1,3-diisopropylbenzene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Read across to isomer 1,4-diisopropylbenzene:

No effects on fertility were found in an OECD 422 reproductive screening study. The NOAEL was the high dose (and limit dose) of 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied. Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action.
The source substance ‘1,4-diisopropylbenzene' is a structural isomer of the target substance. The physico-chemical properties are highly equivalent based on the high structural similarity. As a conclusion, it is scientifically justified to address the endpoint Toxicity tto reproduction with data on this substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: 1,4-diisopropylbenzene (p-Diisopropylbenzene), CAS no. 100-12-5
structural formula: C12H18
Smiles: CC(C)c1ccc(cc1)C(C)C
Molecular weight: 162 g/mol
CAS 100-12-5
EC No 202-826-9
purity: not specified

target substance: 1.3-diisopropylbenzene (or m-DIPB)
structural formula: C12H18
Smiles: CC(C)c1cccc(c1)C(C)C
Molecular weight: 162 g/mol
CAS 99-62-7
EC No 202-773-1
purity: not specified

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".


4. DATA MATRIX
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females survived to the scheduled necropsy.
Test item-related clinical observations of red and/or clear material around the mouth were noted for males and females in all test item-treated groups approximately 1 hour following dose administration. These observations were noted throughout the treatment period and occurred in a dose-related manner. However, the material observations did not persist to the weekly examinations and were considered nonadverse in the absence of other signs of systemic toxicity (body weight and food consumption decrements). There were no test item-related clinical observations noted at the weekly examinations.
Observations noted in the test item-treated groups, including hair loss on various body surfaces and yellow material in the urogenital area, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All males and females survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
Mean body weight gain in the 1000 mg/kg/day group males was similar to the control group during the pre-mating treatment period (study days 0-13) and during study days 13-21. A test item-related, significantly (p < 0.01) lower mean body weight gain was noted in this group during study days 21-27 compared to the control group. As a result, a lower mean body weight gain was noted in the 1000 mg/kg/day group when the entire treatment period (study days 0-27) was evaluated; the difference from the control group was significant (p < 0.05). The effect on mean body weight gain during the last week of treatment was not of sufficient magnitude to affect absolute mean body weights for this group, and therefore, was not considered adverse. Mean body weights and body weight gains in the 100 and 300 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.

FEMALES (pre-mating)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

.MALES
Mean food consumption, evaluated as g/animal/day, in the 1000 mg/kg/day group males was significantly (p < 0.01) lower (2 g/animal/day) than the control group during the first week of treatment (study days 0-7). This decrement was noted in the absence of an effect on mean body weight gain during this same interval. Mean food consumption in the 1000 mg/kg/day group was similar to the control group during study days 7-13, and therefore the effect on mean food consumption noted during the first week of test item administration was considered transient and not test item-related. Mean food consumption in the 100 and 300 mg/kg/day group males was similar to that in the control group throughout the study. None of the differences from the control group were statistically significant.

FEMALES (pre-mating)
Mean food consumption, evaluated as g/animal/day, in the 100, 300, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. Differences from the control group were not statistically significant, occurred in a manner that was not dose-related, and/or were transient.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in hematology and coagulation parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher cholesterol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and bile acids (1000 mg/kg/day group females), lower glucose (1000 mg/kg/day group males and females), higher phosphorus (300 and 1000 mg/kg/day group males, 1000 mg/kg/day group females) and higher urea nitrogen (1000 mg/kg/day group females) values were noted at study week 4 (males) or lactation day 14 (females). Minimal to mild elevations of cholesterol, ALP, ALT, and bile acids in 1000 mg/kg/day group females were coincident with test item-related hepatocellular hypertrophy; differences from the control group were significant (p < 0.05 or p < 0.01) for cholesterol, ALT, and bile acids. Minimally lower glucose was not associated with lower food consumption. Minimally to mildly higher phosphorus (significant at p < 0.01 or p < 0.05, males and p < 0.05, females) and urea nitrogen (significant at p < 0.01, females) were suggestive of dehydration but urinalysis was not performed in the present study. Glucose, phosphorous and urea changes were considered nonadverse given the magnitude of change.
There were no other test item-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower urea nitrogen in the 100 mg/kg/day group males was not considered test item-related given the lack of a dose related response and a high control group mean and individual values. There were no test item-related changes in T4 levels in males.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

OPEN FIELD OBSERVATIONS
Open field parameters were unaffected by test item administration. A significantly (p < 0.05) higher mean rearing count was noted in the 1000 mg/kg/day group males during study week 3 compared to the control group. However, in the absence of effects on any other effects on CNS activity, the higher rearing count was not considered to be test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. A significantly (p < 0.05) lower mean hindlimb grip strength value was noted in the 300 mg/kg/day group males during study week 3 compared to the control group value. However, no dose-response was evident, and therefore this difference was not considered test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

MOTOR ACTIVITY
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 3 (males) and on lactation day 13 (females). Significantly (p ≤ 0.008) lower mean total counts were noted in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval. However, the differences were the result of a greater habituation response in these groups relative to the control group during the first 20 minutes of testing. When rats are placed in a novel environment, exploratory behavior (resulting in a high level of activity counts) followed by habituation (resulting in a reduced level of activity counts) is an expected response. In addition, there were no statistically significant differences in the cumulative total counts at these dosage levels. Therefore, the lower activity counts in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval were attributed to biologic variability and were not considered test item-related. All other values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL Research historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL Research historical control data ranges, and/or did not occur in a dose-related manner. Other than the greater habituation response described previously for the 300 and 1000 mg/kg/day males during the 11-20 minute subinterval, no remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 3 or lactation day 13.

