4,4'-cyclohexylidenedi-o-cresol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty virgin male and 50 virgin female Crl:CD®(SD) rats were received from the supplier on May 14, 2001. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 63 days old upon receipt and 10-11 weeks old at initiation of test article administration. The range of F0 male body weights at the start of the prebreed exposure period was 315.0 to 361.7 g. The range for F0 females was 213.2 to 248.5 g. During the one-week acclimation the animals were examined by a veterinarian, weighed, and representative animals were subjected to faecal examination and serum viral antibody analysis. In addition, one rat per sex was selected as a sentinel which remained in the study room under the identical conditions of the study animals and was sacrificed and subjected to faecal examination and serum viral antibody analysis and the end of the study. All evaluations were negative and the animals assigned to study were deemed healthy and appropriate for use.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt, all F0 parental test animals were housed individually, except during the mating period when they were house two per cage, in clean, solid-bottom polycarbonate cages with stainless-steel wire lids. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The animal room was on a 12-hour light cycle with a temperature range of 68.0 to 73.6 °F and a relative humidity range of 45.8 to 66.6 % throughout the study. Certified rodent chow and city tap water were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose (CAS No. 9004-67-5; 4000 centipoises, Fisher Scientific)

Details on oral exposure:

DMBPC was weighed into colour-coded glass beakers for each concentration. The formulations were not corrected for the purity of the test substance. Approximately 400 mL of vehicle was added to 2-litre calibrated beakers to a 1-litre mark. With the vehicle in the 2-litre beakers being stirred on magnetic stir plates, DMBPC was slowly added and the suspensions were stirred for at least 15 minutes. Additional vehicle was added to the calibration line. All test article formulations were then sonicated for 30 minutes, then stirred at least 5 minutes, allowed to rest for 2.5 hours, then stirred for an additional time (30 minutes to 2.5 hours), depending on the formulation, prior to analytical and archival sampling.

Prior to and during the study, dose formulations encompassing the range of dose concentrations employed in the study (2 and 200 mg/mL) were found to be homogeneous, stable for four hours under room temperature (to simulate daily dosing periods), and stable for at least 32 days in amber bottles under refrigerated conditions (approximately 4 °C). Based on these stability results, formulations were used within the stability limits established and were stored under refrigeration. Dose formulations were originally analysed as 91.3 - 110 % of nominal concentrations, although an error was subsequently detected after the formulations were presented to the animals, which indicated that the top dose formulated on June 28, 2001, was not 91.3 % of nominal but 86.7 % of nominal. The use of this formulation was unavoidable and had no effect on the study. No DMBPC was detected in the vehicle control formulations, with an estimated limit of detection of 0.4 mg/mL.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC (CAS No. 2362-14-3) orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each dose formulation prepared for use on study were analysed by single injection using high performance liquid chromatography (HPLC) with a Model 680 Automated Gradient Controller (Waters Corporation), a Model 510 pump and a Model 710B WISP autosampler. The column was a Zorbax SB-C18 4.6 x 50 mm; 3.5 µm (Agilent), and the guard column was a Zorbax SB-C18 (Agilent) 4.6 x 12.5mm; 5 µm. The detector was a Model 757 absorbance detector (Applied Biosystems). The regression equation for the vehicle standard data was calculated, and the data were plotted for a visual evaluation of linearity. The concentration of DMBPC was calculated in the dose formulation samples (mg/mL) from the peak area ratio for each sample and the linear regression equation.

Standards for acceptable accuracy of formulations were the mean of the analysed samples was within ± 10 % of nominal, and the % RSD (Relative Standard Deviation) for triplicate samples did not exceed 10 %. If one or more of these standards were not met, the dosing formulations were not administered to the animals until the problem was resolved by analysis of the archived sample of the dosing formulations and/or after reformulation and reanalysis. All dose formulations used in the study had analytical values of 91.4 to 110 % of target concentrations with one dose of 86.7 %. Vehicle control formulations contained no DMBPC, with an estimated detection limit of 0.4 mg/mL.

Prior to and during the performance of this study, aliquots of doses, encompassing the range of concentrations that were to be used in the study (2 and 200 mg/mL), were used to assess stability of the dose suspensions. Triplicate one-mL aliquots were collected from the top, middle, and bottom of the prepared concentrations and analysed for homogeneity by HPLC.

For stability studies, the doses mixed for the low and high homogeneity study were removed from the refrigerator on the day of sampling (3, 9-10, 21, and 32 days) and brought to ambient conditions. The formulations were stirred for two hours prior to sampling. Triplicate 1-g aliquots were removed and analysed by high performance liquid chromatography. Dose formulations at 2 and 200 mg/mL were stable for 32 days when stored in the refrigerator.

