs in all dose groups and variations of the sternebrae in all groups, exluding the 30 mg/kg bw/day dose level. All observed variations were either incidental findings, not dose-related or its occurrence did not differ significantly between dose groups and were thus not considered as treatment-related. Visceral findings included a single incidence of discolored intestine (gross finding) in the 100 mg/kg bw/day dose group and a single incidence of discolored liver (variation) in the 30 mg/kg bw/day dose group. Both findings were considered incidental and thus not treatment-related.

Taken together, there were no test substance-related fetal malformations or variations observed at any dose level tested. The fetal alterations that were observed were unremarkable and occurred with low frequency across the dose levels tested.

 

Based on the above reported findings a NOAEL for maternal (general and developmental) toxicity and fetal developmental toxicity was determined at 1000 mg/kg bw/day, the highest dose level tested.

 

 

In addition, developmental toxicity was tested in a GLP compliant study in rabbits (New Zealand White) according to OECD 414 (2011 o).In this study 22 timed-pregnant rabbits per dose level were treated daily by gavage with 100, 300, 500 and 1000 mg test substance /kg bw/day suspended in 0.5% methylcellulose at a dosage volume of 4 mL/kg bw from gestation day 7 to day 28. The animals of the control group received vehicle only (0.5% methylcellulose). To achieve targeted concentrations of active ingredient, the formulations were adjusted for sample purity (92.2%). This purity was revised by a later GLP analytical study and reported to be 90.5%. This reduction in determined purity of 1.7% was considered to habe only minimal impact on the reported doses and on the values utilized for risk assessments. Therefore, the reported doses have not been adjusted. Test substance formulations met the homogeneity requirements and mean concentrations were within acceptable limits (85% to 115% of target concentration).

Examinations of maternal animals included clinical observations, mortality/moribundity, body weight, food consumption and gross pathological examinations. In addition, kidneys, liver, stomach and all gross lesions were preserved for possible histopathologic examinations. On gestation day 29 a laparohysterectomy was performed on each surviving female. The number of pregnant females and females that aborted or delivered were determined. The uteri, placentae and ovaries were examined, and the numbers of live and dead fetuses, early and late resorptions, pre- and post-implantation loss and implantation sites and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and

developmental variations.

In the highest dose group (1000 mg/kg bw/day), one test substance-related death and 2 test substance-related abortions occurred on gestation day 20 and 26. These females had clinical findings of increased respiration, rales and/or decreased defecation and mean body weight losses (approximately 19% and 17%) and severely reduced food consumption. The abortions were considered to be test substance-related but likely secondary to the decreased food consumption. Increased risk of abortion has been associated with decreased food consumption and greater than 10% body weight loss in rabbits.

An abortion on gestation day 28 in the 300 mg/kg bw/day dose group was not considered treatment-related, due to absence of a dose response. There were no other test substance-related mortalities. Increased occurrences of soft stool at 500 and 1000 mg/kg/day and hair loss at 1000 mg/kg/day were noted beginning on gestation day 18 until end of the study. Other clinical findings occurred infrequently, in a manner that was not dose-related and were thus not considered treatment-related.

Test substance-related lower mean body weight gains and mean food consumption were noted in the 500 and 1000 mg/kg/day groups compared to the control group when the entire treatment period was evaluated primarily due to changes noted during the latter part of gestation. Statistically significant reductions in body weight and food consumption were only achieved in the highest dose group on gestation days 14-17. Effects observed at 500 mg/kg bw/day were, although not statistically significant, considered treatment-related, because of the observed dose-response

There were no test substance-related effects on mean body weights, body weight gains and food consumption in the 100 and 300 mg/kg/day groups. Kidney and liver weights did not differ across the different dose groups. At scheduled necropsy on gestation day 29, 1 female in the control, 1 female in the 100 mg/kg/day group and 2 females in the 1000 mg/kg/day group were determined to be non-gravid. All macroscopic findings were not considered test substance-related. The number of corpora lutea, early and late resorptions, pre- and post- implantation loss were comparable among dose goups.

The number of viable and dead pups and sex ratio were comparable across the different dose groups. At 500 and 100 mg/kg bw/day fetal weights were slightly reduced compared to controls. Since this observation was not statistically significant and no dose-response was visible it was not considered as treatment-related.