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male in the 1000 mg/kg/day group did not sire a litter, as the female that this male was paired with was nongravid. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
The mean gestation length in the 1000 mg/kg/day group (22.3 days) was significantly (p < 0.05) longer than the concurrent control group (21.7 days). The 1000 mg/kg/day group gestation length value was within the WIL Research historical control range (21.3 to 22.3 days) and was primarily attributed to a single female (no. 2852) that delivered on gestation day 24 (and subsequently had a total litter loss on lactation day 0). Therefore, the longer mean gestation length at 1000 mg/kg/day was not considered test item-related.
Mean gestation lengths in the 100 and 300 mg/kg/day groups (21.7 and 22.0 days, respectively) were similar to those in the concurrent control group. No signs of dystocia were noted in any group.

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 item-related microscopic findings were noted in the adrenal cortex of the 1000 mg/kg/day group males and liver of the 1000 mg/kg/day group males and females.

Liver: Minimal to mild hepatocellular hypertrophy, characterized by increased amounts of cytoplasm, was noted in the centrilobular region of the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females. Angiectasis, characterized by focally extensive dilation of sinuosoids, was observed in a single male each in the 300 and 1000 mg/kg/day groups (male nos. 2827 and 2797, respectively) and correlated with dark red discoloration in the liver. Angiectasis was differentiated from routine terminal congestion that was noted in the control and all test item-treated groups and was characterized by blood in the sinusoids.

Adrenal cortex: There was an increased incidence and severity of cytoplasmic vacuolation of the adrenal cortex in the 1000 mg/kg/day group males. Vacuolation was characterized by variably sized, well demarcated vacuoles in the zona fasciculata and was not considered adverse. Vacuolation was also noted in females but vacuoles were uniform and there was no test item-related difference.

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Female no. 2852, which had a total litter loss, had mild myeloid hyperplasia of the bone marrow and a moderate increase in splenic extramedullary hematopoeisis which were both related to moderate uterine subacute inflammation with endometrial hyperplasia and mild mixed cell infiltrate in the vagina. Female no. 2846, which failed to conceive, was in early diestrus at the time of necropsy (study day 39).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male in the 1000 mg/kg/day group did not sire a litter, as the female that this male was paired with was nongravid. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

CLINICAL OBSERVATIONS AND SURVIVAL
Test item-related clinical observations of red and/or clear material around the mouth were noted for males and females in all test item-treated groups approximately 1 hour following dose administration. These observations were noted throughout the treatment period and occurred in a dose-related manner. However, the material observations did not persist to the weekly examinations and were considered nonadverse in the absence of other signs of systemic toxicity (body weight and food consumption decrements. There were no test item-related clinical observations noted at the weekly examinations. Observations noted in the test item-treated groups, including hair loss on various body surfaces and yellow material in the urogenital area, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

BODY WEIGHTS
MALES
Mean body weight gain in the 1000 mg/kg/day group males was similar to the control group during the pre-mating treatment period (study days 0-13) and during study days 13-21. A test item-related, significantly (p < 0.01) lower mean body weight gain was noted in this group during study days 21-27 compared to the control group. As a result, a lower mean body weight gain was noted in the 1000 mg/kg/day group when the entire treatment period (study days 0-27) was evaluated; the difference from the control group was significant (p < 0.05). The effect on mean body weight gain during the last week of treatment was not of sufficient magnitude to affect absolute mean body weights for this group, and therefore, was not considered adverse. Mean body weights and body weight gains in the 100 and 300 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.

BODY WEIGHTS
FEMALES (pre-mating)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

FOOD CONSUMPTION
MALES
Mean food consumption, evaluated as g/animal/day, in the 1000 mg/kg/day group males was significantly (p < 0.01) lower (2 g/animal/day) than the control group during the first week of treatment (study days 0-7). This decrement was noted in the absence of an effect on mean body weight gain during this same interval. Mean food consumption in the 1000 mg/kg/day group was similar to the control group during study days 7-13, and therefore the effect on mean food consumption noted during the first week of test item administration was considered transient and not test item-related. Mean food consumption in the 100 and 300 mg/kg/day group males was similar to that in the control group throughout the study. None of the differences from the control group were statistically significant.

FOOD CONSUMPTION
FEMALES (pre-mating)
Mean food consumption, evaluated as g/animal/day, in the 100, 300, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. Differences from the control group were not statistically significant, occurred in a manner that was not dose-related, and/or were transient.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

OPEN FIELD OBSERVATIONS
Open field parameters were unaffected by test item administration. A significantly (p < 0.05) higher mean rearing count was noted in the 1000 mg/kg/day group males during study week 3 compared to the control group. However, in the absence of effects on any other effects on CNS activity, the higher rearing count was not considered to be test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. A significantly (p < 0.05) lower mean hindlimb grip strength value was noted in the 300 mg/kg/day group males during study week 3 compared to the control group value. However, no dose-response was evident, and therefore this difference was not considered test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

MOTOR ACTIVITY
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 3 (males) and on lactation day 13 (females). Significantly (p ≤ 0.008) lower mean total counts were noted in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval. However, the differences were the result of a greater habituation response in these groups relative to the control group during the first 20 minutes of testing. When rats are placed in a novel environment, exploratory behavior (resulting in a high level of activity counts) followed by habituation (resulting in a reduced level of activity counts) is an expected response. In addition, there were no statistically significant differences in the cumulative total counts at these dosage levels. Therefore, the lower activity counts in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval were attributed to biologic variability and were not considered test item-related. All other values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL Research historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL Research historical control data ranges, and/or did not occur in a dose-related manner. Other than the greater habituation response described previously for the 300 and 1000 mg/kg/day males during the 11-20 minute subinterval, no remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 3 or lactation day 13.