Duration of treatment / exposure:

Males: Two weeks prior to mating and during the two week mating period (for a total of 28 doses)
Females: Two weeks prior to mating, during the two week mating period, through three weeks of gestation and through lactation day 3 (the day prior to necropsy)

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/group; 5 additional males in the control and high concentration (recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were chosen based on a range-finding study, employing doses of 0, 50, 200, and 1000 mg/kg/day, administered by oral gavage at 5 mL/kg for 14 days.

- Details on study schedule: Males received a total of 14 or 15 doses prior to mating (one dose per day). Males were dosed throughout the mating period through the day prior to euthanasia for a total of 28 doses. Females received 14 doses prior to pairing (one dose per day) and were dosed through lactation day 3 (the day prior to necropsy). After the two-week prebreed exposure period, animals were randomly mated within treatment groups for a two-week mating period to produce the F1 generation, with exposure continuing. Five additional males per group in the control and high dose (1000 mg/kg/day) groups were retained as recovery animals for two weeks after the end of the dosing period to evaluate recovery from any possible treatment-related effects identified in the high dose group.

Positive control:

none

Observations and examinations performed and frequency:

Parental animals: Observations and examinations: Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the time of onset and degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behaviour pattern.

The body weights of the F0 male rats were determined and recorded initially and weekly through mating. The body weights of F0 female rats were recorded in the same manner until confirmation of mating. During gestation, F0 females were weighed on gestational day (GD) 0, 7, 14, and 20. Dams producing litters were weighed on PND 0 and 4, and body weight gains were computed.

Feed consumption measurements were recorded weekly for all F0 parental study animals during the prebreed treatment period. During pregnancy of F0 females, feed consumption was recorded for GD 0-7, 7-14, and 14-20. During lactation of the F1 litters, maternal feed consumption was measured for PND 0-4. Feed consumption was not measured during the cohabitation period, since two adult animals (breeding pair) were in the same cage. Feed consumption collection periods corresponded to the collection of the animals' body weight data.

A Functional Observational Battery (FOB), including home cage observations, handling observations, and open field observations, was performed on all animals at least once per week during prebreed, mating (both sexes), gestation and lactation (females) treatment periods.
Five F0 males and five females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy.
In addition, haematology, clinical biochemistry, and urinalysis (males only) assays were performed at necropsy for five randomly selected parental F0 males and females per dose group.
- Haematology Parameters Examined: Red blood cell (nucleated RBC), Red cell distribution width, RBC Morphology, Mean platelet volume, White blood cell (Total/differential), Mean corpuscular volume, Mean corpuscular haemoglobin, Platelet counts, Mean corpuscular haemoglobin concentration , Haemoglobin concentration and Haematocrit.
- Clinical Chemistry Parameters Examined: Albumin, Creatinine, Aspartate aminotransferase, Glucose, Alanine aminotransferase, Total protein and sodium, Urea nitrogen, Potassium, Total cholesterol and Chloride.


Litter observations: Beginning on GD 20, each female was observed twice daily (a.m. and p.m.) for evidence of littering. On the day of birth (PND 0), anogenital distance was measured and body weight recorded for all live F1 pups in all litters. Body weight was recorded for all live pups on PND 4 prior to euthanasia. Any female which did not show evidence of successful mating after 14 days of cohabitation was weighed weekly, and treatment continued until GD 26 or delivery occurred. If a female without a confirmed GD 0 date was, in fact, pregnant and delivered a litter, her lactational information was collected as described below.

All F1 pups were sexed and examined as soon as possible after birth (PND 0) to determine the number of viable and stillborn from each litter. Thereafter, litters were evaluated for survival through PND 4. Individual anogenital distance and body weight were recorded on PND 0 for all F1 offspring. All live pups were counted, sexed, weighed individually, and examined grossly at birth and on PND 4. The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified. All pups were examined for physical abnormalities at birth and on PND 4. All pups dying during the early lactation period were necropsied, when possible, to investigate the cause of death.

Sacrifice and pathology:

Postmortem examinations (Parental animals): A complete necropsy was conducted on all F0 animals, including recovery males, at termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. The number of nidation scars and corpora lutea were recorded for all F0 females. The fixed (buffered neutral 10 % formalin) uteri from any females of the F0 generation failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy. This staining procedure did not interfere with subsequent histopathologic evaluation. The ovaries, uterus (with cervix and vagina), prostate, epididymides, testes and seminal vesicles (with coagulating glands and their fluids) were weighed and retained for all F0 animals. The liver, heart, kidneys, adrenals, brain, spleen and thymus were weighed and retained from 5 selected male and female animals/group. In addition, the spinal cord, thyroid, stomach, urinary bladder, sciatic nerve, femur, small and large intestines (including Peyer’s patches), trachea and lungs, lymph nodes and all gross lesions were retained from 5 F0 adult males and females/group including all recovery males. Full histopathology of the retained organs was performed on five high dose and control F0 animals.

