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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 OCT 2005 to 25 JUN 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS (Crl:CD (SD))
- Source: Charles River Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 207 g to 263 g ( group means: 235-237 g); Females: 151 to 201 g (group means: 169-171 g)
- Fasting period before study: no
- Housing: 3 per cage (by sex) for the initial acclimation period; individually thereafter except for mating period and after parturition; following weaning dams were individually housed until scheduled necropsy; weaned pups were housed by litter in plastic cages for 1 week, beginning on PND 28, the F1 and F2 pups were inidvidually housed until the start of the mating period (F1) or until euthanasia (F2)
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002; ad libitum except during exposure periods
- Water: reverse osmosis-purified drinking water; ad libitum except during exposure periods
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2 cubic metre stainless steel and glass whole body exposure chambers/group
- Method of holding animals in test chamber: individually housed in stainless steel wire-mesh caging suspended over cageboard during exposure
- Source and rate of air: provided from a HEPA- and charcoal-filtered, temperature and humidity controlled source
- Method of conditioning air: test article vapour was directed to the exposure chamber inlet where the vapour concentration was reduced to the desired level by mixing with the chamber ventilation air
- System of generating vapour: glass-bead column-type vaporization system
- Temperature, humidity, pressure in air chamber: daily mean chamber temperature: 21 to 22°C; daily mean chamber humidity: 43 to 56%; slight negative pressure
- Air flow rate: mean values: 430 to 475 litters per minute
- Air change rate: at least 12 to 15 per hour
- Treatment of exhaust air: charcoal and HEPA filtration


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatograph (GC)
- Samples taken from breathing zone: yes; approximately every 35 minute


Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for an additional 7 days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: plastic maternity cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- gas chromatography
Duration of treatment / exposure:
- 6 hours per day for at least 70 consecutive days prior to mating
- F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20; inhalation exposure was suspended from gestation day 21 through lactation day 4
- on lactation days 1 to 4 F0 and F1 maternal animals were exposed via oral gavage
- inhalation exposure of F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia
- inhalation exposure of the F1 was initiated on PND 22
Frequency of treatment:
7 days per week
Details on study schedule:
- F1 parental animals not mated until 2-3 weeks after selected from the F1 litters.
- Selection of parents from F1 and F2 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: prior to the F0 pairing (study week 10) the animals were approximately 17 weeks old; prior to the F1 pairing (study week 29) the animals were approximately 15-16 weeks old
Dose / conc.:
750 ppm (nominal)
Remarks:
1125 mg/kg bw/day actual ingested
F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
Dose / conc.:
1 500 ppm (nominal)
Remarks:
2250 mg/kg bw/day actual ingested
F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
Dose / conc.:
2 000 ppm (nominal)
Remarks:
3000 mg/kg bw/day actual ingested
F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
No. of animals per sex per dose:
30/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on range finding study (see section 7.8.1)
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations:
F0 and F1 males: weekly
F0 and F1 females: weekly until evidence of copulation; on gestation day 0, 4, 7, 11, 14, 20 and on lactation day 1, 4, 5 (pre-exposure), 7, 14, 21; weekly after weaning until scheduled necropsy
F2 males and females: weekly beginning on PND 28 through euthanasia


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations:
F0 and F1 males: weekly (not recorded during mating period)
F0 and F1 females: weekly until evidence of copulation (not recorded during mating period); on gestation day 0, 4, 7, 11, 14, 20 and on lactation day 1, 4, 5 (pre-exposure), 7, 14, 21; weekly after weaning until scheduled necropsy
F2 males and females: weekly beginning on PND 28 through euthanasia
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female for 21 days prior to pairing and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, cauda epididymis weight, sperm count in testes, calculation of sperm production rate, sperm count in epididymides, sperm motility, progressive motility and sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weights, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F0 and F1 animals were euthanized following completion of the parturition period.
- Maternal animals: All surviving F0 and F1 animals that delivered were euthanized between 6 and 10 days after wening of their litters. F0 and F1 females that mated but did not give birth were euthanized on presumed gestation day 25. F0 and F1 females that experienced total litter loss were euthanized within 24 hours. F0 and F1 females that failed to mate within the first 14 days of the breeding period were euthanized on gestation day 15 (females that mated with the second male) or post-cohabitation day 15 (females that did not mate with the second male).
Animals underwent complete necropsy and selective histopathologic examination.

GROSS NECROPSY (F0, F1 and F2 adults)
- Gross necropsy consisted of examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females euthanized on gestation day 15. For females that failed to deliver, a pregnancy status was determined,
and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.


HISTOPATHOLOGY (F0 and F1 parental animals)
Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high-exposure groups:

Adrenals Prostate
Coagulating glands Seminal vesicles
Kidneys Spleen
Liver Testis and epididymis (right)
Lungs Thyroid
Nasal passages (additionally investigated in animals of the 750 ppm group) Uterus
Ovaries Vagina
Oviducts Gross lesions
Pituitary

In addition, microscopic evaluations were performed on the following tissues for F0 and F1 parental animals from the low- and mid-exposure groups that did not mate or produce offspring.

Males Females

Epididymis a Ovaries
Prostate Oviducts
Seminal vesicles Uterus
Testis a Pituitary
Pituitary

a = Right testis and epididymis only.


ORGAN WEIGHTS (F0 and F1 adults)
Adrenals Prostate
Brain Seminal vesicles (with coagulating glands and accessory fluids)

Epididymisa (total and cauda)
Kidneys Spleen
Liver Testes
Lungs (prior to inflation with fixative) Thyroid
Ovaries Uterus (with oviducts and cervix)
Pituitary



Postmortem examinations (offspring):
SACRIFICE
All surviving F1 and F2 pups not selected for test article exposure were euthanized on PND 21 by carbon dioxide inhalation. F1 and F2 weanlings exposed to the test article beginning on PND 22 but were not selected to continue exposure into the respective maturation phases were euthanized by carbon dioxide inhalation followed by exsanguination between PND 36-47 (F1 generation) and on PND 36 (F2 generation).
Animals underwent necropsy and/or organ weight determination.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY (F1 and F2 pups/weanlings)
Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on nonselected F1 and F2 pups euthanized on PND 21, on the F1 weanlings from dam no. 99179 in the 1500 ppm group that were euthanized on PND 28, and on the extra weanlings selected for inhalation exposure beginning on PND 22 that were euthanized between PND 36-47 (F1 generation) and PND 36 (F2 generation).
The following F1 and F2 organs were collected from 3 pups/sex/litter that survived to their
scheduled termination on PND 21. Only gross lesions were collected and preserved for
the F1 and F2 weanlings exposed up to PND 47 or PND 36.

All organs were placed in 10% neutral-buffered formalin (except as noted):

Adrenal glands (2) Seminal vesicles (2)
Brain Spleen
Coagulating glands (2) Testes and epididymides a (2)
Kidneys (2) Thymus
Liver Uterus with cervix
Ovaries (2) Vagina
Pituitary Gross lesions
Prostate

a = If retained, testis and epididymis were fixed in Bouin’ s solution.


HISTOPATHOLOGY
not performed


ORGAN WEIGTHS (F2 adults and F1 and F2 pups)
Brain Thymus
Pituitary Testes
Spleen Uterus
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5%, comparing each test article-exposed group to the control group by sex. Where applicable, the litter was used as the experimental unit.
Parental mating, copulation, conception and fertility indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (premating, gestation and lactation) and body weight gains, mean parental food consumption and food efficiency (at each interval), implantation sites, number of unaccounted-for sites, pre-coital intervals, estrous cyclicity, follicular counts (ovary), gestation length, mean day of attainment of pre-weaning and post-weaning developmental landmarks, mean weight on the day of attainment of post-weaning developmental landmarks, mean pup body weights (postnatal period), mean number of pups born, live litter size, organ weights (absolute, relative to body and relative to brain) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences (Snedecor and Cochran, 1980). If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group.
Mean litter proportions (percent per litter) of postnatal pup viability, pup sex ratio at birth, percentage of motile and progressively motile sperm and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-treated groups to the control group. Epididymal and testicular sperm numbers and sperm production rates were analyzed by a t-test (Steel and Torrie, 1980).
Reproductive indices:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Females Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100

Male Copulation Indes (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100
Offspring viability indices:
Mean Live Litter Size = Total Viable Pups on PND 0 / No. Litters with Viable Pups on PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = Summ (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/ No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Summ (Viable Pups Per Litter at End of Interval N/Viable Pups per Litter at Start of Interval N) / No. of Litters Per Group x 100