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male in the 1000 mg/kg/day group did not sire a litter, as the female that this male was paired with was nongravid. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
The mean gestation length in the 1000 mg/kg/day group (22.3 days) was significantly (p < 0.05) longer than the concurrent control group (21.7 days). The 1000 mg/kg/day group gestation length value was within the WIL Research historical control range (21.3 to 22.3 days) and was primarily attributed to a single female (no. 2852) that delivered on gestation day 24 (and subsequently had a total litter loss on lactation day 0). Therefore, the longer mean gestation length at 1000 mg/kg/day was not considered test item-related. Mean gestation lengths in the 100 and 300 mg/kg/day groups (21.7 and 22.0 days, respectively) were similar to those in the concurrent control group. No signs of dystocia were noted in any group.

CLINICAL PATHOLOGY
SERUM CHEMISTRY
Test item-related higher cholesterol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and bile acids (1000 mg/kg/day group females), lower glucose (1000 mg/kg/day group males and females), higher phosphorus (300 and 1000 mg/kg/day group males, 1000 mg/kg/day group females) and higher urea nitrogen (1000 mg/kg/day group females) values were noted at study week 4 (males) or lactation day 14 (females). Minimal to mild elevations of cholesterol, ALP, ALT, and bile acids in 1000 mg/kg/day group females were coincident with test item-related hepatocellular hypertrophy; differences from the control group were significant (p < 0.05 or p < 0.01) for cholesterol, ALT, and bile acids. Minimally lower glucose was not associated with lower food consumption. Minimally to mildly higher phosphorus (significant at p < 0.01 or p < 0.05, males and p < 0.05, females) and urea nitrogen (significant at p < 0.01, females) were suggestive of dehydration but urinalysis was not performed in the present study. Glucose, phosphorous and urea changes were considered nonadverse given the magnitude of change. There were no other test item-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower urea nitrogen in the 100 mg/kg/day group males was not considered test item-related given the lack of a dose related response and a high control group mean and individual values.

MACROSCOPIC EXAMINATIONS
Test item-related dark red discoloration of the liver was noted in 100, 300, and 1000 mg/kg/day group males and correlated with angiectasis in single males in the 300 (no. 2827) and 1000 (no. 2797) mg/kg/day groups. Dark red discoloration in remaining animals in the 100, 300, and 1000 mg/kg/day groups correlated with sinusoidal congestion or was not associated with a relevant gross finding in 1 male (no. 2813) in the 1000 mg/kg/day group. The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values.

ORGAN WEIGHTS
Test item-related higher liver weights were noted in the 1000 mg/kg/day group males and females. Higher liver weights (absolute, relative to body and brain weight) were associated with test item-related hepatocellular hypertrophy and the weight relative to body weight was significant (p < 0.01) in males. Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a non-test item-related higher body weight. The thyroid/parathryoid weight relative to brain weight was higher in 1000 mg/kg/day group females.
There were no other test item-related effects on organ weights. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Higher mean Cowper’s gland weights were noted in the 100 (absolute, relative to brain and body weight), 300 (absolute and relative to body weight), and 1000 (relative to body weight) mg/kg/day group males but were not considered test item-related given the lack of a dose-related response and lack of correlating microscopic changes.

MICROSCOPIC EXAMINATIONS
Test item-related microscopic findings were noted in the adrenal cortex of the 1000 mg/kg/day group males and liver of the 1000 mg/kg/day group males and females. Liver: Minimal to mild hepatocellular hypertrophy, characterized by increased amounts of cytoplasm, was noted in the centrilobular region of the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females. Angiectasis, characterized by focally extensive dilation of sinuosoids, was observed in a single male each in the 300 and 1000 mg/kg/day groups (male nos. 2827 and 2797, respectively) and correlated with dark red discoloration in the liver. Angiectasis was differentiated from routine terminal congestion that was noted in the control and all test item-treated groups and was characterized by blood in the sinusoids. Adrenal cortex: There was an increased incidence and severity of cytoplasmic vacuolation of the adrenal cortex in the 1000 mg/kg/day group males. Vacuolation was characterized by variably sized, well demarcated vacuoles in the zona fasciculata and was not considered adverse. Vacuolation was also noted in females but vacuoles were uniform and there was no test item-related difference. There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Female no. 2852, which had a total litter loss, had mild myeloid hyperplasia of the bone marrow and a moderate increase in splenic extramedullary hematopoeisis which were both related to moderate uterine subacute inflammation with endometrial hyperplasia and mild mixed cell infiltrate in the vagina. Female no. 2846, which failed to conceive, was in early diestrus at the time of necropsy (study day 39).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: NOAEL is the highest dose (limit dose) of 1000 mg/kg bw/day.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10, 10-13, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 13 were unaffected by test item administration at all dosage levels. Differences form the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner. Slightly lower postnatal survival was noted during PND 0 relative to birth and when birth to PND 4 was evaluated in the 1000 mg/kg/day group; however, these differences were not statistically significant compared to the control group and were attributed to 1 female (no. 2852) in this group that had a total litter loss on PND 0.

GENERAL PHYSICAL CONDITION
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups (litters) that were found dead numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. One, 2, and 2 pups in the control, 300, and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

ANOGENITAL DISTANCE
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4-7 (post-selection), 7-10, 10-13, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 13 were unaffected by test item administration at all dosage levels.
Pups (litters) that were found dead numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. One, 2, and 2 pups in the control, 300, and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1-13 in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental administration of the test item. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

F1 pups were sacrificed prior to weaning.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Description (incidence and severity):

Not a valid metric in this study design

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was not affected by parental administration of the test item. The test item group values for males were not statistically significantly different from the control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related changes in thyroid/parathyroid weights for F1 pups at 100, 300, and 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Aside from the presence or absence of milk in the stomach, the following internal findings were noted. Pup no. 2882-10 in the 1000 mg/kg/day group was noted with a malformation consisting of a right-sided aortic arch (aortic arch and descending aorta coursed to the right of the vertebral column; the right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]; and the left carotid and subclavian arteries arose from a common vessel from the aortic arch). Dark red subcutis contents were noted for pup no. 2871-01 in the 1000 mg/kg/day group. In the 100 mg/kg/day group, pup no. 2873-15 was noted with clear fluid in the abdominal cavity and renal papilla(e) not developed.

