2-butyl-6-(butylamino)-1H-benz[de]isoquinoline-1,3(2H)-dione

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from HPVIS document

Data source

Reference

Reference Type:

secondary source

Title:

Reproductive toxicity for CAS 550-77-4

Author:

U.S Environmental Protection agency

Year:

2017

Bibliographic source:

High Production Volume Information System (HPVIS)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N-methylphthalimide

EC Number:

208-982-4

EC Name:

N-methylphthalimide

Cas Number:

550-44-7

Molecular formula:

C9H7NO2

IUPAC Name:

2-methyl-1H-isoindole-1,3(2H)-dione

Test animals

Species:

rat

Strain:

other: CD® (SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical signs, Mortality, Body weight, Food consumption, Organ weights

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

yes

Postmortem examinations (parental animals):

Gross pathology and Histopathology

Postmortem examinations (offspring):

Gross pathology and Histopathology

Reproductive indices:

yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ataxia, audible breathing, and piloerection were observed in the high-dose group. "Rooting" and salivation were seen in the mid- and high-dose groups and considered to be a response to taste aversion to the dosing solutions and not an indication of toxicity

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 4 unscheduled, treatment related deaths during the peri-parturitional period for the F0 females at 600 mg/kg/day and most likely due to significant overdosing of the F0 dams during that period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 females exhibited reduced body weights during gestation and lactation at 600 mg/kg/day. Weight gain during gestation was also reduced at 600 mg/kg/day, but weight gain during lactation was significantly increased at 150 and 600 mg/kg/day, indicating possible rebound even with continued exposure.
F0 females exhibited reduced body weights during gestation and lactation at 600 mg/kg/day. Weight gain during gestation was also reduced at 600 mg/kg/day, but weight gain during lactation was significantly increased at 150 and 600 mg/kg/day, indicating possible rebound even with continued exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption, was decreased at 600 mg/kg/day during prebreed in F0 males.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Average pupil size score was reduced in wk 1 (prebreed) at 600 mg/kg/day, and the percentages of F0 females with abnormal arousal were increased at 600 mg/kg/day for wks 3 and 4 (mating and early gestation). The relationship and biological significance of changes in these two parameters at 600 mg/kg/day are unknown.

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total cholesterol values were significantly increased at 600 mg/kg/day. Average pupil size score was reduced in wk 1 (prebreed) at 600 mg/kg/day, and the percentages of F0 females with abnormal arousal were increased at 600 mg/kg/day for wks 3 and 4 (mating and early gestation). The relationship and biological significance of changes in these two parameters at 600 mg/kg/day are unknown.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

centrilobular hepatocellular hypertrophy in the liver at 600 mg/kg/day (but not at 150 or 0 mg/kg/day), which was consistent with the increased liver weights at this dose in both sexes, and hyaline droplets in the kidneys of all males at 600 mg/kg/day (none noted at 0 or 150 mg/kg/day).

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The deaths of 4 dams (out of 10 pregnant) at 600 mg/kg/day, one each on gd 22 and lactational days 2, 3, and 6 are considered treatment related and evidence that this dose exceeded the maximum tolerated dose in this study design of F0 female reproductive toxicity. Precoital interval was significantly decreased at 600 mg/kg/day (the significance, if any, of a shorter precoital interval of 1.4 days is not known). There were no effects on gestational length or reproductive indices. There were significant decreases for the number of total implantation sites per litter and the number of total and live pups per litter at birth (pnd 0) at 600 mg/kg/day.