N = PND 0-1, 1-4 (Pre Selection, 4 (Post Selection)-7, 7-14, 14-21 or 4 (Post Selection)-21
Clinical signs:
no effects observed
Description (incidence and severity):
- One F0 female in the 750 ppm group experienced a total litter loss on PND 7; this was not considered related to test article exposure in the absence on an exposure-response.
- One F0 female each in the 750 ppm and 2000 ppm groups failed to deliver and were necropsied on post-mating day 25. In addition, 2 females each in the control and 750 ppm groups and 1 female in the 1500 ppm group were necropsied on gestation day or post-cohabitation day 15. All remaining F0 parental animals survived to the scheduled necropsy.
- No test article-related clinical findings were noted for animals that died or that survived to the scheduled necropsies.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One F0 female in the 2000 ppm group was found dead approximately 3 hours after the first oral gavage dose on lactation day 3. Based on the macroscopic and microscopic examinations, the mortalities in the control group and the lack of mortalities in the 2000 ppm male group, this single death was not considered test article-related.
- One female and one male in the control group were euthanized in extremis on study day 37 and study day 106 (study week 15), respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean cumulative body weight gains in the 1500 ppm and 2000 ppm male groups were statistically significantly lower than the control group when the entire pre-mating period (191 g and 174 g, respectively, compared to 250 g in the control group during study weeks 0-10) and entire generation (224 g and 203 g, respectively, compared to 304 g in the control group during study weeks 0-15) were evaluated. In addition, mean male body weights were 5.2% to 15.0% and 4.2% to 18.7% lower in these same respective groups when compared to the control group.
- Mean body weight gains in the 1500 ppm and 2000 ppm F0 female groups resulted in lower mean cumulative body weight gains (86 g and 79 g, respectively, compared to 106 g in the control group) when the entire pre-mating period (study weeks 0-10) was evaluated. In addition, mean F0 female body weights were 4.4% to 7.4% and 5.3% to 9.3% lower (statistically significant) in these same respective groups when compared to the control group beginning on study weeks 6 and 5, respectively, and continuing throughout the remainder of the pre-mating period
- A slight decreasing trend in mean body weight gain was noted in the 750 ppm F0 male and female groups when compared to the control group. Although statistical significance for lower mean body weight gains was only achieved during study weeks 8-9 for males and study weeks 0-1 and 7-8 for females (none in a manner that was exposure-dependent), the mean cumulative body weight gains for the males (235 g) and females (93 g) in this group were lower (statistically significant for females) than the control group (250 g and 106 g, respectively) during the pre-mating period (study weeks 0-10). The higher (statistically significant) mean body weight gain noted for the 750 ppm F0 males during study week 11-12 was of no toxicological significance. In addition, the slightly lower mean body weight gains noted for both sexes in this group were not of sufficient magnitude to affect mean body weights when evaluated during the pre-mating (males and females) and post-mating (males) exposure periods.
- Mean body weight gains in the 1500 ppm and 2000 ppm groups were generally similar to the control group values during gestation days 0-4, 4-7, 7-11 and 11-14; no statistically significant differences were noted. However, due to statistically significantly lower mean body weight gains noted for both groups during gestation days 14-20, mean body weight gains in these groups were lower than the control group when the entire gestation exposure period (gestation days 0-20) was evaluated; the difference was statistically significant at 2000 ppm.
- Mean F0 maternal body weights in the 1500 ppm and 2000 ppmgroups were statistically significantly lower (5.4% to 8.4% and 9.1% to 11.3%, respectively) throughout lactation when compared to the control group. However, mean body weight gains in these groups were generally similar (and not statistically significant) to the control group when the overall lactation period was evaluated. Therefore, the decreased mean body weights at 1500 ppm and 2000 ppm were considered a continuation of the decreased mean body weights noted during the pre-mating and gestation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean weekly food consumption (evaluated as g/animal/day and g/kg/day) in the 1500 ppm and 2000 ppm F0 male groups was lower than the control group throughout the generation. For both groups, the g/animal/day differences were statistically significant throughout the exposure period, and the g/kg/day differences were statistically significant during study weeks 0-1 through 4-5, 7-8 and 8-9. No test article-related effects on mean weekly food consumption and food efficiency were noted for F0 males in the 750 ppm group.
- Mean weekly food consumption in the 1500 ppm and 2000 ppm F0 female groups was lower than the control group throughout the pre-mating period; the differences were generally statistically significant. Mean weekly food consumption in the 750 ppm F0 female group was slightly lower than the control group during study week 0-1; the g/kg/day difference was statistically significant, and this reduction corresponded to the effect noted on body weight gain during this interval. With the exception of lower food consumption noted during study weeks 7-8 and 8-9 (generally statistically significant), mean food consumption for the females in this group was generally similar to the control group for the remainder of the pre-mating period. The lower food consumption noted for the 750 ppm female group corresponded to the slightly lower body weight gain noted when the entire pre-mating period was evaluated.
- Mean F0 maternal food consumption in the 1500 and 2000 ppm groups were statistically significantly lower than the control group throughout the gestation period. However, there was no corresponding effect on food efficiency at either exposure level.
- During lactation mean food consumption in the 2000 ppm group was lower than the control group, there was no corresponding effect on food efficiency.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test article-related histologic observations were restricted to the nasal cavity and were observed in F0 and F1 males and females at the 750 and 2000 ppm exposure levels; tissues from the 1500 ppm exposure group were not examined histologically. In the high-exposure (2000 ppm level) animals, degeneration of the olfactory epithelium was most severe and widely distributed at level II, with the dorsal meatus generally affected diffusely and bilaterally, and with multifocal involvement of the nasoturbinates, typically at the tips. Histologic findings were similar at the 750 ppm exposure level, although lesions were less severe and less widely distributed. In both the 750 and 2000 ppm exposure groups, lesions occurred on a gradient, i.e. decreased in incidence, severity and distribution moving caudally within the nasal cavity.
- There were no other test article-related histologic changes in the F0 and F1 generation. Remaining histologic findings were considered to be incidental, manifestations of spontaneous diseases or related to some aspect of experimental manipulation other than exposure to the test article. There was no test article-related alteration in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- No test article-related effects on F0 reproductive performance were observed at any exposure level.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- No test article-related effects were observed on F0 spermatogenesis endpoints in males at any exposure level.
Reproductive performance:
no effects observed
Description (incidence and severity):
- No test article-related effects on F0 mean gestation length or the process of parturition were observed at any exposure level.
Dose descriptor:
LOAEC
Remarks:
local effects, adult rats
Effect level:
750 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: degeneration of the olfactory epithelium in the nasal cavity at 750 and 2000 ppm in F0 and F1 males and females (1500 ppm group not investigated)
Remarks on result:
other: Generation: F0, F1
Dose descriptor:
NOAEC
Remarks:
systemic toxicity, adult rats
Effect level:
750 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Decrements in mean body weights, body weight gains and/or food consumptions at 1500 and 2000 ppm
Remarks on result:
other: Generation: F0, F1, F2
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Effect level:
750 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F1, F2
Dose descriptor:
NOAEC
Remarks:
fertility
Effect level:
2 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: No functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, spermatogenic endpoints and gestation length) in any test article-exposed group in the F0 or F1 generations
Remarks on result:
other: Generation: F0, F1
Clinical signs:
no effects observed
Description (incidence and severity):
- No test article-related clinical findings were noted for animals that died or that survived to the scheduled necropsy.
-The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test article exposure. Pups (litters) that were found dead numbered 11(7), 22(10), 11(7) and 17(7) in the control, 750, 1500 and 2000 ppm groups, respectively. Four (4), 3(3), 4(3) and 4(3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Fifteen pups in the litter of female no. 99210 in the 2000 ppm/3000 mg/kg/day group were euthanized and discarded without examination due to the death of their dam on PND 3.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
-One female in the control group, 2 males in the 1500 ppm group, 1 female in the 1500 ppm group and 2 females in the 2000 ppm group were found dead between PND 24 and PND 28. In addition, 2 females in the 1500 ppm group were found dead on lactation day 3 or 4. None of these deaths were considered test article-related because all F1 animals found dead during the week following weaning, with the exception of the 2 males in the 1500 ppm group, weighed at least 19.3% less than their exposed littermate of the same sex and at least 14.3% less than the mean value for their same sex littermates on PND 21 and the deaths during lactation did not occur in an exposure-related manner.
- Five, 4, 0 and 1 females in the control, 750 ppm, 1500 ppm and 2000 ppm group, respectively, failed to deliver and were necropsied on post-mating day 25; an additional female in the 1500 ppm group was euthanized on presumed post-mating day 25. Two and 1 females in the 750 ppm and 1500 ppm groups, respectively, were necropsied on post-cohabitation day or gestation day 15. All remaining F1 parental animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Test article-related lower mean F1 male body weight gains were noted in the 1500 ppm and 2000 ppm groups. The lower mean body weight gains noted in these groups, though not always exposure-dependent, were generally statistically significant. Mean cumulative body weight gains in the 1500 ppm and 2000 ppm groups were statistically significantly lower than the control group when the entire pre-mating period (318 g and 304 g, respectively, compared to 388 g in the control group during study weeks 18-29) and entire generation (359 g and 341 g, respectively, compared to 449 g in the control group during study weeks 18-35) were evaluated.
- F1 females in the 1500 ppm and 2000 ppm group had reduced body weight gain during the pre-mating period. During the entire pre-mating period (study weeks 18-29) mean cumulative body weight gains in the 1500 ppm (163 g) and 2000 ppm (159 g) groups were statistically significantly lower than the control group (183 g).
- Mean cumulative body weight gains in the F1 males in the 750 ppm group were lower than the control group when the entire pre-mating period (360 g compared to 388 g in the control group during study weeks 18-29) and entire generation (418 g compared to 449 g in the control group during study weeks 18-35) were evaluated; the difference during the pre-mating period was statistically significant. Mean body weights were 3.7% to 7.7% lower in this group when compared to the control group.

- Lower (statistically significant) mean F1 maternal body weight gains were noted in the 2000 ppm group throughout the gestation period, with the exception of gestation days 11-14, when mean body weight gain was similar (and not statistically different from) the control group. Mean body weights in this group were 13.1% to 16.9% lower than the control group during gestation days 0 to 20; differences from the control group were statistically significant. Mean body weights in the 1500 ppm group were also 8.5% to 10.8% lower (statistically significant) than the control group throughout gestation, with lower (occasionally statistically significant) mean body weight gains during gestation days 0-11 and 14-20. In addition, mean cumulative body weight gains during gestation days 0-20 in the 1500 ppm and 2000 ppm groups (112 g and 100 g, respectively) were statistically significantly lower than the control group value (133 g). Mean body weight gains and body weights of the 750 ppm F1 females were not affected.

- Mean F1 maternal body weights were statistically significantly lower in the 1500 ppm and 2000 ppm groups throughout lactation (5.7% to 8.1% and 12.0% to 14.1%, respectively) when compared to the control group. However, mean body weight gains in these groups were generally similar to the control group when the overall lactation period was evaluated. The only statistically significant differences were lower mean body weight gains noted for both groups during lactation days 7-14. The decreased mean body weights at 1500 ppm and 2000 ppm were considered a continuation of the decreased mean body weights noted during the pre-mating and/or gestation periods.

- Mean F1 male and female pup body weights in the 2000 ppm group were 7.0% and 7.5% lower, respectively, than the control group values on PND 1; the difference for the females was statistically significant. Mean body weight gains in the male and female pups in the 2000 ppm group were generally lower (occasionally statistically significant) compared to the control group throughout the pre-weaning period. As a result, mean F1 male and female pup body weights in this group were 7.9% to 15.1% and 9.3% to 17.5% lower, respectively, than the control group values from PND 4-21; the differences were generally statistically significant.
- Mean F1 male and female pup body weight gains in the 1500 ppm group were lower than the control group values during PND 1-4, 7-14 (statistically significant) and 14-21 and similar to the control group during PND 4-7. As a result, mean F1 male and female pup body weights in the 1500 ppm groups were 5.1% to 11.2% and 6.2% to 12.2% lower, respectively than the control group values during PND 4-21; statistical significance was achieved on PND 14 and 21.
- Mean F1 male and female pup body weight gains and body weights in the 750 ppm group were similar to (and not statistically significantly different from) the control group during the first week of the postnatal period and during PND 14-21. During PND 7-14, mean F1 pup body weight gains in these males and females were slightly but statistically significantly lower than the control group values. These lower mean body weight gains were not of sufficient magnitude to affect mean body weights. Differences in mean pup body weights in the 750 ppm group were slight and not statistically significant compared to the control group.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean weekly food consumption (evaluated as g/animal/day) in the 1500 ppm and 2000 ppm F1 male groups was lower than the control group during study weeks 18-19 through 34-35. Differences from the control group were generally statistically significant. Lower food consumption was also noted at a lesser extent at 750 ppm with statistically significantly lower food consumption noted from study weeks 18-19 through 22-23, 24-25 and 34-3 5. Statistically significant increases in g/kg/day food consumption were noted at 2000 ppm during study weeks 17-18 and 32-33.
- Mean weekly food consumption (evaluated as g/animal/day) in the 1500 ppm and 2000 ppm F1 female groups was generally lower than the control group during the pre-mating period (study weeks 18-19 through 28-29). Differences from the control group were generally statistically significant. Statistically significant differences in g/kg/day food consumption were noted at 1500 ppm during study week 27-28 (lower than control group and did not occur in an exposure-dependent manner) and at 2000 ppm during study weeks 20-2 1 (higher than control group due to the lower body weights). Mean food consumption and food efficiency in the 750 ppm F1 female group were unaffected by test article exposure. - Mean F1 maternal food consumption (evaluated as g/animal/day) in the 1500 ppm and 2000 ppm groups was lower (generally statistically significant) than the control group throughout the gestation period. Mean food consumption and food efficiency in the 750 ppm group were unaffected by test article exposure during gestation.
- Mean F1 maternal food consumption (evaluated as g/animal/day) at 750 ppm, 1500 ppm and 2000 ppm was generally similar to the control group during the first week of lactation. During . lactation days 7-14 and 14-2 1, mean food consumption in these groups was lower (generally statistically significant) than the control group. However, there was no corresponding effect on g/kg/day food or food efficiency at any exposure level. When the entire lactation period (days 1-21) was evaluated, mean food consumption at all exposure levels was lower than the control group
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):
- balanopreputial separation: mean age of attainment of the F1 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 45.6, 47.8*., 46.6 and 47.2 at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and did not follow a clear dose response; mean weights at attainment were lower compared to the control group (221.8, 219.8, 200.*1, 195.8* g at 0, 750, 1500 and 2000 ppm); these effects were attributed to the lower body weights noted during the pre-and post-weaning periods.
- vaginal patency: not affected
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
-Mean relative (to body weight) lung weight was statistically significantly higher as compared to the control values in test article-exposed F1 males (males, relative to body weight: 6.8%, 13.3%, 13.0%, respectively at 750, 1500 and 2000 ppm). Absolute lung weights were generally lower than controls throughout test article-exposed groups in the presence of lower final body weights. Lung weights relative to body weight were increased in F1 females(2.6%, 3.3%, 5.1%, respectively at 750, 1500 and 2000 ppm), but decreased relative to brain weight. There were no histologic correlates, therefore the effects on lung weight were not considered to be adverse.
- Further organ weight changes for which a statistically significant weight change was observed (e.g. epididymis, prostate, seminal vesicles, testis, thyroid) were considered by the author of the study report to be secondary to changes in body weight.
- Significantly lower thymus weights (absolute, relative to final body and/or brain weights) were noted in all dose groups for both males and females on PND 21. As there was no clear dose-response relationship (thymus weights relative to body weight (g/100g) males: 0.438, 0.398, 0.375, 0.400 at 0, 750, 1500 and 2000 ppm, respectively; females: 0.452, 0.418, 0.400, 0.427 at 0, 750, 1500 and 2000 ppm, respectively) and the body weight of the animals was also lower in comparison to the controls it remains unclear whether this effect is test item related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test article-related gross necropsy observations in the F1 generation.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- No test article-related effects on F1 reproductive performance were observed at any exposure level.
- No test article-related effects were observed on F1 spermatogenesis endpoints in males at any exposure level.
- No test article-related effects on F1 mean gestation length or the process of parturition were observed at any exposure level.
- Pinnal detachment: not affected
- Incisor eruption: not affected
- eye opening: not affected
VIABILITY (OFFSPRING)
F1 and F2 litter:
- The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival were unaffected by the test article at all exposure levels. Differences from the control group were slight, were not statistically significant and/or did not occur in an exposure-dependent manner.