Histopathological findings:

not examined

Other effects:

not specified

Description (incidence and severity):

THYROID HORMONE ANALYSIS (PND 13)
There were no test item-related changes in T4 levels in F1 males or females on PND 13.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10, 10-13, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 13 were unaffected by test item administration at all dosage levels. Differences form the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner. Slightly lower postnatal survival was noted during PND 0 relative to birth and when birth to PND 4 was evaluated in the 1000 mg/kg/day group; however, these differences were not statistically significant compared to the control group and were attributed to 1 female (no. 2852) in this group that had a total litter loss on PND 0.

GENERAL PHYSICAL CONDITION
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups (litters) that were found dead numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. One, 2, and 2 pups in the control, 300, and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

ANOGENITAL DISTANCE
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes during PND 1-13 in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental administration of the test item. No statistically significant differences from the control group were noted.

AREOLAE/NIPPLE ANLAGEN
Areolae/nipple anlagen in the F1 male pups was not affected by parental administration of the test item. The test item group values for males were not statistically significantly different from the control group.

THYROID HORMONE ANALYSIS (PND 13)
There were no test item-related changes in T4 levels in F1 males or females on PND 13.

ORGAN WEIGHTS (PND 13)
There were no test item-related changes in thyroid/parathyroid weights for F1 pups at 100, 300, and 1000 mg/kg/day.

NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead during PND 0-13 numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. No internal findings that could be attributed to parental test item administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, the following internal findings were noted. Pup no. 2882-10 in the 1000 mg/kg/day group was noted with a malformation consisting of a right-sided aortic arch (aortic arch and descending aorta coursed to the right of the vertebral column; the right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]; and the left carotid and subclavian arteries arose from a common vessel from the aortic arch). Dark red subcutis contents were noted for pup no. 2871-01 in the 1000 mg/kg/day group. In the 100 mg/kg/day group, pup no. 2873-15 was noted with clear fluid in the abdominal cavity and renal papilla(e) not developed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

gross pathology

Remarks on result:

other: NOAEL is the highest dose (limit dose) of 1000 mg/kg bw/day.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of 1,4-diisopropylbenzene when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test item-related clinical observations or effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones, or macroscopic/microscopic alterations in F0 males and females at any dosage levels. Therefore, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for postnatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

The objective of this study according to OECD Guideline 422 and GLP was to evaluate the potential toxic effects of the test item, 1,4-diisopropylbenzene, when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

STUDY DESIGN: The test item, 1,4-diisopropylbenzene, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dose volumes of 0.12, 0.35, and 1.18 mL/kg. A concurrent control group of 10 rats/sex received the control item (deionized water) at a dose volume of 1.18 mL/kg on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28-29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 49-59 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following 23 days of dose administration and for 5 females/group on lactation day 13. All F0 females were allowed to deliver and rear their pups until lactation day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (1/sex/litter) but were not analyzed. All F1 male pups were evaluated for areolae/nipple anlagen on PND 12. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0 animals/sex/group just prior to scheduled necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females prior to euthanasia; only male samples were analyzed. F0 males were euthanized following completion of the mating period and F0 females were euthanized on lactation day 14 for females that delivered or post-mating day 25 for females that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and 1000 mg/kg/day groups. In addition, the adrenal cortex (males) and liver (males and females) were identified as potential target tissues and were examined from all animals.

RESULTS: All F0 males and females survived to the scheduled necropsies. Red and/or clear material around the mouth was noted in a dose-related manner for males and females in all test item-treated groups approximately 1 hour following dose administration throughout the treatment period. The aforementioned material observations did not persist to the weekly examinations and were considered nonadverse in the absence of other signs of systemic toxicity (body weight and food consumption decrements). No test item-related clinical observations were noted for F0 males and females at the weekly examinations at any dosage level. Mean body weight gain in the 1000 mg/kg/day group males was similar to the control group during the pre-mating treatment period (study days 0-13) and during study days 13-21. A test item-related, lower mean body weight gain was noted in this group during study days 21-27 and when the entire treatment period (study days 0-27) was evaluated. However, the effect on mean body weight gain during the last week of treatment was not of sufficient magnitude to affect absolute mean body weights, and therefore, was not considered adverse. Mean body weights, body weight gains, and food consumption for F0 males at 100 and 300 mg/kg/day and females at 100, 300, and 1000 mg/kg/day were unaffected by test item administration throughout the study. No test item-related effects were noted during the FOB or motor activity evaluations at any dosage level. There was 1,4 -diisopropylbenzene-related minimal to mild nonadverse hepatocellular hypertrophy in the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females that was associated with higher liver weights in males and females at 1000 mg/kg/day and nonadverse elevations in alkaline phosphatase, alanine aminotransferase, bile acids, and cholesterol in females at 1000 mg/kg/day. Nonadverse angiectasis of the liver was noted in the 300 and 1000 mg/kg/day group males and correlated with gross observations of dark red discoloration. Increased incidence and severity of nonadverse adrenal cortical vacuolation was noted in the 1000 mg/kg/day group males. Other nonadverse findings included lower glucose values (1000 mg/kg/day group males and females), higher urea nitrogen values (1000 mg/kg/day group females), and higher phosphorus (300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females). There were no test item-related changes in hematology and coagulation parameters for F0 males and females. T4 levels in F0 males were unaffected by test item administration. F0 male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. There were no test item-related effects on the mean number of implantation sites and unaccounted-for sites at any dosage level.