Details on results (P0)

F0 Adult Systemic Toxicity:
Males: No mortality. Clinical signs: ataxia, audible breathing, and piloerection were observed in the high-dose group. "Rooting" and salivation were seen in the mid- and high-dose groups and considered to be a response to taste aversion to the dosing solutions and not an indication of toxicity, per se. Since there was a dose-related response, it is presumed that PI, was resulting in the adverse taste. Rooting in bedding, especially postdosing, is observed frequently in gavage studies in the laboratory in a dose-related incidence, consistent with dose-related taste aversion. Feed consumption, was decreased at 600 mg/kg/day during prebreed in F0 males. Liver weights, both absolute and relative to terminal body and brain weight, were significantly increased at 600 mg/kg/day in F0 males. Other potentially treatment-related findings in the F0 males included significantly decreased prostate weight, absolute and relative to terminal body weight at 600 mg/kg/day, and total cholesterol values significantly increased at 600 mg/kg/day. There were no treatment-related effects for the gross necropsy findings. Histological findings included centrilobular hepatocellular hypertrophy in the liver at 600 mg/kg/day (but not at 150 or 0 mg/kg/day), which was consistent with the increased liver weights at this dose in both sexes, and hyaline droplets in the kidneys of all males at 600 mg/kg/day (none noted at 0 or 150 mg/kg/day). All other statistically significant changes were not considered treatment related, based on the lack of dose response. In the male recovery group, body weights were decreased by day 28 (lower than control) but not statistically significantly at the end of recovery. No other signs of toxicity were observed for the recovery group males.
Females: There were 4 unscheduled, treatment related deaths during the peri-parturitional period for the F0 females at 600 mg/kg/day and most likely due to significant overdosing of the F0 dams during that period. Dosing volumes were adjusted during pregnancy, based on each dam's most recent body weight on gd 0, 7, 14, and 20, but the large "maternal" weight gain from gd 14 to 20 (~ 30%) was due to the growth of the uterine contents and not to any appreciable weight gain of the extra-uterine maternal animal. Therefore, the dosing volume set on gd 20, and used until pnd 0, was greatly increased (~ 30%) over the volume used from gd 14-20 and most likely overwhelmed the metabolic capacity of the maternal liver, confounded by increased absorption (from reduced motility and therefore longer transit time) from the gastrointestinal tract late in gestation. F0 females exhibited reduced body weights during gestation and lactation at 600 mg/kg/day. Weight gain during gestation was also reduced at 600 mg/kg/day, but weight gain during lactation was significantly increased at 150 and 600 mg/kg/day, indicating possible rebound even with continued exposure. Feed consumption was significantly decreased during prebreeding, gestation, and lactation at 600 mg/kg/day, but decreased at 150 mg/kg/day only during prebreeding. Total cholesterol values were significantly increased at 600 mg/kg/day. Average pupil size score was reduced in wk 1 (prebreed) at 600 mg/kg/day, and the percentages of F0 females with abnormal arousal were increased at 600 mg/kg/day for wks 3 and 4 (mating and early gestation). The relationship and biological significance of changes in these two parameters at 600 mg/kg/day are unknown.
F0 Parental Reproductive Toxicity: The table below presents a summary of F0 parental reproductive toxicity. The deaths of 4 dams (out of 10 pregnant) at 600 mg/kg/day, one each on gd 22 and lactational days 2, 3, and 6 are considered treatment related and evidence that this dose exceeded the maximum tolerated dose in this study design of F0 female reproductive toxicity. Precoital interval was significantly decreased at 600 mg/kg/day (the significance, if any, of a shorter precoital interval of 1.4 days is not known). There were no effects on gestational length or reproductive indices. There were significant decreases for the number of total implantation sites per litter and the number of total and live pups per litter at birth (pnd 0) at 600 mg/kg/day.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