Postmortem examinations (Offspring): All F1 pups were examined externally for gross abnormalities, euthanised on PND 4 and examined viscerally.

Other examinations:

Reproductive indices: The following reproductive indices were calculated: Male Mating Index (%); Male Fertility Index (%); Male Pregnancy Index (%); Female Mating Index (%); Female Fertility Index (%): and Female Gestational Index (%).
 
Offspring viability indices: The following litter parameters were calculated: Live birth index (%); Day 4 Survival Index (%, survival between PND 0 and 4); and Mean Live Litter Size.

Statistics:

The unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using either parametric ANOVA under the standard assumptions or robust regression method. The homogeneity of variance assumption was examined via Levene's Test. If Levene's Test indicated lack of homogeneity of variance (p<0.05), robust regression methods were used to test all treatment effects. The presence of linear trends was analysed by GLM procedures for homogenous data or by robust regression methods for nonhomogenous data. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS 6.12 was used to evaluate the overall effect of treatment and when a significant treatment effect was present, to compare each exposed group to control via Dunnett's Test. For the litter-derived percentage data (e.g., PND 0-4 pup survival indices), the ANOVA was weighted according to litter size. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for parental and pup body weights, parental organ weight parameters and feed consumption, percent F1 males per litter, and F1 anogenital distance. For any endpoints which involved only two groups (e.g., for F0 recovery males, etc.), Student’s t-test was used for the two-group comparisons.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

No parental animals died during the test.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, F0 male body weights were significantly reduced on study day (SD) 14 and 21 and male body weight changes were significantly reduced for SD 7 to 14 and SD 0 to 28. Absolute body weights for recovery males at 1000 mg/kg/day remained lower throughout the two week recovery period; however, body weight gain was comparable to the recovery control males. F0 female body weight for SD 14 and body weight changes for SD 0-14 were significantly reduced at 1000 mg/kg/day. F0 maternal body weights during gestation were significantly reduced at 200 and 1000 mg/kg/day for gestation day (GD) 7 and 14, whereas body weight change showed no effect of treatment among groups. During lactation, F0 maternal body weights were significantly reduced on post natal day (PND) 0 and 4 at 1000 mg/kg/day. Maternal body weight change was not affected during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption expressed as g/day was significantly reduced for the F0 females at 200 and 1000 mg/kg/day for SD 0-7. When expressed as g/kg/day, feed consumption was significantly reduced at 200 mg/kg/day for SD 0-7. During gestation, maternal feed consumption was significantly reduced when expressed as g/day for GD 0-7 and GD 7-14 at 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haemoglobin was significantly reduced at 50, 200, and 1000 mg/kg/day, the haematocrit was reduced at 50 and 200 (but not 1000) mg/kg/day, and there was a reduced red blood cell count at 50 (but not 200 or 1000) mg/kg/day. Red blood cell distribution width (a measure of variation in red blood cell size) was significantly increased at 50, 200, and 1000 mg/kg/day, but there was no change in mean corpuscular volume (the size of the red blood cell) at any dose, and there was no evidence of extramedullary haematopoiesis in the livers or spleens (expected if there were decreases in circulating erythrocytes) of females (or males) at 1000 mg/kg/day. Due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters are considered incidental, due to biologic variation, and not treatment related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The F0 males’ sodium level was significantly lower at 200 mg/kg/day, and chloride was significantly higher at 200 and 1000 mg/kg/day. Clinical chemistry values for F0 females were unaffected, except alanine aminotransferase was significantly increased at 1000 mg/kg/day.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, paired testes weight, relative to terminal body weight, was significantly increased at 1000 mg/kg/day; however, organ weights, relative to terminal brain weights, were unaffected across groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic findings.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of F1 offspring toxicity at any dose, either pre- or postnatally, with no effects on offspring survival, anogenital distance, sex ratio (% males) per litter, or body weights per litter. The one exception is reduced body weight per litter at 1000 mg/kg/day on PND 4 for males only, with no effects on body weights per litter of females or all pups combined.