There were no test item-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 clinical observations, postnatal survival and growth, anogenital distance, or areolae/nipple anlagen. There were no test item-related macroscopic findings for F1 pups that were found dead at any dosage level. There were no test item-related changes in T4 levels or thyroid/parathyroid weights in F1 males or females on PND 13.

 

In summary, under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test item-related clinical observations or effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones, or macroscopic/microscopic alterations in F0 males and females at any dosage levels. Therefore, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for postnatal toxicity and for reproductive performance was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 422, GLP with the source substance 1,4 -diisopropylbenzene

Under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of 1,4-diisopropylbenzene when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test item-related clinical observations or effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones, or macroscopic/microscopic alterations in F0 males and females at any dosage levels. Therefore, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for postnatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Effects on developmental toxicity

Description of key information

No developmental toxicity was found in an OECD 414 prenatal developmental toxicity study. The NOAEL was the high dose (and limit dose) of 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, on 21-May-2015.
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are fully accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
- Diet: ad libitum, the basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Water: ad libitum, municipal water supplying delivered by an automatic watering system
- Acclimation period: a minimum of 12 days for acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3°C to 22.3°C
- Humidity (%): 46.0% to 57.5%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

The control item was prepared approximately weekly for administration to the control group (Group 1); aliquots were prepared for daily dispensation to the control group and stored at room temperature.

GroupNumber Treatment Dosage Level (mg/kg/day) Dosage Volume (mL/kg)
1 CONTROL 0 1.16
2 m-DIPB 100 0.12
3 m-DIPB 300 0.35
4 m-DIPB 1000 1.16

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dosage levels were selected based on the results of a previous 28-day study. Analyses to demonstrate the homogeneity, stability, and concentration of the test item were not conducted in this study. The test item was expected to be stable for the duration of testing. Characterization of the test item was conducted previously and the test item was applied in pure form.

Details on mating procedure:

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated. The experimental design consisted of 3 test item-treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony. Body weight values ranged from 220 g to 284 g on gestation day 0.

Duration of treatment / exposure:

19 days

Frequency of treatment:

once daily

Duration of test:

19 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

Timed-pregnant female rats were treated with the test article by oral gavage from gestation day 6 to 19, at which point a cessarian section was performed and data on the dams and fetuses was obtained.

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Individual maternal body weights were recorded on gestation days 0-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 1-3, 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” on the individual report tables. Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 0-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Ovaries and uterine content:

UNSCHEDULED DEATHS
A gross necropsy was performed on females that were found dead during the course of the study. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally. The females and all products of conception were discarded.

GESTATION DAY 20 LAPAROHYSTERECTOMY
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss
(Salewski, 1964). The kidney, liver, and stomach from all females euthanized at the scheduled necropsy were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Other maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Fetal examinations:

FETAL MORPHOLOGICAL EXAMINATION
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976) as described in the protocol. Fetuses were then examined for skeletal malformations and developmental variations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the
means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20 with 13 dead fetuses (with no apparent malformations) in utero; this female was noted with rales on gestation day 2 but had no remarkable clinical findings between gestation day 2 through the day of death. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 (pregnant) was found dead on gestation day 12 following intermittent body weight losses during gestation day 1-11 (9.2% body weight loss). During gestation day 10-11, this animal consumed only 1 g of feed (with corresponding decreased defecation). At necropsy, this female had lungs that were not fully collapsed, suggesting that the death was likely related to the dosing procedure, and therefore was not considered test item-related. Female no. 8129 (pregnant) was found dead on gestation day 12; at necropsy, this animal had lungs that were not fully collapsed (with dark red areas) and foamy contents in the trachea. The death of female no. 8129 was likely related to the dosing procedure, and therefore was not considered test item-related. All other females survived to the scheduled necropsy.
Increased incidences of clear and/or red material around the mouth were noted at 1 hour following dose administration for the surviving 1000 mg/kg/day group females when compared to the control group. These findings were noted as early as gestation day 1 and persisted through gestation day 19. Additionally, a slight increase of these same observations were noted sporadically in the 300 mg/kg day group females when compared to the control group. There were no test item-related clinical observations noted in the 100 mg/kg/day group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Due to a significant (p<0.01) mean maternal body weight loss in the 1000 mg/kg/day group following the first day of treatment (gestation day 1-2), mean maternal body weight gain in this group during gestation days 1-3 was significantly (p<0.01) lower than the control group value (1 g compared to 10 g in the control group). Mean body weight gains in the 1000 mg/kg/day group were similar to the control group values during gestation days 3-6, 6-9, 9-12, and 12-15. Significantly (p<0.05) lower mean body weight gains compared to the control group were noted in the 1000 mg/kg/day group during gestation days 15-20 and when the overall treatment period (gestation days 1-20; 13.1% lower) was evaluated. As a result, mean body weights in the 1000 mg/kg/day group during gestation days 18-20 were slightly lower (up to 5.3%) than the control group values; the differences for gestation days 18 and 19 were significant (p<0.05).
Although not statistically significant, mean net body weight gain (12.5%) in this group were lower compared to the control group. The aforementioned changes were considered test item-related. The lower mean net body weight gain in the 1000 mg/kg/day group was not of sufficient magnitude to result in a lower mean net body weight. As a result of the lower mean number of viable fetuses in this group, gravid uterine weight was 12.2% lower than in the control group. Because the lower number of viable fetuses was not considered to be test item-related, the lower gravid uterine weight was not considered to be test item-related.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 300 mg/kg/day groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was lower than the control group values during gestation days 1-3; the differences were significant (p<0.01). The lower food consumption values
in this group during the first 3 days of treatment corresponded to lower body weight gains, and were therefore also considered test item-related. With the exception of slightly higher mean g/kg/day food consumption values during gestation days 9-12 and 15-20 (significant at p<0.05), mean food consumption values in the 1000 mg/kg/day group were similar to the control group values throughout the remainder of the treatment period (gestation days 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20). Test item-related significantly (p<0.05 or p<0.01) lower mean food consumption was noted in the 100 and 300 mg/kg/day groups during gestation days 1-3. In the absence of an effect on body weights and body weight gains in these groups, the initial food consumption decrements were considered nonadverse. Mean food consumption in these groups was generally similar to the control group for the remainder of the treatment period, with the following exception, higher mean food consumption was noted during gestation days 12-15 (g/animal/day and g/kg/ day) and 15-20 (g/kg/day only) in the 300 mg/kg/day group; differences from the control group were significant (p<0.01 or p<0.05). The aforementioned higher mean food consumption was not attributed to test item administration because the direction of change was not considered toxicologically relevant.