clinical biochemistry

2

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

3

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was 1 unscheduled death each for adult F1 males at 50 and 600 mg/kg/day. Both unscheduled deaths were due to a dosing error and were not considered related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were significant reductions in body weight and weight gain for the F1 males during the postweaning period (from pnd 22 to 71) at all dose levels. However, there was no dose-response relationship in the body weight reductions for the low- and mid-dose groups; the males at the low dose exhibited lower body weights than the males at the mid dose. Consistent with the reduced body weights at all doses for F1 males, the absolute and adjusted ages at acquisition of puberty (PPS) were also significantly delayed in all treated groups. These slight but statistically significant delays for absolute age (2.3, 4.1, and 5.0) and for adjusted age (2.3, 3.3, and 5.2) at 50, 150, and 600 mg/kg/day, respectively, are most likely due to the reduced body weights during this time in all PI groups.
Significantly lower mean body weights occurred at 150 and 600 mg/kg/day for the F1 females throughout the postweaning period (from pnd 22 to 71). Consistent with the reduced body weights at 600 mg/kg/day, age at acquisition of puberty (VP) was also significantly delayed at 600 mg/kg/day by 3.0 days for absolute age and by 3.1 days for adjusted age. This delay at 600 mg/kg/day is most likely due to the reduced body weights at this dose during this time. Body weight gains were significantly decreased at 150 mg/kg/day for the F1 females only during the first week of the postweaning period (from pnd 22 to 29).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption, expressed as g/day, was significantly decreased at all dose levels during the postweaning period. However, there were no significant changes in feed consumption expressed as g/kg/day at any dose level, suggesting the reduced feed consumption was correlated with (likely due to) the reduced body weights in all groups. As with the males, there were no significant differences in feed consumption values expressed as g/kg/day.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

hemoglobin values were statistically increased at 600 mg/kg/day. Platelet count values were statistically decreased at 150 and 600 mg/kg/day, and the mean platelet volume values were statistically decreased at all dose levels.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total cholesterol was increased at 600 mg/kg/day for the F1 females.

Urinalysis findings:

not specified

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Delayed acquisition of puberty in both F1 sexes

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At scheduled necropsy, body weights were significantly decreased at 50, 150, and 600 mg/kg/day. Reduced absolute organ weights were likely due to the reduced body weights, since organ weights relative to body or brain weights were either unaffected or increased. The increase in liver weights, relative to terminal body weights at 600 mg/kg/day, was most likely related to the lower body weights in this group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects for the gross necropsy findings.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histological findings included centrilobular hepatocellular hypertrophy in all 5 F1 male livers at 600 mg/kg/day (but not at 0 or 150 mg/kg/day), although there was no associated liver weight increase. In addition, hyaline droplets in all 5 F1 male kidneys at 600 mg/kg/day (but not at 0 or 150 mg/kg/day) were considered treatment related. Histopathological findings included minimal centrilobular hepatocellular hypertrophy in all 5 F1 female livers at 600 mg/kg/day, but no such findings at 0 or 150 mg/kg/day.