CLINICAL SIGNS (OFFSPRING)
F1 litter:
-The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test article exposure. Pups (litters) that were found dead numbered 11(7), 22(10), 11(7) and 17(7) in the control, 750, 1500 and 2000 ppm groups, respectively. Four (4), 3(3), 4(3) and 4(3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Fifteen pups in the litter of female no. 99210 in the 2000 ppm/3000 mg/kg/day group were euthanized and discarded without examination due to the death of their dam on PND 3.

F2 litter:
- The numbers of F2 pups found dead and/or missing, as well as the general physical condition of all F2 pups in this study were unaffected by F1 parental test article exposure. Pups (litters) that were found dead numbered 17(9), 29(12), 5(5) and 15(7) in the control, 750, 1500 and 2000 ppm groups, respectively. Two (2), 6(5), 1(1) and 0(0) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Sixteen and 10 pups in the litters of female nos. 99137-11 and 99196-12, respectively, in the 1500 ppm group were euthanized and necropsied due to the death of their dam on PND 3 and 4, respectively.

F2 adults:
- One male and one female in the 2000 ppm group were found dead on PND 25 and 22, respectively. Two males and one female in the 750 ppm group were found dead between PND 23 and 31. There were no test article-related clinical findings noted prior to the death of these animals, and based on macroscopic examination, a cause of death could not be determined for these animals. All remaining F2 animals in the control, 750, 1500 and 2000 ppm groups survived to the scheduled necropsy.

BODY WEIGHT (OFFSPRING)
F1 litter:
- Mean F1 male and female pup body weights in the 2000 ppm group were 7.0% and 7.5% lower, respectively, than the control group values on PND 1; the difference for the females was statistically significant. Mean body weight gains in the male and female pups in the 2000 ppm group were generally lower (occasionally statistically significant) compared to the control group throughout the pre-weaning period. As a result, mean F1 male and female pup body weights in this group were 7.9% to 15.1% and 9.3% to 17.5% lower, respectively, than the control group values from PND 4-21; the differences were generally statistically significant.
- Mean F1 male and female pup body weight gains in the 1500 ppm group were lower than the control group values during PND 1-4, 7-14 (statistically significant) and 14-21 and similar to the control group during PND 4-7. As a result, mean F1 male and female pup body weights in the 1500 ppm groups were 5.1% to 11.2% and 6.2% to 12.2% lower, respectively than the control group values during PND 4-21; statistical significance was achieved on PND 14 and 21.
- Mean F1 male and female pup body weight gains and body weights in the 750 ppm group were similar to (and not statistically significantly different from) the control group during the first week of the postnatal period and during PND 14-21. During PND 7-14, mean F1 pup body weight gains in these males and females were slightly but statistically significantly lower than the control group values. These lower mean body weight gains were not of sufficient magnitude to affect mean body weights. Differences in mean pup body weights in the 750 ppm group were slight and not statistically significant compared to the control group.

F2 litter:
- Mean F2 male and female body weights in the 2000 ppm group were 5.5% and 4.3% lower, respectively, than the control group on PND 1. Mean body weight gains in the 2000 ppm F2 male and female groups were similar to the control group during PND 1-4 (period of oral gavage administration to the F1 maternal animals), but test article-related lower mean body weight gains were noted for both sexes throughout the remainder of the pre-weaning period (PND 4 to 21). The differences from the control group were statistically significant during PND 4-7, PND 7-14, and PND 14-21 for the body weight gains and from PND7 through 21 for body weights. Mean F2 male and female body weights in this group were similar to the control group on PND 4, but were also 8.1% to 14.6% and 9.2% to 15.7% lower, respectively, than the control group during PND 7-21.
- In the 1500 ppm F2 male and female groups, mean pre-weaning body weights and body weight gains were generally similar to the control group during the first week of the pre-weaning period. However, during PND 4-14, mean body weight gains for both sexes in this group were lower than the control group. Statistical significance was achieved during PND 4-7 (females) and 7-14 (both sexes). Mean male and female body weights in the 1500 ppm group were also up to 6.8% and 7.8% lower, respectively, than the control group during PND 14 through 21; the differences were statistically significant for females on both days. Mean male and female F2 pup body weight gains during PND 14-21 were similar to the control group.
- Mean F2 male and female pup body weights and body weight gains in the 750 ppm group were unaffected by test article exposure. No statistically significant differences from the control group were noted.

F2 adults:
- Mean F2 male and female body weight gains in the 2000 ppm group were lower than the control group during the post-weaning direct exposure period. The differences in the males were generally statistically significant and observed throughout the exposure period (PND 21-28 through 49-56), while the statistically significant differences in the females were observed immediately following direct exposure to the test article (PND 2 1-28 and 28-35). As a result, statistically significantly lower mean cumulative body weight gains were noted for the 2000 ppm F2 males (274 g compared to 312 g in the control group during PND 21-63) and females (172 g compared to 191 g in the control group during PND 21-63) compared to the control group. In addition, mean male and female body weights in this group were 12.4% to 15.4% and 10.8% to 17.1% lower, respectively, (statistically significant) than the control group through PND 63.
- Mean body weight gains in the 1500 ppm F2 female group were statistically significantly lower than the control group values during PND 35-42 and 42-49. These transient differences were not observed in an exposure-dependent manner, but were of sufficient magnitude to cause mean cumulative body weight gain in the 1500 ppm F2 females (174 g) to be statistically significantly lower than the control group value (191 g). Although there were no statistically significant differences in mean body weight gains for the F2 males at 1500 ppm during the exposure period, the mean cumulative body weight gain in this group (297 g) was slightly lower (but not statistically significantly different) than the control group (312 g). Mean body weights in the F2 males and females in the 1500 ppm group were up to 6.4% and 9.1% lower, respectively, than the control group values during PND 21-63; the differences in the females were statistically significant during PND 3 5-63.
- Mean body weights, body weight gains and cumulative body weight gains in the 750 ppm F2 male and female groups were generally similar compared to the control group during the entire post-weaning direct exposure period. Differences from the control group were slight and/or not observed in an exposure-dependent manner. Mean F2 male and female body weights in the 750 ppm group were up to 7.7% and 9.8% lower, respectively, than the control group values during PND 2 1-35; the difference on PND 21 was statistically significant for the females. These differences in mean body weights in the 750 ppm F2 male and female groups were attributed to slightly lower mean body weight gains in these groups prior to weaning and direct exposure. Mean body weights in these animals were similar to the control group values for the remainder of the direct exposure period.

FOOD CONSUMPTION F2 adults:
- Mean food consumption, evaluated as g/animal/day, in the 1500 and 2000 ppm F2 male and female groups was generally lower than the control group throughout the direct exposure period (PND 28-63). The differences from the control group were generally statistically significant. However, there was no corresponding effect on g/kg/day food consumption or food efficiency noted for either sex at 1500 or 2000 ppm. The only statistically significant difference in g/kg/day food consumption and food efficiency was a lower mean g/kg/day food consumption value in the 1500 ppm F2 male group during PND 42-49 compared to the control group; this difference did not occur in an exposure-dependent manner. These food consumption/food efficiency data indicate that body weight gains were consistent with the amount of food consumed.
- Mean food consumption and food efficiency in the 750 ppm F2 male and female groups were generally similar to the control group during the direct exposure period. Statistically significant differences from the control group were noted for the females during PND 5 6-63 (lower g/animal/day food consumption with no corresponding effect on body weight) and during PND 28-35 (higher g/kg/day food consumption that did not occur in an exposure-dependent manner).

SEXUAL MATURATION (OFFSPRING)
F1 litter:
- balanopreputial separation: mean age of attainment of the F1 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 45.6, 47.8*., 46.6 and 47.2 at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and did not follow a clear dose response; mean weights at attainment were lower compared to the control group (221.8, 219.8, 200.*1, 195.8* g at 0, 750, 1500 and 2000 ppm); these effects were attributed to the lower body weights noted during the pre-and post-weaning periods.
- vaginal patency: not affected

F2 litter:
- balanopreputial separation: mean age of attainment of the F2 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 44.6, 45.8, 44.4, 46.4* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and were not dose dependent; the mean weight at attainment was reduced in the 1500 and 2000 ppm group, statistically significant only at 2000 ppm(218.2, 220.4, 207.7, 200.9* g at 0, 750, 1500 and 2000 ppm, respectively). The higher mean age of attainment of balanopreputial separation in the 2000 ppm group was considered to be secondary to lower body weights during pre- and post-weaning periods.
- vaginal patency: age of attainment of the F2 females in the 2000 ppm group was higher than the concurrent control group (mean age at attainment (PN): 33.6, 34.4, 34.4, 35.2* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 32.5 - 38.8); the authors considered the effect as secondary to lower body weights noted in this group during pre-and post-weaning.
*: statistically significant

ORGAN WEIGHTS (OFFSPRING)
F1 litter:
- Significantly lower thymus weights (absolute, relative to final body and/or brain weights) were noted in all dose groups for both males and females on PND 21. As there was no clear dose-response relationship (thymus weights relative to body weight (g/100g) males: 0.438, 0.398, 0.375, 0.400 at 0, 750, 1500 and 2000 ppm, respectively; females: 0.452, 0.418, 0.400, 0.427 at 0, 750, 1500 and 2000 ppm, respectively) and the body weight of the animals was also lower in comparison to the controls it remains unclear whether this effect is test item related.

F2 litter:
- Significantly lower thymus weights (absolute, relative to final body weight and relative to brain weight) were noted in all dose groups. As there was no clear dose-response relationship (thymus weights relative to body weight (g/100g) males: 0.401, 0.384, 0.359, 0.365 at 0, 750, 1500 and 2000 ppm, respectively; females: 0.426, 0.404, 0.397, 0.395 at 0, 750, 1500 and 2000 ppm, respectively() and the body weight of the animals was also lower in comparison to the controls it remains unclear whether this effect is test item related. Organ weight changes of brain and spleen were attributed to lower final body weights by the author.

F2 adults:
- Several differences in mean organ weights in the F2 males and females were observed at the end of the 7-week exposure period that were considered secondary to the lower mean final body weights in these groups: e.g. Mean brain relative to final body weight was higher for males at 2000 ppm and females at 1500 and 2000 ppm, while absolute brain weight was lower in both sexes at 2000 ppm. Lower mean absolute pituitary weight were noted for males at 2000 ppm and for females at 1500 and 2000 ppm while lower mean pituitary relative ot brain weights were noted for males at 2000 ppm.
- reduction of thymus weights was only observed in females, but without clear dose-response relationship (thymus weights relative to body weight (g/100g) females: 0.230, 0.229, 0.218, 0.223 at 0, 750, 1500 and 2000 ppm, respectively)


GROSS PATHOLOGY (OFFSPRING)
F1 and F2 litter:
- No internal findings that could be attributed to parental exposure to the test article were noted at the necropsies of pups that were found dead or pups weaned at PND 21.


HISTOPATHOLOGY (OFFSPRING)
- not examined

OTHER FINDINGS (OFFSPRING)
F1 and F2 litter:
- Pinnal detachment: not affected
- Incisor eruption: not affected
- eye opening: not affected

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
750 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: growth retardation (lower pup bws and bw gains) and delays in attainment of developmental landmarks (secondary to reduced bw) at 1500 and 2000 ppm were only observed in association with maternal toxicity; no effects on fertility at any dose level.
Description (incidence and severity):
- The numbers of F2 pups found dead and/or missing, as well as the general physical condition of all F2 pups in this study were unaffected by F1 parental test article exposure. Pups (litters) that were found dead numbered 17(9), 29(12), 5(5) and 15(7) in the control, 750, 1500 and 2000 ppm groups, respectively. Two (2), 6(5), 1(1) and 0(0) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Sixteen and 10 pups in the litters of female nos. 99137-11 and 99196-12, respectively, in the 1500 ppm group were euthanized and necropsied due to the death of their dam on PND 3 and 4, respectively.
Mortality / viability:
no mortality observed
Description (incidence and severity):
- The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival were unaffected by the test article at all exposure levels. Differences from the control group were slight, were not statistically significant and/or did not occur in an exposure-dependent manner.