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

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20; at necropsy, this animal had 13 dead fetuses (with no apparent malformations) in utero. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 was found dead on gestation day 12; this animal had lungs that were not fully collapsed and 12 normally developing implantations in utero. Female no. 8129 (also found dead on gestation day 12) had lungs that were not fully collapsed (with dark red areas), foamy contents in the trachea, and 16 normally developing implantations in utero. All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 100, 300, and 1000 mg/kg/day. Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

None found

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

None found

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was not a significant change in early or late resorptions

Dead fetuses:

no effects observed

Description (incidence and severity):

None

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not a valid metric of the study

Changes in number of pregnant:

not examined

Description (incidence and severity):

Not a valid metric of the study

Other effects:

not specified

Details on maternal toxic effects:

MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20 with 13 dead fetuses (with no apparent malformations) in utero; this female was noted with rales on gestation day 2 but had no remarkable clinical findings between gestation day 2 through the day of death. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 (pregnant) was found dead on gestation day 12 following intermittent body weight losses during gestation day 1-11 (9.2% body weight loss). During gestation day 10-11, this animal consumed only 1 g of feed (with corresponding decreased defecation). At necropsy, this female had lungs that were not fully collapsed, suggesting that the death was likely related to the dosing procedure, and therefore was not considered test item-related. Female no. 8129 (pregnant) was found dead on gestation day 12; at necropsy, this animal had lungs that were not fully collapsed
(with dark red areas) and foamy contents in the trachea. The death of female no. 8129 was likely related to the dosing procedure, and therefore was not considered test item-related. All other females survived to the scheduled necropsy. Increased incidences of clear and/or red material around the mouth were noted at 1 hour following dose administration for the surviving 1000 mg/kg/day group females when compared to the control group. These findings were noted as early as gestation day 1 and persisted through gestation day 19. Additionally, a slight increase of these same observations were noted sporadically in the 300 mg/kg day group females when compared to the control group. There were no test item-related clinical observations noted in the 100 mg/kg/day group.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Due to a significant (p<0.01) mean maternal body weight loss in the 1000 mg/kg/day group following the first day of treatment (gestation day 1-2), mean maternal body weight gain in this group during gestation days 1-3 was significantly (p<0.01) lower than the control group value (1 g compared to 10 g in the control group). Mean body weight gains in the 1000 mg/kg/day group were similar to the control group values during gestation days 3-6, 6-9, 9-12, and 12-15. Significantly (p<0.05) lower mean body weight gains compared to the control group were noted in the 1000 mg/kg/day group during gestation days 15-20 and when the overall treatment period (gestation days 1-20; 13.1% lower) was evaluated. As a result, mean body weights in the 1000 mg/kg/day group during gestation days 18-20 were slightly lower (up to 5.3%) than the control group values; the differences for gestation days 18 and 19 were significant (p<0.05). Although not statistically significant, mean net body weight gain (12.5%) in this group were lower compared to the control group. The aforementioned changes were considered test item-related. The lower mean net body weight gain in the 1000 mg/kg/day group was not of sufficient magnitude to result in a lower mean net body weight. As a result of the lower mean number of viable fetuses (see Section 6.5.) in this group, gravid uterine weight was 12.2% lower than in the control group. Because the lower number of viable fetuses was not considered to be test item-related, the lower gravid uterine weight was not considered to be test item-related. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 300 mg/kg/day groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

MATERNAL FOOD CONSUMPTION
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was lower than the control group values during gestation days 1-3; the differences were significant (p<0.01). The lower food consumption values in this group during the first 3 days of treatment corresponded to lower body weight gains, and were therefore also considered test item-related. With the exception of slightly higher mean g/kg/day food consumption values during gestation days 9-12 and 15-20 (significant at p<0.05), mean food consumption values in the 1000 mg/kg/day group were similar to the control group values throughout the remainder of the treatment period (gestation days 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20). Test item-related significantly (p<0.05 or p<0.01) lower mean food consumption was noted in the 100 and 300 mg/kg/day groups during gestation days 1-3. In the absence of an effect on body weights and body weight gains in these groups, the initial food consumption decrements were considered nonadverse. Mean food consumption in these groups was generally similar to the control group for the remainder of the treatment period, with the following exception, higher mean food consumption was noted during gestation days 12-15 (g/animal/day and g/kg/ day) and 15-20 (g/kg/day only) in the 300 mg/kg/day group; differences from the control group were significant (p<0.01 or p<0.05). The aforementioned higher mean food consumption was not attributed to test item administration because the direction of change was not considered toxicologically
relevant.