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

F1 Adult Systemic Toxicity: Males: There was 1 unscheduled death each for adult F1 males at 50 and 600 mg/kg/day. Both unscheduled deaths were due to a dosing error and were not considered related to treatment. There were significant reductions in body weight and weight gain for the F1 males during the postweaning period (from pnd 22 to 71) at all dose levels. However, there was no dose-response relationship in the body weight reductions for the low- and mid-dose groups; the males at the low dose exhibited lower body weights than the males at the mid dose. Consistent with the reduced body weights at all doses for F1 males, the absolute and adjusted ages at acquisition of puberty (PPS) were also significantly delayed in all treated groups. These slight but statistically significant delays for absolute age (2.3, 4.1, and 5.0) and for adjusted age (2.3, 3.3, and 5.2) at 50, 150, and 600 mg/kg/day, respectively, are most likely due to the reduced body weights during this time in all PI groups. Feed consumption, expressed as g/day, was significantly decreased at all dose levels during the postweaning period. However, there were no significant changes in feed consumption expressed as g/kg/day at any dose level, suggesting the reduced feed consumption was correlated with (likely due to) the reduced body weights in all groups. For the F1 males, hemoglobin values were statistically increased at 600 mg/kg/day. Platelet count values were statistically decreased at 150 and 600 mg/kg/day, and the mean platelet volume values were statistically decreased at all dose levels. There were no treatment-related changes in FOB measurements. Other statistically significant differences were considered unrelated to treatment, based on the lack of dose response. At scheduled necropsy, body weights were significantly decreased at 50, 150, and 600 mg/kg/day. Reduced absolute organ weights were likely due to the reduced body weights, since organ weights relative to body or brain weights were either unaffected or increased. There were no treatment-related effects for the gross necropsy findings. However, histological findings included centrilobular hepatocellular hypertrophy in all 5 F1 male livers at 600 mg/kg/day (but not at 0 or 150 mg/kg/day), although there was no associated liver weight increase. In addition, hyaline droplets in all 5 F1 male kidneys at 600 mg/kg/day (but not at 0 or 150 mg/kg/day) were considered treatment related. Andrological evaluations included cauda epididymal sperm motility and progressive motility in all groups, cauda epididymal sperm concentration in all groups, and percent abnormal epididymal sperm at 0 and 600 mg/kg/day, which were all unaffected. Also evaluated initially at 0 and 600 mg/kg/day was testicular homogenization-resistant spermatid head concentration. Values for this parameter were then used to calculate daily sperm production and efficiency of daily sperm production. These last parameters were significantly increased at 600 mg/kg/day, so the 0 and 600 mg/kg/day samples were reanalyzed along with the samples at 150 mg/kg/day. The reanalysis indicated no effects at 600 mg/kg/day, but significantly increased values at 150 mg/kg/day. These findings are con sidered unrelated to treatment or dose and not adverse, since increases in these parameters do not indicate damage to the testicular spermatogenesis machinery (decreases in these parameters would indicate possible damage to the testis) and were not dose related.
Females: There were no unscheduled deaths for the adult F1 females. Significantly lower mean body weights occurred at 150 and 600 mg/kg/day for the F1 females throughout the postweaning period (from pnd 22 to 71). Consistent with the reduced body weights at 600 mg/kg/day, age at acquisition of puberty (VP) was also significantly delayed at 600 mg/kg/day by 3.0 days for absolute age and by 3.1 days for adjusted age. This delay at 600 mg/kg/day is most likely due to the reduced body weights at this dose during this time. Body weight gains were significantly decreased at 150 mg/kg/day for the F1 females only during the first week of the postweaning period (from pnd 22 to 29). Feed consumption values expressed as g/day were decreased at 150 and 600 mg/kg/day. As with the males, there were no significant differences in feed consumption values expressed as g/kg/day. In addition, there were no significant differences in the duration of estrous cycles across all groups and no treatment-related changes in FOB measurements. At necropsy, terminal body weights were significantly decreased at 600 mg/kg/day. The increase in liver weights, relative to terminal body weights at 600 mg/kg/day, was most likely related to the lower body weights in this group. Total cholesterol was increased at 600 mg/kg/day for the F1 females. There were no treatment-related effects for the gross necropsy findings. Histopathological findings included minimal centrilobular hepatocellular hypertrophy in all 5 F1 female livers at 600 mg/kg/day, but no such findings at 0 or 150 mg/kg/day.
F1 Offspring Toxicity: There was evidence of F1 offspring toxicity at 600 mg/kg/day, expressed as reduced pup body weights per litter on pnd 7 (males only), 14 (males, females, and all pups), and 21 (males and all pups). The stillbirth index at 600 mg/kg/day was increased, but not statistically significantly, due to inclusion of a litter born dead on pnd 0. Therefore, the live birth index at 600 mg/kg/day was also decreased through lactation, but not statistically significantly. There were no effects on anogenital distance at birth, sex ratio (% males) per litter, or retention of nipples/areolae for preweanling male pups on pnd 11-13. Acquisition of F1 male PPS was significantly delayed across all dose groups and was considered treatment related. F1 female vaginal opening was significantly delayed at 600 mg/kg/day and was considered treatment related. Delayed acquisition of puberty in both F1 sexes was most likely due to the reduced body weights for F1 postweanling males in all PI-exposed groups and the reduced body weights for F1 postweanling females at 600 mg/kg/day during the peripubertal period. There were significant decreases in body weights at sacrifice observed at 600 mg/kg/day in both F1 males and females on pnd 21. Absolute spleen weights were significantly decreased in the F1 males on pnd 21. Brain weights, relative to terminal body weights, were significantly increased at 600 mg/kg/day (but not at any other dose level) for the F1 males. Paired epididymal weights, relative to body weight, were significantly increased at 600 mg/kg/day for the F1 males. Absolute thymus and spleen weights were significantly decreased at 600 mg/kg/day for the F1 females. Brain weight, relative to body weight, was significantly increased at 600 mg/kg/day for the F1 females. Thymus and spleen weights, relative to terminal brain weights, were significantly decreased for the F1 females at 600 mg/kg/day. There were no treatment-related findings at necropsy of the F1 pups on pnd 21.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