F2 adults:
- One male and one female in the 2000 ppm group were found dead on PND 25 and 22, respectively. Two males and one female in the 750 ppm group were found dead between PND 23 and 31. There were no test article-related clinical findings noted prior to the death of these animals, and based on macroscopic examination, a cause of death could not be determined for these animals. All remaining F2 animals in the control, 750, 1500 and 2000 ppm groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 litter:
- Mean F2 male and female body weights in the 2000 ppm group were 5.5% and 4.3% lower, respectively, than the control group on PND 1. Mean body weight gains in the 2000 ppm F2 male and female groups were similar to the control group during PND 1-4 (period of oral gavage administration to the F1 maternal animals), but test article-related lower mean body weight gains were noted for both sexes throughout the remainder of the pre-weaning period (PND 4 to 21). The differences from the control group were statistically significant during PND 4-7, PND 7-14, and PND 14-21 for the body weight gains and from PND7 through 21 for body weights. Mean F2 male and female body weights in this group were similar to the control group on PND 4, but were also 8.1% to 14.6% and 9.2% to 15.7% lower, respectively, than the control group during PND 7-21.
- In the 1500 ppm F2 male and female groups, mean pre-weaning body weights and body weight gains were generally similar to the control group during the first week of the pre-weaning period. However, during PND 4-14, mean body weight gains for both sexes in this group were lower than the control group. Statistical significance was achieved during PND 4-7 (females) and 7-14 (both sexes). Mean male and female body weights in the 1500 ppm group were also up to 6.8% and 7.8% lower, respectively, than the control group during PND 14 through 21; the differences were statistically significant for females on both days. Mean male and female F2 pup body weight gains during PND 14-21 were similar to the control group.
- Mean F2 male and female pup body weights and body weight gains in the 750 ppm group were unaffected by test article exposure. No statistically significant differences from the control group were noted.

F2 adults:
- Mean F2 male and female body weight gains in the 2000 ppm group were lower than the control group during the post-weaning direct exposure period. The differences in the males were generally statistically significant and observed throughout the exposure period (PND 21-28 through 49-56), while the statistically significant differences in the females were observed immediately following direct exposure to the test article (PND 2 1-28 and 28-35). As a result, statistically significantly lower mean cumulative body weight gains were noted for the 2000 ppm F2 males (274 g compared to 312 g in the control group during PND 21-63) and females (172 g compared to 191 g in the control group during PND 21-63) compared to the control group. In addition, mean male and female body weights in this group were 12.4% to 15.4% and 10.8% to 17.1% lower, respectively, (statistically significant) than the control group through PND 63.
- Mean body weight gains in the 1500 ppm F2 female group were statistically significantly lower than the control group values during PND 35-42 and 42-49. These transient differences were not observed in an exposure-dependent manner, but were of sufficient magnitude to cause mean cumulative body weight gain in the 1500 ppm F2 females (174 g) to be statistically significantly lower than the control group value (191 g). Although there were no statistically significant differences in mean body weight gains for the F2 males at 1500 ppm during the exposure period, the mean cumulative body weight gain in this group (297 g) was slightly lower (but not statistically significantly different) than the control group (312 g). Mean body weights in the F2 males and females in the 1500 ppm group were up to 6.4% and 9.1% lower, respectively, than the control group values during PND 21-63; the differences in the females were statistically significant during PND 3 5-63.
- Mean body weights, body weight gains and cumulative body weight gains in the 750 ppm F2 male and female groups were generally similar compared to the control group during the entire post-weaning direct exposure period. Differences from the control group were slight and/or not observed in an exposure-dependent manner. Mean F2 male and female body weights in the 750 ppm group were up to 7.7% and 9.8% lower, respectively, than the control group values during PND 2 1-35; the difference on PND 21 was statistically significant for the females. These differences in mean body weights in the 750 ppm F2 male and female groups were attributed to slightly lower mean body weight gains in these groups prior to weaning and direct exposure. Mean body weights in these animals were similar to the control group values for the remainder of the direct exposure period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean food consumption, evaluated as g/animal/day, in the 1500 and 2000 ppm F2 male and female groups was generally lower than the control group throughout the direct exposure period (PND 28-63). The differences from the control group were generally statistically significant. However, there was no corresponding effect on g/kg/day food consumption or food efficiency noted for either sex at 1500 or 2000 ppm. The only statistically significant difference in g/kg/day food consumption and food efficiency was a lower mean g/kg/day food consumption value in the 1500 ppm F2 male group during PND 42-49 compared to the control group; this difference did not occur in an exposure-dependent manner. These food consumption/food efficiency data indicate that body weight gains were consistent with the amount of food consumed.
- Mean food consumption and food efficiency in the 750 ppm F2 male and female groups were generally similar to the control group during the direct exposure period. Statistically significant differences from the control group were noted for the females during PND 5 6-63 (lower g/animal/day food consumption with no corresponding effect on body weight) and during PND 28-35 (higher g/kg/day food consumption that did not occur in an exposure-dependent manner).
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):
- balanopreputial separation: mean age of attainment of the F2 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 44.6, 45.8, 44.4, 46.4* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and were not dose dependent; the mean weight at attainment was reduced in the 1500 and 2000 ppm group, statistically significant only at 2000 ppm(218.2, 220.4, 207.7, 200.9* g at 0, 750, 1500 and 2000 ppm, respectively). The higher mean age of attainment of balanopreputial separation in the 2000 ppm group was considered to be secondary to lower body weights during pre- and post-weaning periods.
- vaginal patency: age of attainment of the F2 females in the 2000 ppm group was higher than the concurrent control group (mean age at attainment (PN): 33.6, 34.4, 34.4, 35.2* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 32.5 - 38.8); the authors considered the effect as secondary to lower body weights noted in this group during pre-and post-weaning.
*: statistically significant
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F2 litter:

- Significantly lower thymus weights (absolute, relative to final body weight and relative to brain weight) were noted in all dose groups. As there was no clear dose-response relationship (thymus weights relative to body weight (g/100g) males: 0.401, 0.384, 0.359, 0.365 at 0, 750, 1500 and 2000 ppm, respectively; females: 0.426, 0.404, 0.397, 0.395 at 0, 750, 1500 and 2000 ppm, respectively() and the body weight of the animals was also lower in comparison to the controls it remains unclear whether this effect is test item related. Organ weight changes of brain and spleen were attributed to lower final body weights by the author.

F2 adults:
- Several differences in mean organ weights in the F2 males and females were observed at the end of the 7-week exposure period that were considered secondary to the lower mean final body weights in these groups: e.g. Mean brain relative to final body weight was higher for males at 2000 ppm and females at 1500 and 2000 ppm, while absolute brain weight was lower in both sexes at 2000 ppm. Lower mean absolute pituitary weight were noted for males at 2000 ppm and for females at 1500 and 2000 ppm while lower mean pituitary relative ot brain weights were noted for males at 2000 ppm.
- reduction of thymus weights was only observed in females, but without clear dose-response relationship (thymus weights relative to body weight (g/100g) females: 0.230, 0.229, 0.218, 0.223 at 0, 750, 1500 and 2000 ppm, respectively)
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test article-related gross necropsy observations in the F2 generation.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Pinnal detachment: not affected
- Incisor eruption: not affected
- eye opening: not affected
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
750 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: growth retardation (lower pup bws and bw gains) and delays in attainment of developmental landmarks (secondary to reduced bw) at 1500 and 2000 ppm were only observed in association with maternal toxicity; no effects on fertility at any dose level.
Reproductive effects observed:
no

RESULTS

Summary of mean measured test article exposure concentration


Mean nominal concentrations (ppm) by generation

Target concentration (ppm):

0

750

1500

2000

F0 generation

0

749.4

1495.8

2018.1

F1generation

0

752.8

1495.5

2008.1

F2generation

0

760.8

1506.3

2009.6

Conclusions:
Inhalation exposure of rats to 750, 1500 or 2000 ppm n-butyl acetate in a two generation study did not affect reproductive performance. Effects on body weight/body weight gain and food consumption at 1500 and 2000 ppm as well as histopathological changes of the nasal cavity (already at 750 ppm) but no other histopathological effects were observed in parental animals. Adverse effects on pups born to dams exposed to the test article were limited to exposure -related growth retardation (lower mean pup body weights and body weight gains at 1500 and 2000 ppm) and delays in attainment of post-weaning developmental landmarks at 2000 ppm (an effect considered to be secondary to reduced body weight). No teratogenicity was observed. The NOEC for fertility was 2000 ppm, the NOAEC for developmental toxicity 750 ppm and the NOAEC for systemic toxicity 750 ppm.
Executive summary:

A two generation study was performed with rats which were exposed to n-butyl acetate. Four groups of male and female Crl:CD(SD) rats (30/sex/group) were exposed to either clean filtered air or vapour atmospheres of the test article, n-butyl acetate, for 6 hours per day (7 days per week) for at least 70 consecutive days prior to mating (whole body exposure). Target test article concentrations were 750, 1500 and 2000 ppm for the F0, F1and F2 generations. Exposures were initiated when the F0 animals were approximately 7 weeks of age. Exposure of the F0 and F1 males continued throughout mating, and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. Exposure was suspended from gestation day 21 through lactation day 4 to avoid the confounding effects on nesting and nursing behavior caused by separation of dams from their litters. Therefore, on lactation days 1-4, the F0 and F1 maternal animals received deionized water or the test article via oral gavage at dosage levels of 0, 1125, 2250 and 3000 mg/kg/day (0, 375, 750 and 1000 mg/kg/dose, respectively, administered as 3 equal doses, approximately 2 hours apart), to mimic the internal dose expected from inhalation exposure. Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. Inhalation exposure of the F1 animals was initiated on PND 22 (2 weanlings/sex/litter, when possible), and in order to determine if differences from the control group in mean body weights, body weight gains and food consumption noted in F1 males at 750 ppm during the post-weaning period were reproducible, F1 animals (2 weanlings/sex/litter, when possible) were directly exposed to the test article beginning on PND 22. Following approximately 2-3 weeks of exposure (PND 36-47) of the F1 generation and on PND 36 for the F2 generation, offspring (minimum of 1 animal/sex/litter, to a total of 30/sex/group) were selected for the maturation phase of each generation. F0 males and females were exposed for 113-114 and 121-127 consecutive days, respectively, and F1 males and females were exposed for 134-135 and 140-153 consecutive days, respectively. F2 males and females were exposed for 47-50 consecutive days.

There were no functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, spermatogenic endpoints and gestation length) in any test article-exposed group in the F0 or F1 generations. Adverse effects on pups born to dams exposed to the test article were limited to exposure-related growth retardation, as evidenced by lower mean pup body weights, body weight gains at 1500 and 2000 ppm; delays in attainment of post-weaning developmental landmarks were also observed at 1500 and 2000 ppm, which were considered to be secondary to lower body weights noted in this groups during the pre- and post-weaning periods. F1and F2 pup survival was unaffected by the test article at all exposure levels. Therefore, in this study an exposure level of 2000 ppm was considered to be the no-observed-adverse-effect concentration (NOAEC) for fertility and 750 ppm is the NOAEC for developmental toxicity.

General systemic toxicity was evident in the 1500 and 2000 ppm group F0, F1and F2 adult males and females. Decrements in mean body weights, body weight gains and food consumption were observed at 1500 and 2000 ppm. Although body weights, body weight gains and food consumption were reduced in the 750 ppm males of the F1 generation, direct inhalation exposure of F2 animals on a comparable regimen did not reproduce this response. Additionally, the magnitude and temporal pattern of the reductions in the F1males at 750 ppm were not noted in the F0 generation. Therefore, mean body weights, body weight gains and food consumption at 750 ppm were considered to be unaffected by test article exposure. Degeneration of the olfactory epithelium in the nasal cavity was noted at 750 and 2000 ppm in F0 and F1 males and females (endpoint not investigated at 1500 ppm). This finding was considered to be a site-of-contact irritant effect and not indicative of systemic toxicity. Statistically significantly higher mean lung weights (relative to final body weight) in F0 males at 1500 and 2000 ppm, in F1 males at 750, 1500 and 2000 ppm and in F0 females at 2000 ppm and adrenal weights (absolute, relative to brain and/or body weight) in F0 at 1500 and 2000 ppm were noted; all were observed without correlating histologic findings and were considered a stress-related response (adrenal weights) or not adverse (lung weights). Therefore, the NOAEC for systemic toxicity in adult rats was considered to be 750 ppm under the conditions of this study.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no repeat-dose inhalation reproductive toxicity studies for n-propyl acetate.