MATERNAL NECROPSY DATA
Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20; at necropsy, this animal had 13 dead fetuses (with no apparent malformations) in utero. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 was found dead on gestation day 12; this animal had lungs that were not fully collapsed and 12 normally developing implantations in utero. Female no. 8129 (also found dead on gestation day 12) had lungs that were not fully collapsed (with dark red areas), foamy contents in the trachea, and 16 normally developing implantations in utero. All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 100, 300, and 1000 mg/kg/day. Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group (3.8, 3.6, and 3.7 g, respectively) were slightly lower than the concurrent control group values (4.0, 3.8, and 3.9 g, respectively); the difference for females was significant (p<0.05). However, all high-dosage group values were equal to the respective mean values in the WIL Research historical control data; therefore, the slightly lower mean fetal body weights in the 1000 mg/kg/day group were not considered test item-related.
Mean fetal body weights in the 100 and 300 mg/kg/day groups were similar to the control group values; none of the differences were statistically significant. Intrauterine growth and survival was unaffected by test item administration at dosage levels of 100 and 300 mg/kg/day.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No changes found

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not a valid metric for this study design

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were noted in 1 fetus in the 100 mg/kg/day group. Fetus no. 8092-05 in the 100 mg/kg/day group was observed with macroglossia and a palate with a high arch. Based on the occurrence in a single fetus and the absence of a
dose-response relationship, these malformations were not considered test item-related. No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in 2(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fetus no. 8074-09 in the 1000 mg/kg/day group (previously noted with visceral malformations) had malaligned sternebrae (nos. 2 and 3 [moderate] and 4 and 5 [severe]). Fetus nos. 8086-11 and 8086-13 in the control group and no. 8152-05 in the 100 mg/kg/day group had costal cartilage anomalies (fused, bifurcated, and/or malpositioned costal cartilages). The skeletal malformations noted in the 100 and 1000 mg/kg/day groups were not attributed to the test item because they occurred in single fetuses and/or similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. No test item-related skeletal developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data for definitive studies

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were noted in 3(2), 0(0), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Interventricular septal defects (≤ 2-mm in diameter opening in the anterior or posterior portion of the septum) were noted for fetus nos. 8086-04 and 8114-01 in the control group and fetus no. 8074-09 in the 1000 mg/kg/day group. Fetus no. 8074-09 in the 1000 mg/kg/day group also had a malpositioned atrium (the left atrium was located more dorsal than normal). The occurrences of interventricular septal defect and malpositioned atrium in the 1000 mg/kg/day group were not attributed to the test item because they occurred in a single fetus and/or similarly in the concurrent control group and the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group. In addition, the mean litter proportion of interventricular septal defect was within the range of the WIL Research historical control data for definitive studies, whereas malpositioned atrium has not been previously observed in the WIL Research historical control data. Fetus no. 8086-04 in the control group was also noted with an absent left kidney, ureter, and horn of the uterus and transposition of the great vessels. Another fetus in the same control litter (no. 8086-16) had a malpositioned right testis and epididymis (located more anterior than normal). Fetus no. 8114-01 in the control group also had lobular dysgenesis of the lungs (1 lobe was present bilaterally). No test item-related visceral developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.
Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) were noted for 9, 5, 3, and 3 fetuses in the control, 100, 300, and 1000 mg/kg/day groups, respectively. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test item-related because they occurred infrequently in the test item-treated groups, at a higher frequency in the control group, and in a manner that was not dose-related.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
Although the mean litter proportion of pre-implantation loss was unaffected by test itemadministration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item
administration. Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group (3.8, 3.6, and 3.7 g, respectively) were slightly lower than the concurrent control group values (4.0, 3.8, and 3.9 g, respectively); the difference for females was significant (p<0.05). However, all high-dosage group values were equal to the respective mean values in the WIL Research historical control data; therefore, the slightly lower mean fetal body weights in the 1000 mg/kg/day group were not considered test item-related. Mean fetal body weights in the 100 and 300 mg/kg/day groups were similar to the control group values; none of the differences were statistically significant. Intrauterine growth and survival was unaffected by test item administration at dosage levels of 100 and 300 mg/kg/day. Parameters evaluated included pre-implantation loss, postimplantation loss, live litter size, and fetal sex ratios.

FETAL MORPHOLOGICAL DATA
The numbers of fetuses (litters) available for morphological evaluation were 359(25), 349(25), 312(23), and 271(21) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Malformations were observed in 5(2), 2(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

EXTERNAL MALFORMATIONS AND VARIATIONS
External malformations were noted in 1 fetus in the 100 mg/kg/day group. Fetus no. 8092-05 in the 100 mg/kg/day group was observed with macroglossia and a palate with a high arch. Based on the occurrence in a single fetus and the absence of a dose-response relationship, these malformations were not considered test item-related. No external developmental variations were observed in fetuses in this study.