Key result

Dose descriptor:

NOAEL

Remarks:

for F0 generation

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

for F1 generation

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at the maximum dose tested

Dose descriptor:

NOAEL

Remarks:

for risk assessments

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight and body weight gain reductions lower than 10 %

Key result

Critical effects observed:

no

Body Weights/Body Weight Changes - Males (g)

Dose (mg/kg/day)	0	50	200	1000
Body Weights
Day 14
Mean	419.2	411.2	401.9	393.7**
S.D.	6.1	6.6	5.1	5.0
N	10	10	10	10
Day 21
Mean	439.2	431.0	417.7	412.6*
S.D.	6.8	8.6	5.6	5.8
N	10	10	10	10
Body Weight Changes
Days 7 to 14
Mean	36.2	31.1	28.2	20.6**
S.D.	2.5	2.7	1.8	4.4
N	10	10	10	10
Days 0 to 28
Mean	125.9	117.1	104.1	97.4*
S.D.	6.1	9.6	5.2	4.8
N	10	10	10	10
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

 

 

Body Weights and Body Weight Changes – Females (g)

Dose (mg/kg/day)	0	50	200	1000
Body Weights (Prebreed)
Day 14
Mean	257.4	257.8	247.3	239.4*
S.D.	4.1	6.4	4.8	4.4
N	10	10	10	10
Body Weight Changes (prebreed)
Days 0-14
Mean	25.4	23.5	16.4	10.1*
S.D.	2.1	5.0	3.1	4.0
N	10	10	10	10
Body Weights (Gestation)
GD 7
Mean	297.4	297.2	275.0*	273.7*
S.D.	3.1	7.6	7.1	5.0
N	10	9	7	10
GD 14
Mean	333.4	334.6	307.3*	307.1*
S.D.	3.5	8.8	9.9	4.4
N	10	9	7	10
Body Weights (Lactation)
Lactation day 0
Mean	320.3	317.3	297.9	297.1*
S.D.	5.5	5.8	8.8	5.9
N	10	9	7	10
Lactation day 4
Mean	334.5	339.5	316.3	309.1*
S.D.	4.9	9.0	9.5	5.8
N	10	9	7	10
* Significantly different from the control group at 0.05 using Dunnett’s test

 

Food Consumption – Females

Group (mg/kg/day)	0	50	200	1000
Prebreed
Days 0-7
Mean	21.1	21.4	18.9*	19.1*
S.D.	0.5	0.4	0.7	0.6
N	10	10	10	10
Gestation
GD 0 to 7
Mean	23.6	25.7	21.4	21.0*
S.D.	0.6	0.7	1.1	0.7
N	10	9	7	10
GD 7 to 14
Mean	25.3	27.5	23.8	21.9*
S.D.	0.7	1.0	1.2	0.8
N	10	9	7	10
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Conclusions:

Under the conditions of this study, the NOAEL for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.

Executive summary:

The toxicity of the test material was assessed in a combined repeated dose and reproduction / developmental screening study conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day in 0.5 % aqueous methyl cellulose.

Ten/animals/sex/dose were treated for two weeks pre-breeding (males and females), two weeks of mating (males and females), and three weeks of gestation and lactation each (females). Five additional males per group from the control and 1000 mg/kg/day groups were designated as recovery animals and held without dosing for two weeks after the male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high dose group.

Body weights and feed consumption were recorded at least weekly during the pre-breed period for both sexes and for females during gestation and lactation; clinical signs were recorded at least once daily. A Functional Observational Battery (FOB) was performed on all animals at least once per week during pre-breed, mating (both sexes), gestation, and lactation (females) treatment periods. Five F0 males and five females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy. F0 males were necropsied after the mating period with complete histologic evaluation of five males in the 0 and 1000 mg/kg/day groups. On the day of birth, F0 females were necropsied with complete histologic evaluation of five females in the 0 and 1000 mg/kg/day groups. In addition, haematology, clinical biochemistry, and urinalysis (males only) assays were performed at necropsy for five randomly selected parental F0 males and females per dose group.

No parental animals died during the test. No test substance-related clinical observations were noted throughout the study. In F0 males, a statistically significant decrease in body weight was found at the highest dose on study day 14 (p<0.01) and study day 21 (p<0.05); however, there was not a statistically significant difference in body weight between the controls and the high dose on study day 28. Similarly while the body weight change in F0 males was statistically significant between the control and 1000 mg/kg bw/d dose group from day 7 to day 14 and from day 0 to day 28, there was not a statistically significant difference from day 0 to 7, 14 to 21, or from day 21 to 28. In addition, no statistically significant effects in body weight or body weight change were observed between controls and 1000 mg/kg bw/d dose groups used for recovery animals at any point of the study. Alterations in haematology parameters were observed during the study, but due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters were considered incidental, due to biologic variation, and not treatment related. There were no significant alterations in clinical chemistry parameters. At necropsy, paired testes weight, relative to terminal body weight, was significantly increased at 1000 mg/kg bw/d in parental males; however, organ weights, relative to terminal brain weights, were unaffected across parental groups. All other absolute organ weights and organ weights relative to terminal body weight were not affected by treatment. There were no treatment-related gross or histopathological findings in parental animals and no neurobehavioral alterations or significant effects on reproductive success at any dose.