Information is available, however, for n-propyl alcohol as metabolite and n-butyl acetate as analogous substance.

The effect of inhalation exposure to n-propyl alcohol on fertility was investigated in male and female rats (Nelson et al. 1989). Male animals (N=18/group) were treated for 42 days, then mated (1:1) with untreated virgin females. Resulting pregnant females were sham-exposed on gestation days 1-19 (experiment 1). Another group of untreated males and females were mated. Resulting pregnant females (target N=15/group) were then exposed to the test substance on gestation days 1-19 (experiment 2). The incidence of successful breeding was evaluated among males and females that had been exposed to propyl alcohol as well as sham-exposed rats.

Because infertility was observed in high dose males, a recovery group of six males from the 7000 ppm (17.220 mg/L) group was retained and remated at biweekly intervals to determine if infertility was reversible.

Maternal/paternal effects: Weight gain was reduced in males and females exposed to n-propyl

alcohol for 42 days; food intake was reduced in females exposed to 7000 ppm (17.9 mg/L). There were no significant effects on food intake or body weight gains at 3500 ppm (8.9 mg/L).

Maternal/Paternal Fertility: Male rats exposed to 7000 ppm (17.9 mg/L) displayed reduced fertility; in spite of sperm plugs apparent in 16 paternally-exposed males (one died because of fighting, and another did not mate), only two litters resulted with populations of 12 and 2 pups, respectively. No resorption sites were noted in mated, non-pregnant females, indicating that pregnancy had not occurred.  There was no adverse effect on male or female fertility in rats exposed to 3500 ppm (8.9 mg/L). At 7000 ppm, the number pregnant/number bred were 17/17 for maternally exposed rats, 2/16 for paternally exposed rats, 18/18 for controls and 38/40 for forster rats in the 7000 ppm group. Loss of male fertility was reversible within 13 weeks.

Offspring:  Examination of offspring revealed that 2 of 15 litters from the 7000 ppm maternally exposed group had pups with crooked tails (noted soon after birth) and these defects persisted. Sex differences in weights became apparent on day 28.

The NOAEL for paternal toxicity (fertility) was determined to be 3500 ppm (8.9 mg/L). The NOAEL for maternal toxicity (fertility) was determined to be 7000 ppm (17.9 mg/L).

A reliable (RL1) two generation inhalation study was performed with Sprague Dawleyrats (whole body exposure, 6 h/d, 7 d/w,30/sex/group) which were exposed to 0,750, 1500 or 2000 ppm. Exposure was suspended from gestation day 21 through lactation day 4 to avoid the confounding effects on nesting and nursing behavior caused by separation of dams from their litters.Therefore, on lactation days 1-4, the F0 and F1 maternal animals received deionized water or the test article via oral gavage at dosage levels of 0, 1125, 2250 or 3000 mg/kg/day. F0 males and females were exposed for 113-114 and 121-127 consecutive days, respectively, and F1 males and females were exposed for 134-135 and 140-153 consecutive days, respectively. F2 males and females were exposedfor 47-50 consecutive days.

There were no functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, spermatogenic endpoints and gestation length) in any test article-exposed group in the F0 or F1 generations.The exposure level of 2000 ppm (9640 mg/m3) was considered to be the no-observed-adverse-effect concentration (NOAEC) for reproductive toxicity (fertility).

General systemic toxicity was evident in the 1500 and 2000 ppm group F0, F1 and F2 adult males and females. Decrements in mean body weights, body weight gains and food consumption were observed at 1500 and 2000 ppm. Although body weights, body weight gains and food consumption were reduced in the 750 ppm males of the F1 generation, direct inhalation exposure of F2 animals on a comparable regimen did not reproduce this response. Additionally, the magnitude and temporal pattern of the reductions in the F1 males at 750 ppm were not noted in the F0 generation. Therefore, mean body weights, body weight gains and food consumption at 750 ppm were considered to be unaffected by test article exposure. Degeneration of the olfactory epithelium in the nasal cavity was noted at 750 and 2000 ppm in F0 and F1 males and females (1500 ppm group not investigated). This finding was considered to be a site-of-contact irritant effect and not indicative of systemic toxicity. Statistically significantly higher mean lung weights (relative to final body weight) in F0 males at 1500 and 2000 ppm, in F1 males at 750, 1500 and 2000 ppm and in F0 females at 2000 ppm and adrenal weights (absolute, relative to brain and/or body weight) in F0 at 1500 and 2000 ppm were noted; all were observed without correlating histologic findings and were considered a stress-related response (adrenal weights) or not adverse (lung weights). Therefore, the NOAEC for systemic toxicity in adult rats was considered to be 750 ppm under the conditions of this study (Nemec, 2010).

 

 

Read across justification to propan-1-ol and n-butyl acetate for filling data gaps of n-propyl acetate:

For each of the source substances a reliable study is available which is considered appropriate to fulfil the standard information requirements of Annex X, Section 8.7.3 of the REACH Regulation according to the provisions of Annex XI, 1.5 of the REACH Regulation.

As indicated by toxicokinetic studies (see chapter on toxicokinetics, metabolism and distribution), n-propyl acetate is rapidly hydrolyzed to propan-1-ol and acetate (acetic acid). Available data on propan-1-ol is therefore suitable for filling data gaps of n-propyl acetate.

N-propyl acetate and n-butyl acetate differ structurally by only one –CH2 group and both substances have a similar toxicological profile. The available data for n-butyl acetate is therefore suitable for filling the data gaps of n-propyl acetate due to structural similarities.

For a detailed justification of read-across, please refer to IUCLID section 13.


Short description of key information:
Propyl acetate
no data available

Analogous substances

Propan-1-ol
NOAEL fertility rat inhalation 42 d prior to mating: 8.9 mg/L (3500 ppm) based on paternal fertility effects; NOEL parental: 8.9 mg/L based on reduced body weight (Nelson et al. 1989)

Butyl acetate
NOAEL fertility rat inhalation from a 2-Gen study = 2000 ppm (ACC, 2010)

Effects on developmental toxicity

Description of key information

- OECD 414, GLP, 25 female rats/dose, 100 - 1000 mg/kg bw/day, exposure from GD 6 -19. No maternal and fetal developmental toxicity (2018)

- OECD 414, GLP, 24 female rabbits/dose, 100 - 1000 mg/kg/bw/day, exposure from GD 7 -27. No maternal and fetal developmental toxicity (2019)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep - Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Expiration date of the batch: 08 Nov 2018
- Purity: 99.9%
- Identitiy: Confirmed
- Physical state/appearance: liquid/colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions (in the vehicle): Guaranteed by analytical verifications in corn oil over a period of seven days at room temperature
- Homogeneity: given (visually)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer or by shaking until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI[Han]
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 144.5 - 195.6 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm illumination)

IN-LIFE DATES: From: 1st Cohort: 28 Sep 2017; 2nd Cohort: 29 Sep 2017 To: 1st Cohort: 18 Oct 2017; 2nd Cohort: 19 Oct 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Volume applied: 4ml/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): at the beginning and at intervals thereafter
- Mixing appropriate amounts: test substance was weighed, topped up with corn oil and intensely mixed.
- Storage temperature: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): non given
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent thrice to the analytical laboratory for verification of the concentrations. Control procedures were Gas chromatography (GC) and H-NMR spectrum.
Details on mating procedure:
Animals were paired by the breeder ("time-mated"); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
daily
Duration of test:
14 days
Dose / conc.:
100 mg/kg bw/day
Remarks:
2.5 g/100 ml
Dose / conc.:
300 mg/kg bw/day
Remarks:
7.5 g/100 ml
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
25.00 g/100 ml
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Reason for species selection:
The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.
Maternal examinations:
MORTALITY: Yes
- Time schedule: twice daily (Mo-Fr), once a day (Sat, Sun)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19, and 20

FOOD CONSUMPTION: Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No data
- Time schedule for examinations: no data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Gross pathology

NECROPSY:
- uteri and ovaries were analyzed: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations
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
- Dead fetuses: Yes
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data
- Weight, sex, external tissues, and all orifices were examined
- Viability
- Condition and weight of placentas, umbilical cords, fetal membranes, and fluids
Statistics:
Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) forthe hypothesis of equal means

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians
Indices:
Conception rate, preimplantation loss, postimplantation loss
Historical control data:
MATERNAL BODY WEIGHTS (RANGE) FROM 973 (Days 1 - 20) AND 974 (Day 0) ANIMALS

Day 0: 140.0 - 196.6 g
Day 1: 152.1 - 208.6
Day 3: 158.7 - 216.0
Day 6: 167.6 - 231.5
Day 8: 174.3 - 242.1
Day 10: 181.6 - 252.9
Day 13: 186.6 - 278.4
Day 15: 192.5 - 290.4
Day 17: 183.3 - 318.4
Day 19: 190.5 - 349.8
Day 20: 193.2 - 364.3

REPRODUCTION DATA

- Females mated: 999 of which pregnant: 976 (98%)
- Aborted: 0
- Premature births: 0
- Dams with viable fetuses: 973
- Dams with all resorptions: 3
- Female mortality: 0%
- Corpora lutea (range): 10.5 - 12.3
- Implantations sites (range): 9.9 - 11.8
- Preimplantation loss (range): 1.4 - 13.3%
- Postimplantation loss (range): 3.2 - 14.0%
- Dams with resorptions: 449 (46%)
- Resorptions (range): 0.3 - 1.5
- Dead fetuses: 0
- Viable fetuses: 9778
- Litter size (range): 9.3 - 11.2
- Placenta weights (range): 0.35 - 1.14 g (males); 0.32 - 1.10 g (females)
- Fetal weights (range): 2.8 - 4.5 g (males); 2.3 - 4.4 g (females)

FETAL MALFORMATIONS:

- External malformations: 13 (9778 evaluated) ; 0.1%
- External variations: 4 (9778 evaluated); 0.04%
- External unclassified findings: 3 (9778 evaluated); 0.03%
- Visceral malformations: 13 (4650 evaluated); 0.3%
- Visceral variations: 156 (4650 evaluated); 3.4%
- Visceral unclassified findings: 1 (4650 evaluated); 0.02%
- Skeletal malformations: 39 (5128 evaluated); 0.8%
- Skeletal variations: 5035 (5128 evaluated); 98.2%
- Skeletal cartilage: 3655 (5218 evaluated); 71.3%
Clinical signs:
no effects observed
Description (incidence and severity):
In 21 out of 25 females occasionally salivation during treatment period in group 1000 mg/kg bw/day. Salivation occurred in the respective animals only shortly, i.e. within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as a sign of systemic toxicity.

No other effects observed
Mortality:
no mortality observed
Description (incidence):
No test substance-related or spontaneous deaths in any females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and average body weight gain of treated groups comparable to control.

The statistically significantly decreased body weight gain value in test group 1 during GD 3-6 (pre-treatment period) was assessed as incidental.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption of treated groups comparable to control.

The statistically significantly decreased food consumption value in the mid-dose dams during GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.
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):
Mean gravid uterus weights not influenced by the test substance. Difference between treated groups and control group revealed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0 and 1000 mg/kg bw/d). These gross findings were:
• Dilated renal pelvis (right) in dam No. 2,
• Diaphragmatic hernia in dam No. 4,
• Stomach: erosion(s) (one) in dam No. 79.
No further necropsy findings which could be attributed to the test substance were seen in any dam.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test substance related differences in conception rates, mean number of corpora lutea and implantation sites.
Two females in the control group and two females in the 300 mg/kg bw/day group did not get pregnant.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no maternal developmental toxicity observed
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Three external malformations were exclusively detected in the control group.