< 50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

other: delays in acquisition of puberty at all doses

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

other: delay in acquisition of puberty at 600 mg/kg/day

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Reproductive toxicity was evaluated for the substance 2-methyl-1H-isoindole-1,3(2H)-dione. The F0 male and female systemic No observable adverse effect level (NOAEL) was 150 mg/kg/day. The F1 male systemic NOAEL was less than 50 mg/kg/day, and the F1 female systemic NOAEL was 50 mg/kg/day (both due to reduced body weights at higher doses). The NOAELs for F0 reproductive toxicity were at or above 600 mg/kg/day for males and 150 mg/kg/day for females (due to periparturitional demise and to reduced total implants/litter and reduced total and live litter sizes at birth at 600 mg/kg/day). The NOAELs for F1 offspring toxicity were less than 50 mg/kg/day for males (due to reduced postweaning body weights and delays in acquisition of puberty at all PI doses) and at 50 mg/kg/day for females (due to reduced postwean body weights at 150 and 600 mg/k g/day and delay in acquisition of puberty at 600 mg/kg/day).

Executive summary:

The substance2-methyl-1H-isoindole-1,3(2H)-dioneadministered by gavage once daily at 0, 50, 150, and 600 mg/kg/day to parental F0 CD® (SD) rats, 10/sex/group, through prebreed, mating, gestation, and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice, resulted in adult F0 parental toxicity at 600 mg/kg/day, which was more severe in females than in males. Toxicity was present in F0 females at 600 mg/kg/day was expressed as periparturtional demise in 4 of the 10 pregnant females and reduced total and live litter size at birth. There was F1 offspring toxicity observed postnatally, expressed as reduced pup body weights per litter during lactation in both sexes, and centrilobular hepatocellular hypertrophy in F1 males and females at 600 mg/kg/day, and hyaline droplets in the kidneys of the F1 adult males at 600 mg/kg/day. In addition, F1 postweanlings exhibited reduced body weights at 150 and 600 mg/kg/day for females and at 50, 150, and 600 mg/kg/day for males. Acquisition of puberty in F1 males was significantly delayed at 50, 150, and 600 mg/kg/day and in F1 females at 600 mg/kg/day, associated with reduced body weights during the postwean retention period at all 3 doses for F1 males and at the top dose for F1 females.The F0 male and female systemic No observable adverse effect level (NOAEL) was 150 mg/kg/day. The F1 male systemic NOAEL was less than 50 mg/kg/day, and the F1 female systemic NOAEL was 50 mg/kg/day (both due to reduced body weights at higher doses). The NOAELs for F0 reproductive toxicity were at or above 600 mg/kg/day for males and 150 mg/kg/day for females (due to periparturitional demise and to reduced total implants/litter and reduced total and live litter sizes at birth at 600 mg/kg/day). The NOAELs for F1 offspring toxicity were less than 50 mg/kg/day for males (due to reduced postweaning body weights and delays in acquisition of puberty at all the doses) and at 50 mg/kg/day for females (due to reduced postwean body weights at 150 and 600 mg/k g/day and delay in acquisition of puberty at 600 mg/kg/day).