VISCERAL MALFORMATIONS AND VARIATIONS
Visceral malformations were noted in 3(2), 0(0), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Interventricular septal defects (≤ 2-mm in diameter opening in the anterior or posterior portion of the septum) were noted for fetus nos. 8086-04 and 8114-01 in the control group and fetus no. 8074-09 in the 1000 mg/kg/day group. Fetus no. 8074-09 in the 1000 mg/kg/day group also had a malpositioned atrium (the left atrium was located more dorsal than normal). The occurrences of interventricular septal defect and malpositioned atrium in the 1000 mg/kg/day group were not attributed to the test item because they occurred in a single fetus and/or similarly in the concurrent control group and the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group. In addition, the mean litter proportion of interventricular septal defect was within the range of the WIL Research historical control data for definitive studies, whereas malpositioned atrium has not been previously observed in the WIL Research historical control data. Fetus no. 8086-04 in the control group was also noted with an absent left kidney, ureter, and horn of the uterus and transposition of the great vessels. Another fetus in the same control litter (no. 8086-16) had a malpositioned right testis and epididymis (located more anterior than normal). Fetus no. 8114-01 in the control group also had lobular dysgenesis of the lungs (1 lobe was present bilaterally). No test item-related visceral developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) were noted for 9, 5, 3, and 3 fetuses in the control, 100, 300, and 1000 mg/kg/day groups, respectively. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test item-related because they occurred infrequently in the test item-treated groups, at a higher frequency in the control group, and in a manner that was not dose-related.

SKELETAL MALFORMATIONS AND VARIATIONS
Skeletal malformations were noted in 2(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fetus no. 8074-09 in the 1000 mg/kg/day group (previously noted with visceral malformations) had malaligned sternebrae (nos. 2 and 3 [moderate] and 4 and 5 [severe]). Fetus nos. 8086-11 and 8086-13 in the control group and no. 8152-05 in the 100 mg/kg/day group had costal cartilage anomalies (fused, bifurcated, and/or malpositioned costal cartilages). The skeletal malformations noted in the 100 and 1000 mg/kg/day groups were not attributed to the test item because they occurred in single fetuses and/or similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. No test item-related skeletal developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data for definitive studies.

SUMMARY OF EXTERNAL, VISCERAL, AND SKELETAL EXAMINATIONS
The numbers of fetuses (litters) available for morphological evaluation were 359(25), 349(25), 312(23), and 271(21) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Malformations were observed in 5(2), 2(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test item-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the WIL Research historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test item.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on fetuses

Remarks on result:

other: (NOAEL is the highest dose (limit dose) of 1000 mg/kg bw/day.)

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Based on maternal toxicity in the 1000 mg/kg/day group, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on a lack of developmental toxicity at all dosage levels, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

The developmental toxicity test was performed according to OECD guideline 414 with GLP compliance.The test item was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.12, 0.35, and 1.16 mL/kg, respectively.

Based on maternal toxicity in the 1000 mg/kg/day group, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on a lack of developmental toxicity at all dosage levels, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when test item was administered orally by gavage to bred Crl:CD(SD) rats.

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:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414, GLP

m-DIPB was evaluated in an OECD 414 study, where the material was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.12, 0.35, and 1.16 mL/kg, respectively. A concurrent control group composed of 25 bred females received the control item, deionized water, on a comparable regimen at a dosage volume of 1.16 mL/kg. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. One female was found dead on gestation day 20 with 13 dead fetuses (with no apparent malformations) in utero; this female was noted with rales on gestation day 2 but had no remarkable clinical findings thereafter. At necropsy, the cause of death was not determined but a relationship to the test item cannot be ruled out. Two pregnant females were found dead on gestation day 12 with necropsy findings (lungs that were not fully collapsed and/or foamy contents in the trachea) that were suggestive of the deaths being related to the dosing procedure, and therefore were not considered test item-related. All other females survived to the scheduled necropsy; no test item-related macroscopic findings were observed at any dosage level. Dose-related increased incidences of clear and/or red material around the mouth were noted at 1 hour following dose administration in the 300 and 1000 mg/kg/day groups when compared to the control group. These findings were noted as early as gestation day 1 and persisted through gestation day 19. There were no test item-related clinical observations noted in the 100 mg/kg/day group. A test item-related mean maternal body weight loss was noted in the 1000 mg/kg/day group following the first day of treatment (gestation day 1-2) resulting in lower mean maternal body weight gain during gestation days 1-3 (1 g compared to 10 g in the control group) and 15-20, resulting in a lower mean body weight gain for the overall treatment period (gestation days 1-20) and slightly lower mean body weights (up to 5.3 % lower) during gestation days 18-20 compared to the control group. The lower weight gain in the 1000 mg/kg/day group during gestation days 1-3 correlated with test item-related lower food consumption during this same interval. Although not statistically significant, mean net body weight gain (12.5 %) was lower than in the control group. The lower mean net body weight gain in the 1000 mg/kg/day group was not of sufficient magnitude to result in a lower mean net body weight. Lower mean food consumption was noted in the 100 and 300 mg/kg/day groups following the initiation of dose administration (gestation days 1-3). Mean food consumption in these group was similar to the control group for the remainder of the treatment period. In the absence of an effect on maternal mean body weights or body weight gains in the 100 and 300 mg/kg/day group, the initial decrements in mean food consumption were considered nonadverse. There were no test item-related effects on mean net body weights, net body weight gains, or gravid uterine weights, at 100 and 300 mg/kg/day. Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group was lower than the concurrent control group value. As a result, the mean number of viable fetuses in the 1000 mg/kg/day group was lower than the concurrent control group. The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group compared to the control group. Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group were slightly lower than the concurrent control group values. However, all high-dosage group values were equal to the respective mean values in the WIL Research historical control data; therefore, the slightly lower mean fetal body weights in the 1000 mg/kg/day group were not considered test item-related. Intrauterine growth and survival was unaffected by test item administration at dosage levels of 100 and 300 mg/kg/day. No test item-related fetal malformations or developmental variations were noted at any dosage level.

 

Based on maternal toxicity in the 1000 mg/kg/day group, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on a lack of developmental toxicity at all dosage levels, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when 1,3-diisopropylbenzene (DIPB) was administered orally by gavage to bred Crl:CD(SD) rats.

Justification for classification or non-classification

Based on the available data the registered substance is not classified in accordance with Regulation (EC) No 1272/2008 .