No fetal external variations were reported. No fetal external unclassified findings were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in two fetuses of the control group and in one fetus, each, of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control fetus had associated external malformations. The incidences of these malformations were neither
statistically significantly different from control nor dose-dependent. The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without relation to dose. The mean value of the incidences in test group 3 was within the historical
control range. Therefore, it was not assessed as treatment-related, adverse finding.
All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to dose and were single, isolated cases which were mostly covered by the historical control data.
The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and adverse.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The
overall incidences of skeletal variations were comparable to the historical control data.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations were recorded for two control, one mid-dose and three high-dose fetuses. The finding situs inversus in the fetuses of test groups 300 mg/kg bw/day and 1000 mg/kg bw/day were single events in individual fetuses and the mean values were within the historical control data. The findings absent subclavian and anophthalmia were observed in one fetus each of test group 1000 mg/kg bw/day and the mean values were within the historical control ranges.
All these findings were single cases, they were within the historical control ranges.
No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring. Therefore, the observed malformations were not assessed as treatment-related, adverse findings.

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently altered. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.

No fetal visceral unclassified findings were reported.
Other effects:
no effects observed
Description (incidence and severity):
Mean placental weights comparable among groups.
Details on embryotoxic / teratogenic effects:
No teratogenic effects due to treatment were recorded.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity and teratogenic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

RESULTS

ANALYSES

Stability analysis

The stability of the test substance in corn oil over a period of 7 days at room temperature was

demonstrated before the start of the study.

Homogeneity analysis of the test substance preparations

Given that the test substance is completely miscible with corn oil, solutions were considered to

be homogenous without further analysis.

Concentration control analysis of the test substance preparations

The results of the analysis of the test substance preparations confirmed the correctness of the

prepared concentrations of the mid- and high-dose (samples 04 and 05, respectively). These

analytical values of the samples corresponded to the expected values within the limits of the

analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Concerning the lowest dose (100 mg/kg bw/d), the values of samples 03, 03R and 08 new

(149.8, 139 and 124%, respectively) were outside the range of 90% - 110% of the nominal

concentrations. A mean value for all three measurements of around 138% corresponds to actual

dose of 138 mg/kg bw/d for the lowest test group. This has no influence of the validity of

the study.

Food analyses

On the basis of the duration of use and the analytical findings with respect to chemical and

microbiological contaminants, the food was found to be suitable. The EPA Fed. Reg. of 09 May

1979 (Vol. 44, No. 91, p. 27354) served as a guideline for maximum tolerable chemical contaminants.

The amount of microorganisms did not exceed 1*105/g feed.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Drinking water analyses

On the basis of the analytical findings, the drinking water was found to be suitable. The German

Drinking Water Regulation (“Trinkwasserverordnung”) served as the guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Bedding and enrichment analyses

On the basis of the analytical findings, the bedding and the enrichment were found to be suitable.

Levels given in Lab Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Clinical examinations of the dams

Only pregnant dams were used for the calculations of mean maternal food consumption, body

weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were

used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and

summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):

• Females Nos. 13, 14 – not pregnant

Test group 2 (300 mg/kg bw/d):

• Females Nos. 54, 62 – not pregnant

4.2.1.1. Mortality

There were no test substance-related or spontaneous mortalities in any females of all test

groups (0, 100, 300 or 1000 mg/kg bw/d).

Clinical symptoms

Nearly all (21 out of 25) high-dose females (1000 mg/kg bw/d) showed occasionally salivation

during the treatment period. Salivation occurred in the respective animals only shortly, i.e.

within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical

signs or changes of general behavior were detected in any female of all test groups beyond 2

hours after treatment. The occasional salivation was most probably caused by the bad taste or

smell of the test substance and was not assessed as a sign of systemic toxicity.

No further clinical signs or changes of general behavior, which may be attributed to the test

substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during

the entire study period.

Food consumption

The mean food consumption of the high-, mid- and low-dose dams (1000, 300 and 100 mg/kg

bw/d) was generally comparable to the concurrent control group throughout the entire study

period.

The statistically significantly decreased food consumption value in the mid-dose dams during

GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.

Body weight data

The mean body weights and the average body weight gain of the low-, mid- and high-dose

dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control

group throughout the entire study period.

The statistically significantly decreased body weight gain value in test group 1 during GD 3-6

(pre-treatment period) was assessed as incidental.

Corrected (net) body weight gain

The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d)

revealed no difference of any biological relevance to the corresponding control group.

Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Terminal examinations of the dams

Uterus weight

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg

bw/d) were not influenced by the test substance. The differences between these groups and

the control group revealed no dose-dependency and were assessed to be without biological

relevance.

Necropsy findings

Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0

and 1000 mg/kg bw/d). These gross findings were:

• Dilated renal pelvis (right) in dam No. 2,

• Diaphragmatic hernia in dam No. 4,

• Stomach: erosion(s) (one) in dam No. 79.

No further necropsy findings which could be attributed to the test substance were seen in any

dam.

Reproduction data

The conception rate was 92% in the control and the mid-dose groups (0 and 300 mg/kg bw/d)

and 100% in the low- and high-dose groups (100 and 1000 mg/kg bw/d). With these rates, a

sufficient number of pregnant females were available for the purpose of this study (according

to the test guidelines listed in chapter 2.3.).

There were no test substance-related and/or biologically relevant differences between the different

test groups in conception rate, in the mean number of corpora lutea and implantation

sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions

and viable fetuses. All observed differences are considered to reflect the normal

range of fluctuations for animals of this strain and age.

EXAMINATION OF THE FETUSES

Sex distribution of the fetuses

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was

comparable to the control fetuses.

Weight of the placentae

The mean placental weights of test groups 1-3 were comparable to the concurrent control

group.

Weight of the fetuses

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance

and did not show any biologically relevant differences in comparison to the concurrent control

group.

Fetal external malformations

Three external malformations were exclusively detected in the control group. In one case, these

external malformations were associated with skeletal malformations. No external malformations

occurred in treated animals.

Tab. 2: Individual fetal external malformations

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

9-03 F

umbilical hernia

15-03 Fa)

gastroschisis, ectrodactyly

1 (100 mg/kg bw/d)

none

 

2 (300 mg/kg bw/d)

none

 

3 (1000 mg/kg bw/d)

none

 

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional skeletal malformations

Tab.3: Total external malformations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

251

25

248

23

229

25

273

 

Fetal incidence

 

N (%)

 

2 (0.8)

 

0.0

 

0.0

 

0.0

 

Litter incidence

 

N (%)

 

2 (8.7)

 

0.0

 

0.0

 

0.0

Affectedfetuses/litter

 

Mean%

 

0.8

 

0.0

 

0.0

 

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal external variations

No external variations were recorded.

Fetal external unclassified observations

No external unclassified observations were recorded.

Fetal soft tissue malformations

Soft tissue malformations were recorded for two control, one mid-dose and three high-dose

fetuses

The finding situs inversus in the fetuses of test groups 2 and 3 were single events in individual

fetuses and the mean values were within the historical control data (affected fetuses per litter, mean: 0.1%, range of 0.0 - 1.4%).

The findings absent subclavian and anophthalmia were observed in one fetus each of test

group 3 and the mean values were within the historical control ranges (anopthalmia: litters,

range of 0.0 - 4.0%, absent subclavian: affected fetuses per litter, range of 0.0 - 1.1%).

All these findings were single cases, they were within the historical control ranges. No ontogenetic

pattern is recognizable for all these individual malformations nor was there any cluster of

any of these individual malformations seen in the other offspring.

Therefore, the observed malformations were not assessed as treatment-related, adverse

findings.

Tab. 4: Individual soft tissue malformations

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

12-02 F

interrupted spinal cord

25-14 F

hydronephrosis, hydroureter

1 (100 mg/kg bw/d)

none

 

2 (300 mg/kg bw/d)

72-06 M

situs inversus

3 (1000 mg/kg bw/d)

76-08 F

situs inversus

86-04 F

absent subclavian

95-09 M

anophthalmia

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Tab. 5: Total soft tissue malformations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

122

25

117

23

110

25

130

 

Fetal incidence

 

N (%)

 

2 (1.6)

 

0.0

 

1 (0.9)

 

3 (2.3)

 

Litter incidence

 

N (%)

 

2 (8.7)

 

0.0

 

1 (4.3)

 

3 (12)

Affectedfetuses/litter

 

Mean%

 

1.5

 

0.0

 

0.9

 

2.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue variations

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal

pelvis and dilated ureter. These variations were neither significantly different from the concurrent

control nor dose-dependently altered. All of them can be found in the historical control data

at comparable incidences. Therefore, they were not assessed as treatment-related.

TAb. 6: Total soft tissue variations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

122

25

117

23

110

25

130

Fetal incidence

 

N (%)

 

3 (2.5)

 

2 (1.7)

 

2 (1.8)

 

4 (3.1)

Litter incidence

 

N (%)

 

3 (13)

 

2 (8.0)

 

2 (8.7)

 

4 (16)

Affectedfetuses/litter

 

Mean%

 

2.6

 

1.3

 

2.2

 

3.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue unclassified observations

No soft tissue unclassified observations were recorded.

Fetal skeletal malformations

Skeletal malformations were noted in two fetuses of the control group and in one fetus, each,

of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control

fetus had associated external malformations. The incidences of these malformations were neither

statistically significantly different from control nor dose-dependent.

The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without

relation to dose. The mean value of the incidences in test group 3 was within the historical

control range (HCD: affected fetuses per litter, range of 0.0 – 0.7%). Therefore, it was not

assessed as treatment-related, adverse finding.

All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to

dose and were single, isolated cases which were mostly covered by the historical control data.

The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of

test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and

adverse.

TAb. 7: Individual fetal skeletal malformations

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

11-04 F

malpositioned and bipartite sternebra

15-03 Fa)

absent forepaw phalanx, severely malformed sternum

1 (100 mg/kg bw/d)

33-10 F

bipartite basisphenoid, misshapen presphenoidal, severely malformed vertebral column and/or ribs

2 (300 mg/kg bw/d)

none

 

3 (1000 mg/kg bw/d)

91-07 M

bipartite basisphenoid

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional external malformations

Tab.8: Total skeletal malformations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

129

25

131

23

119

25

143

Fetal incidence

 

N (%)

 

2 (1.6)

 

1 (0.8)

 

0.0

 

1 (0.7)

Litter incidence

 

N (%)

 

2 (8.7)

 

1 (4.0)

 

0.0

 

1 (4.0)

Affectedfetuses/litter

 

Mean%

 

2.0

 

0.8

 

0.0

 

0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal skeletal variations

For all test groups, skeletal variations of different bone structures were observed, with or

without effects on corresponding cartilages. The observed skeletal variations were related to

several parts of fetal skeletons and appeared without a relation to dose. The

overall incidences of skeletal variations were comparable to the historical control data.

Tab. 9: Total fetal skeletal varations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

129

25

131

23

119

25

143

Fetal incidence

 

N (%)

 

124 (96)

 

126 (96)

 

117 (98)

 

140 (98)

Litter incidence

 

N (%)

 

23 (100)

 

25 (100)

 

23 (100)

 

25 (100)

Affectedfetuses/litter

 

Mean%

 

96.5

 

96.5

 

98.0

 

97.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For a better overview, all skeletal variations with statistically significant differences between

the control and any treated group were compiled in the table below. All

incidences were expressed on a fetus per litter basis.

Tab. 10: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

 

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of supraoccipital; unchanged cartilage

 

20.2

 

16.8

 

34.2*

 

18.9

 

32.2

(14.5 – 63.5)

 

Misshapen sacral vertebra

 

1.3

 

3.2

 

0.9

 

5.5*

 

4.1

(0.7 – 9.8)

 

Unossified sternebra; unchanged cartilage

 

2.1

 

2.3

 

3.4

 

9.1*

 

4.3

(0.0 – 9.7)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p 0.05 (Wilcoxon-test [one-sided]) ** = p 0.01 (Wilcoxon-test [one-sided])

The increased incidences of skeletal variations were not

related to the dose or they were clearly inside the historical control range. Therefore, they are

not considered as treatment-related and adverse.

Fetal skeletal unclassified cartilage observations

Additionally, some isolated cartilage findings without impact on the respective bony structures,

which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and

the sternum and did not show any relation to dosing.