Single incidences of external malformations did not differ statistically significantly from the control group. At 300 mg/kg bw/day, single incidences of protruding tongue (malformation), anopthalmia (malformation) and small eye bulge (gross finding) were observed, but considered an incidental finding and not considered adverse or treatment-related. Skeletal malformations occurred at all dose levels, but none of the observed malformations was statistically significant. In addition, each malformation occurred in a single animal or litter or in a manner that was not dose-related. Therefore, malformations were not considered to be related to test substance administration.

At 1000 mg/kg bw/day two fetuses had only 6 cervical vertebrae present. The mean litter proportion of extra site of ossification (4.2% per litter) was higher (not statistically significant) than in the concurrent control group (0.0% per litter). Although this value exceeded the maximum mean value of the historical control data range (2.3% per litter), it was not considered test substance-related since this finding was primarily observed in five fetuses in one litter. Thus, biological significance of this rather litter-specific finding remains unclear. Other skeletal developmental variations occurred similarly in the control group, were not observed in a dose-related manner and/or occurred in single fetuses and were thus not considered treatment-related.

One animal of the control, 100 and 500 mg/kg bw/day dose group had visceral malformations (persistent truncus arteriosus (control); retroesophageal aortic arch (100 mg/kg bw/day); hydrocephaly (500 mg/kg bw/day)). Evaluated on a percent per litter basis, none of the differences from the concurrent control group were statistically significant. Furthermore, these visceral malformations occurred in single animals or litters or in a manner that was not dose-related and therefore, were not considered to be test substance-related. Soft tissue developmental variations were noted in all groups, but were not considered treatment-related, since they occurred similarly among the dose groups, were not observed in a dose-related manner and/or the mean litter proportions did not differ significantly from controls. Taken together, there were no test substance-related fetal external, visceral or skeletal malformations or developmental variations observed at any dosage level.

 

In summary, there were test substance-related death and abortions observed at 1000 mg/kg bw/day in rabbits. Increased occurrence of soft stool were observed at 500 and 1000 mg/kg bw/day and hair loss at 1000 mg/kg bw/day. Mean body weight gains and food consumption were reduced at 500 and 1000 mg/kg bw/day, respectively.

Based on the above described findings at 500 mg/kg bw/day, a NOAEL for general toxicity in maternal animals was determined at 300 mg/kg bw/day and a LOAEL for general toxicity at 500 mg/kg bw/day.

For developmental effects in maternal animals a NOAEL of 500 mg/kg bw/day and a LOAEL of 1000 mg/kg bw/day were determined based on the observed abortions in the highest dose group that were considered treatment-related.Taking into consideration that systemic toxicity was evident in maternal animals at 500 mg/kg bw/day, the effects observed at 1000 mg/kg bw/day are rather considered as a secondary, non-specific consequence of maternal toxicity and are therefore not considered to meet the classification criteria according to Regulation EC (No) 1272/2009.

A NOAEL of 1000 mg/kg bw/day for fetal developmental effects was determined, based on the absence of any test substance-related fetal external, visceral or skeletal malformations or developmental variations or any other fetal finding at all dose levels.

 

A DNEL for workers was derived for longterm exposure, systemic effects to account for possible effects on body weight parameters and food efficiency which may be linked to maternal toxicity and consequently to secondary non-specific maternal developmental toxicity.

Overall conclusion

In utero exposure to the test substance did not result in developmental toxicity in rats. In rabbits, test substance-related death and abortions occured at 1000 mg/kg bw/day. Taking into consideration that systemic toxicity was evident in maternal animals at 500 mg/kg bw/day, the abortions observed at 1000 mg/kg bw/day are considered as secondary, non-specific consequence of maternal toxicity and not as indication of specific developmental toxicity. In both species, no test substance-related fetal external, visceral or skeletal malformations or developmental variations were observed. In summary, no hazardous properties indicating specific fetal developmental effects as primary consequence were observed in rats and rabbits neither in the two-generation study nor in developmental toxicity studies. Adverse effects observed on fetal body weight and abortions are considered to be related to maternal toxicity. Thus, the test substance does not meet the classification criteria for reproductive/developmental toxicity according to Regulation EC (No) 1272/2008.

Justification for classification or non-classification

No adverse effects on fertility were observed in the available study on reproduction. Developmental effects observed in the offspring and/or maternal animals are considered as a secondary, non-specific consequence of maternal toxicity and not as indication of specific developmental toxicity. In conclusion, the test substance does not meet the classification criteria for reproductive/developmental toxicity according to Regulation EC No 1272/2008.

Additional information