Tab. 11: Total unclassified cartilage observations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

23

129

25

131

23

119

25

143

Fetal incidence

 

N (%)

 

89 (69)

 

79 (60)

 

73 (61)

 

102 (71)

Litter incidence

 

N (%)

 

23 (100)

 

24 (96)

 

22 (96)

 

25 (100)

Affectedfetuses/litter

 

Mean%

 

70.1

 

60.9

 

59.6

 

70.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 100, 300 and 1000 mg/kg bw/d).

Three fetuses carried more than one malformation. Female control fetus No. 15-03 (0 mg/kg

bw/d) had multiple external malformations (i.e. gastroschisis and ectrodactyly) associated with

multiple skeletal malformations (i.e. absent forepaw phalanx and severely malformed sternum).

For female control fetus No. 25-14 hydronephrosis and hydroureter were recorded, while female

low-dose fetus No. 33-10 (100 mg/kg bw/d) had a bipartite basisphenoid, misshapen

presphenoidal and a severely malformed vertebral column and/or ribs (in the region of thoracic

vertebrae). The body weight of this fetus (3.2 g) was lower compared to its group mean value

(females of test group 1: 3.5 g). Further malformations, i.e. umbilical hernia, situs inversus,

interrupted spinal cord, anophthalmia, absent subclavian and malpositioned and bipartite

sternebrae were observed in individual fetuses, unrelated to the dose and, except ‘interrupted

spinal cord’ (control fetus), all of them can be found in the historical control data.

All these findings were single cases, no ontogenetic pattern is recognizable for all these individual

malformations nor was there any cluster of any of these individual malformations seen

in the other offspring of these test groups. They also do neither form a pattern or syndrome

with other minor anomalies which may raise toxicological concern. There is no evidence for

any association of these scattered findings with the treatment.

Tab. 12: Total fetal malformations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

NN

23

251

25

248

23

229

25

273

Fetalincidence

 

N (%)

 

5 (2.0)

 

1 (0.4)

 

1 (0.4)

 

4 (1.5)

Litter

incidence

 

N (%)

 

5 (22)

 

1 (4.0)

 

1 (4.3)

 

4 (16)

Affectedfetuses/litter

 

Mean%

 

2.0

 

0.4

 

0.4

 

1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

External variations did not occur in any of the fetuses in this study. Three soft tissue variations

and a range of skeletal variations were noted

in all test groups including the controls. None of the total incidences showed a relation to dose. The individual variations were equally distributed about the different test

groups, if normal biological variation is taken into account, and can be found in the historical

control data at a comparable frequency.

Tab. 13: Total fetal variations

 

 

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

NN

23

251

25

248

23

229

25

273

Fetalincidence

 

N (%)

 

127 (51)

 

128 (52)

 

119 (52)

 

144 (53)

Litterincidence

 

N (%)

 

23 (100)

 

25 (100)

 

23 (100)

 

25 (100)

Affectedfetuses/litter

 

Mean%

 

50.8

 

51.6

 

52.3

 

52.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

No unclassified external and unclassified soft tissue observations were recorded for any of the

fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage

observations which were observed in several fetuses of all test groups

(0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest

any relation to treatment.

Finally, fetal examinations revealed that there is no effect of the compound on the respective

morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor fetal developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.
Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites. The analytical values of the low-dose samples (100 mg/kg bw/d) were above the expected range of 90% to 110% of the nominal concentrations. The mean value of around 138% of the nominal concentration corresponds to an actual low-dose of 138 mg/kg bw/d. For the mid- and high dose, the correctness of the prepared concentrations was shown. This did not affect the validity of the study. In the following report, the target doses were used.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown (mid- and high-dose). Concentration control analysis of the low-dose: the three analytical samples resulted in a mean value of around 138% of the nominal concentration.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the NOAEL was assessed as 1000 mg/kg bw/day for maternal and fetal developmental toxicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2019 - March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jun 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF Guideline 2-1-18
Version / remarks:
Nov 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Lot No.of test material: D688149955
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: An emulsion was able to be prepared at 250 mg/ml. The test substance was found to be stable at concentrations ranging from 2.5 mg/ml to 250 mg/ml for up to 7 days.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. (CRP), Greenfield, Indiana
- Age at study initiation: sexually mature adults
- Weight at study initiation: 2800-3200 g
- Housing: individually
- Diet: 75 g - 150 g
- Water: ad libitum
- Acclimation period: at least four days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average 20; range 16-22
- Humidity (%): 40-60
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm; illumination)

IN-LIFE DATES: From: 2/18/2019 (Test substance administration ) To: 3/20/2019
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose (METHOCEL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material was prepared by mixing with 0.5% methylcellulose in ultrapure water+Cremophor EL (10 mg/100 ml) at concentrations of 0, 25, 75, or 250 mg/ml.
- Administration volume: 4 ml/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was determined to be insoluble in all vehicles tested other than the prepared emulsion.
- Concentration in vehicle: 0.5% methylcellulose in ultrapure water + Cremophor EL (10 mg/100 ml)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation analyses of all dose levels plus control were determined preexposure. The homogeneity of the low-dose and the high-dose test emulsions was determined concurrent with dose confirmation. The method used for analyzing the test material in 0.5% methylcellulose (METHOCEL™) in ultrapure water +
Cremophor EL (10mg/100mL) was a solvent extraction method followed by gas chromatography-flame ionization detector (GCFID).
Details on mating procedure:
Each sexually mature virgin female was naturally mated with one buck of the same strain at Covence Research Products (CRP). The observed day of breeding was considered GD 0. GD 0 body weights and records of mating pairs were provided by CRP and maintained in the study record.
Rabbits arrived in laboratory on GD 1 or 2.
Duration of treatment / exposure:
20 days
Frequency of treatment:
daily
Duration of test:
GD 7 - 27
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:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of the developmental
toxicity probe study. Due to the highly variable data within each dose group (excluding the GD 7-10 time period), and a lack of a dose response in clinical observations, body weights, body weight gains, and feed consumption in the probe study, these findings were not used to limit the high dose for this study. The high-dose of 1000 mg/kg/day represented a limit dose as defined in the Organization for Economic Co-operation and Development Test Guideline 414 (OECD 414 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high dose group rabbits.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Bodz weights were recorded on GD 0 (by the supplier), daily during the dosing period, and on GD 28 (terminal). Statistical analysis of body weights was performed using data collected on GD 0, 7, 10, 13, 16, 20, 24, and 28. Statistical analysis of body weight gains was conducted for the

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Consumption was recorded and analyzed from GD 4-28

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: stomach, liver, kidney, uterus

OTHER:
- Microscopic examination of tissues was not conducted.
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 with the exception of one litter from Dam #98 of the 1000 mg/kg/day group that was inadvertently not recorded)
- Soft tissue examinations: Yes (with the exception of fetus #5 from Dam #18 of the control group that was inadvertently not recorded)
- Skeletal examinations: Yes
- Head examinations: Yes: [half per litter] with the exception of one litter that was inadvertently not recorded (fetuses from Dam # 14 of the control group).
Statistics:
- Bartlett's test (alpha = 0.01) for Maternal body weights, maternal body weight gains, organ weights (absolute and relative with the exception of only absolute weight for gravid uterus), fetal body weights and feed consumption
- parametric or nonparametric ANOVA. If ANOVA was significant at alpha = 0.05 analysis by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed
- Censored Wilcoxon test with Bonferroni's correction for Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations (if any)
- Nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction for number of corpora lutea, implantations, and litter size
- Fisher exact probability test (alpha = 0.05) with Bonferroni's correction for pregnancy rates
Indices:
Pre-and post-implantation loss
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls. This finding was associated with decreases in feed consumption.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg/day: mean body weight loss of 38.3g, from GD 7-10, and an overall body weight gain decrease of 22.5% from GD 7-28 compared to controls
- 300 mg/kg/day: decrease of 68.4% in body weight gain from GD 7-10 (non-adverse). Body weight gains over entire treatment period (GD 7-28) comparable to controls and thus not adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg/day: decrease in food consumption from GD 7-14 ranging from 6.8%-22.1%
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Scheduled cesarean section
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- no effects on numbers of corpora lutea
- no effects on gravid uterine weights
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: evidence of systemic toxicity at 1000 mg/kg bw/day including decreased body weight gain and feed consumption
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related skeletal alterations in any dose group.
- statistically-identified decrease in the incidence of delayed ossification (DO) of the hyoid in the low and mid dose groups compared to control. No toxicological relevance since incidences were lower than control
- incidental malformations: missing ribs, fused thoracic rib, fused thoracic centra, thoracic hemicentric centra, thoracic hemivertebra and fused lumbar centra bearing no relationship to treatment
- incidental variations: DO pubis, calloused ribs, fused sternebrae, irregular pattern of ossification sternebrae, extra site of ossification sternebrae, DO sternebrae, crooked hyoid, and DO hyoid bearing no relationship to treatment
Given that these observations occurred in the control group, occurred at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically-identified visceral alterations in any dose group.
Incidental findings bearing no relationship to treatment included the malformations of enlarged aorta, ventricular septal wall defect, persistent truncus arteriosus, misshapen heart, and missing gall bladder, and variations of missing caudal lung lobe, liver cyst, right-sided esophagus, hemorrhage thymus, supernumerary hepatic lobule, retrocaval ureter, paraovarian cyst, and paratesticular cyst. Given that these observations occurred in the control group, occurred at low incidences, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
- no craniofacial alterations in any dose group
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, non-treatment-related
Developmental effects observed:
no

RESULTS AND DISCUSSION

Analytical

The dose confirmation and homogeneity report is provided in Appendix A. Analysis of

all dosing emulsions from the first mix revealed mean acceptable concentrations of npropyl

acetate ranging from 89.8% to 97.6% of targeted concentrations.

Analysis of aliquots for the low- and high-dose emulsions indicated that the test material

was homogeneously distributed based on relative standard deviations of 9.15 and 3.43%,

respectively.

In-Life Observations

Clinical and cage-side observations are summarized in Table 2 and individual data are

reported in Appendix Table 1.

There was an increased incidence of transient decreased feces in the 1000 mg/kg/day

dose group compared to controls. This finding was associated with decreases in feed

consumption. All other clinical observations were considered unrelated to treatment due

to the low incidence.

Body Weights/Body Weight Gains

Mean body weight and body weight gain data for pregnant rabbits are summarized in

Tables 3 and 4, individual data for all females are reported in Appendix Tables 2 and 3.

Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss of

38.3g, from GD 7-10, and an overall body weight gain decrease of 22.5% from GD 7-28

compared to controls (Text Table 3). Rabbits administered 300 mg/kg/day had a

treatment-related decrease of 68.4% in body weight gain from GD 7-10 compared to

controls; however, over the entire treatment period (GD 7-28), body weight gains were

similar to controls and were therefore considered non-adverse. Body weights for all

treated groups were similar to controls and there were no treatment-related body weight

gain changes in the 100 mg/kg/day dose group compared to controls.

Text Table 3. Selected Mean Body Weight Gains (g)

Dose (mg/kg/day)

Gestation Day

7-10

Gain

10-13

Gain

7-28

Gain

0

17.4

53.5

307.9

100

18.2

40.4

331.8

300

5.5

60.5

303.2

1000

-38.3*

54.0

238.5

Boldvalues were considered treatment-related effects.

*Statistically Different From Control Mean By Wilcoxon's Test, Alpha=0.05.

Feed Consumption

Mean feed consumption data for pregnant rabbits are summarized in Table 5, individual

data for all females are reported in Appendix Table 4.

Rabbits administered 1000 mg/kg/day had treatment-related decreases in feed

consumption from GD 7-14 compared to controls ranging from 6.8-22.1% (Text Table

4). During the last two weeks of gestation (GD 14-28), the feed consumption amongst all

the dose groups was more variable. However, the feed consumption in the

1000 mg/kg/day dose group was consistently lower than controls across all gestation

days. There were no treatment-related differences in the amount of feed consumed by the

100 or 300 mg/kg/day dose groups when compared to controls.

Text Table 4. Selected Feed Consumption Intervals (g)

Dose (mg/kg/day)

Gestation Day

7-8

8-9

9-10

10-11

11-12

12-13

13-14

0

134.6

134.4

132.6

132.7

129.9

115.5

116.6

100

133.4

134.0

134.6

131.4

127.8

121.1

117.9

300

131.4

128.4

130.6

120.6

123.5

117.2

110.4

1000

104.9

111.5*

114.4*

113.4*

110.3

107.6

103.8

Boldvalues were considered treatment-related effects.

* Statistically Different From Control Mean By Dunnett's Test, Alpha=0.05.

Anatomic Pathology

Organ Weights

Terminal body, liver, and kidney weight data for pregnant rabbits are presented in

Table 6, individual data are reported in Appendix Table 5.

There were no treatment-related or statistically-identified differences in any of the

measured parameters for any treated groups when compared to controls.

Gross Pathology

A summary of gross pathologic observations are presented in Table 7, and

individual data are reported in Appendix Table 6.

There were no treatment-related gross pathologic observations.

Reproductive Parameters

Reproductive parameters measured at necropsy are summarized in Table 8 and individual

data are reported in Appendix Table 7.

There were no treatment-related or statistically-identified effects on pregnancy rates,

resorption rates, litter size, numbers of corpora lutea or implantations, percent

preimplantation loss, percent post-implantation loss, fetal sex ratios, fetal body weights or

gravid uterine weights at any dose level.

Fetal Examination Summary

The incidence of external, visceral, craniofacial, and skeletal variations and

malformations observed among fetal rabbits is summarized in Table 9, while individual

litter data are reported in Appendix Table 8. Malformation historical control data are

presented in Appendix B.

Compared to controls, there were no treatment-related differences in the incidence of any

fetal alteration in any of the treated groups. The small number of alterations observed in

fetuses from dams administered n-propyl acetate either occurred at low frequencies

and/or were not dose related. Details of these findings are described below.

External Examination

There were no alterations in any dose group.

Visceral Examination

There were no treatment-related or statistically-identified visceral alterations in any

dose group. Incidental findings bearing no relationship to treatment included the

malformations of enlarged aorta, ventricular septal wall defect, persistent truncus

arteriosus, misshapen heart, and missing gall bladder, and variations of missing

caudal lung lobe, liver cyst, right-sided esophagus, hemorrhage thymus,

supernumerary hepatic lobule, retrocaval ureter, paraovarian cyst, and paratesticular

cyst. Given that these observations occurred in the control group, occurred at low

incidences, and/or lacked a dose response, these observations were considered

spurious and unrelated to treatment.

Craniofacial Examination

There were no alterations in any dose group.

Skeletal Examination

There were no treatment-related skeletal alterations in any dose group. There was a

statistically-identified decrease in the incidence of delayed ossification (DO) of the

hyoid in the low and mid dose groups compared to control. This finding has no

toxicological significance as the incidences were lower than controls. Other

incidental findings bearing no relationship to treatment included the malformations

missing ribs, fused thoracic rib, fused thoracic centra, thoracic hemicentric centra,

thoracic hemivertebra and fused lumbar centra. Incidental variation findings

bearing no relationship to treatment were DO pubis, calloused ribs, fused

sternebrae, irregular pattern of ossification sternebrae, extra site of ossification

sternebrae, DO sternebrae, crooked hyoid, and DO hyoid. Given that these

observations occurred in the control group, occurred at low frequencies, and/or

lacked a dose response, these observations were considered spurious and unrelated

to treatment.

Conclusions:
Treatment-related clinical observations were limited to an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls.
Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss from GD 7-10 with concomitant decreases in feed consumption. This resulted in an overall 22.5% decrease in body weight gain from GD 7-28 compared to controls. Rabbits administered 300 mg/kg/day had a treatment-related decrease in body weight gain from GD 7-10 compared to controls, however over the entire treatment period (GD 7-28), body weight gains were similar tocontrols and were therefore considered non-adverse.
There were no treatment-related differences in gross pathologic observations or maternal kidney or liver weights for any of the treated groups when compared to controls.
There were no indications of embryo/fetal toxicity or teratogenicity at any dose level.

Under the conditions of this study and based on evidence of systemic toxicity at the high dose level including decreased body weight gain and feed consumption, the no-observedadverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day. The no-observed-effect level (NOEL) for developmental toxicity was 1000 mg/kg/day, the highest dose tested.
Executive summary:

The purpose of this study was to evaluate the potential maternal and developmental toxicity of the test substance in New Zealand White (NZW) rabbits following repeated oral gavage administration. Groups of 24 time-mated female rabbits were administered n-propyl acetate in 0.5% methylcellulose (METHOCEL™) in ultrapure water + Cremophor EL (10mg/100mL) via gavage at dose levels of 0, 100, 300, or 1000 milligrams per kilogram body weight per day (mg/kg/day) on gestation day (GD) 7-27. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption.

On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. Fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.

Treatment-related clinical observations were limited to an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls. Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss from GD 7-10 with concomitant decreases in feed consumption. This resulted in an overall 22.5% decrease in body weight gain from GD 7-28 compared to controls.

Rabbits administered 300 mg/kg/day had a treatment-related decrease in body weight gain from GD 7-10 compared to controls, however over the entire treatment period (GD 7-28), body weight gains were similar to controls and were therefore considered non-adverse.

There were no treatment-related differences in gross pathologic observations or maternal kidney or liver weights for any of the treated groups when compared to controls. There were no indications of embryo/fetal toxicity or teratogenicity at any dose level. Under the conditions of this study and based on evidence of systemic toxicity at the high dose level including decreased body weight gain and feed consumption, the no observed-adverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day. The no-observed-effect level (NOEL) for developmental toxicity was 1000 mg/kg/day, the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
other: rat and rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance propyl acetate was tested for its prenatal developmental toxicity in Wistar rats. The study was conducted according to OECD 414 guideline and GLP. The test substance was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites. The analytical values of the low-dose samples (100 mg/kg bw/d) were above the expected range of 90% to 110% of the nominal concentrations. The mean value of around 138% of the nominal concentration corresponds to an actual low-dose of 138 mg/kg bw/d. For the mid- and high dose, the correctness of the prepared concentrations was shown. This did not affect the validity of the study. In the following report, the target doses were used.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown (mid- and high-dose). Concentration control analysis of the low-dose: the three analytical samples resulted in a mean value of around 138% of the nominal concentration.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the NOAEL was assessed as 1000 mg/kg bw/day for maternal and fetal developmental toxicity.

A GLP-conform study according to OECD 414 was conducted in rabbits (2019). The purpose of this study was to evaluate the potential maternal and developmental toxicity of the test substance in New Zealand White (NZW) rabbits following repeated oral gavage administration. Groups of 24 time-mated female rabbits were administered n-propyl acetate in 0.5% methylcellulose (METHOCEL™) in ultrapure water + Cremophor EL (10mg/100mL) via gavage at dose levels of 0, 100, 300, or 1000 milligrams per kilogram body weight per day (mg/kg/day) on gestation day (GD) 7-27. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption.

On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. Fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.

Treatment-related clinical observations were limited to an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls. Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss from GD 7-10 with concomitant decreases in feed consumption. This resulted in an overall 22.5% decrease in body weight gain from GD 7-28 compared to controls.

Rabbits administered 300 mg/kg/day had a treatment-related decrease in body weight gain from GD 7-10 compared to controls, however over the entire treatment period (GD 7-28), body weight gains were similar to controls and were therefore considered non-adverse.

There were no treatment-related differences in gross pathologic observations or maternal kidney or liver weights for any of the treated groups when compared to controls. There were no indications of embryo/fetal toxicity or teratogenicity at any dose level. Under the conditions of this study and based on evidence of systemic toxicity at the high dose level including decreased body weight gain and feed consumption, the no observed-adverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day. The no-observed-effect level (NOEL) for developmental toxicity/teratogenicity was 1000 mg/kg/day, the highest dose tested.

Developmental toxicity studies have been conducted with the metabolite n-propyl alcohol and the analogous substance n-butyl acetate by the inhalation route of exposure.

n-butyl acetate

Four groups, each containing 37-43 female Sprague-Dawley rats were exposed to n-butyl acetate air concentrations of either 0 or 1,500 ppm (7.050 mg/L) for 7 hours/day (Hackett et al. 1982). Group 1 was not exposed to test material throughout the study and served as the control. Group 2 was exposed to 1,500 ppm (7.05 mg/L) n-butyl acetate from day 7 to 16 of gestation. Group 3 received n-butyl acetate from Day 1 to 16 of gestation and Group 4 was exposed from 3 weeks pre-gestation through day 16 of gestation. On gestation Day 21 (sperm positive = Day 1), the fetuses were collected and examined for both skeletal and visceral malformations.

Maternal toxicity effects were observed as decreased feed consumption and body weight and increased relative kidney and lung weights, with the greatest increase occurring in the animals receiving the longest exposure. There were no changes in histopathology that could be related to n-butyl acetate exposure. Fetal growth measures (fetal body weights and crown-rump growth) and placental weights were lower in Groups 2, 3, and 4. However, the duration of exposure and period of gestation during which exposure occurred did not affect fetal growth indices.

There was no increase in the incidence of malformations in any of the n-butyl acetate exposed groups. There was an increase in the incidence of a skeletal anomaly (total rib dysmorphology) and in a skeletal variation reduced ossification of the pelvis in Groups 2 and 3. There was no increase in the incidence of skeletal anomalies or variations in pups from Group 4 dams, who experienced the longest exposure interval (3 weeks prior to mating and GD 1-16). Group 4 pups displayed an increase in the

incidence of hydroureter as compared to the control group (Group 1), however Groups 2 and 3 were unaffected. The lack of a uniform responses (incidence of a skeletal anomaly,skeletal variation reduced ossification of the pelvis) between treatment groups 2, 3, and 4 should be understood as missing teratogenic effects of n-butyl acetate.

Three groups of 21-25 female New Zealand White rabbits were exposed to n-butyl acetate vapor concentrations of either 0 or 1,500 ppm (7.050 mg/L; Hackett et al. 1982). Rabbits in all groups were placed in exposure chambers for 7-hours per day from study day 1 to 19.

Group 1 received sham exposures to filtered air throughout the study and served as controls. Group 2 was exposed to 1,500 ppm n-butyl acetate from Day 7 to 19 of gestation. Group 3 was exposed to test material from gestation Day 1 to 19. On gestation Day 30 the fetuses were collected and examined for both skeletal and visceral malformations.

Feed consumption and body weight was lowest in the control group. Organ weights and histopathology were unremarkable in animals exposed to n-butyl acetate. Mating and reproductive performance and intrauterine mortality was unaffected by n-butyl acetate exposure. Fetal growth measures (fetal body weights and crown-rump length), placental weights, and sex ratios were not affected by n-butyl acetate exposures. There was no increase in the incidence of malformations in any of the n-butyl acetate exposure groups. There was an increase in skeletal variations or anomalies and ossification retardations which were not regarded as teratogenic effects.

n-propyl alcohol

Groups of 15 mated female Sprague-Dawley rats were exposed to 0, 3500, 7000, or 10,000 ppm (0, 8.9, 17.9 and 25.5 mg/L) n-propyl alcohol vapor for 7 hr per day during gestation days 1 - 19 (Nelson et al. 1990, Nelson et al. 1988, Nelson et al. 1996).

Maternal effects: Food intake was reduced throughout gestation in  females exposed to 10,000 ppm (25.5 mg/L), however, maternal body weight gain was not affected until the end of gestation. Food intake was reduced during the last two weeks of exposure in females exposed to 7,000 ppm (17.9 mg/L), but body weight gain was not significantly affected. In females exposed to 3500 ppm (8.610 mg/L) n-propyl alcohol, food intake was reduced by approximately 10% as compared to controls,

but this effect was not significant and body weight gain was not affected.

Pregnancy/fetal effects: There was an increase in pre- and postimplantation loss among litters from dams exposed to 7000 and 10,000 ppm (17.9 and 25.5 mg/L). Fetal body weights were significantly reduced after maternal exposure to 7000 ppm and 10,000 ppm n-propyl alcohol. Offspring of dams exposed to 10,000 ppm (25.5 mg/L) displayed a significant increase in skeletal and visceral malformations; external malformations were also increased; approximately one-third of the offspring had shortened or missing tails. At 7000 ppm (17.9 mg/L) only the incidence of skeletal malformations (primarily rudimentary cervical ribs) was significantly increased from controls.

These results suggest that inhalation exposure to n-propyl acetate may induce fetal malformations and variations, but only at very high, maternal toxic concentrations. 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification for reproduction toxicity is not warranted.