There was no evidence of F1 offspring toxicity at any dose.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

(screen)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sixty virgin male and 50 virgin female Crl:CD®(SD) rats were received from the supplier on May 14, 2001. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 63 days old upon receipt and 10-11 weeks old at initiation of test article administration. The range of F0 male body weights at the start of the prebreed exposure period was 315.0 to 361.7 g. The range for F0 females was 213.2 to 248.5 g. During the one-week acclimation the animals were examined by a veterinarian, weighed, and representative animals were subjected to faecal examination and serum viral antibody analysis. In addition, one rat per sex was selected as a sentinel which remained in the study room under the identical conditions of the study animals and was sacrificed and subjected to faecal examination and serum viral antibody analysis and the end of the study. All evaluations were negative and the animals assigned to study were deemed healthy and appropriate for use.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt, all F0 parental test animals were housed individually, except during the mating period when they were house two per cage, in clean, solid-bottom polycarbonate cages with stainless-steel wire lids. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The animal room was on a 12-hour light cycle with a temperature range of 68.0 to 73.6 °F and a relative humidity range of 45.8 to 66.6 % throughout the study. Certified rodent chow and city tap water were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose (CAS No. 9004-67-5; 4000 centipoises, Fisher Scientific)

Details on exposure:

DMBPC was weighed into colour-coded glass beakers for each concentration. The formulations were not corrected for the purity of the test substance. Approximately 400 mL of vehicle was added to 2-litre calibrated beakers to a 1-litre mark. With the vehicle in the 2-litre beakers being stirred on magnetic stir plates, DMBPC was slowly added and the suspensions were stirred for at least 15 minutes. Additional vehicle was added to the calibration line. All test article formulations were then sonicated for 30 minutes, then stirred at least 5 minutes, allowed to rest for 2.5 hours, then stirred for an additional time (30 minutes to 2.5 hours), depending on the formulation, prior to analytical and archival sampling.

Prior to and during the study, dose formulations encompassing the range of dose concentrations employed in the study (2 and 200 mg/mL) were found to be homogeneous, stable for four hours under room temperature (to simulate daily dosing periods), and stable for at least 32 days in amber bottles under refrigerated conditions (approximately 4 °C). Based on these stability results, formulations were used within the stability limits established and were stored under refrigeration. Dose formulations were originally analysed as 91.3 - 110 % of nominal concentrations, although an error was subsequently detected after the formulations were presented to the animals, which indicated that the top dose formulated on June 28, 2001, was not 91.3 % of nominal but 86.7 % of nominal. The use of this formulation was unavoidable and had no effect on the study. No DMBPC was detected in the vehicle control formulations, with an estimated limit of detection of 0.4 mg/mL.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC (CAS No. 2362-14-3) orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each dose formulation prepared for use on study were analysed by single injection using high performance liquid chromatography (HPLC) with a Model 680 Automated Gradient Controller (Waters Corporation), a Model 510 pump and a Model 710B WISP autosampler. The column was a Zorbax SB-C18 4.6 x 50 mm; 3.5 µm (Agilent), and the guard column was a Zorbax SB-C18 (Agilent) 4.6 x 12.5mm; 5 µm. The detector was a Model 757 absorbance detector (Applied Biosystems). The regression equation for the vehicle standard data was calculated, and the data were plotted for a visual evaluation of linearity. The concentration of DMBPC was calculated in the dose formulation samples (mg/mL) from the peak area ratio for each sample and the linear regression equation.

Standards for acceptable accuracy of formulations were the mean of the analysed samples was within ± 10 % of nominal, and the % RSD (Relative Standard Deviation) for triplicate samples did not exceed 10 %. If one or more of these standards were not met, the dosing formulations were not administered to the animals until the problem was resolved by analysis of the archived sample of the dosing formulations and/or after reformulation and reanalysis. All dose formulations used in the study had analytical values of 91.4 to 110 % of target concentrations with one dose of 86.7 %. Vehicle control formulations contained no DMBPC, with an estimated detection limit of 0.4 mg/mL.

Prior to and during the performance of this study, aliquots of doses, encompassing the range of concentrations that were to be used in the study (2 and 200 mg/mL), were used to assess stability of the dose suspensions. Triplicate one-mL aliquots were collected from the top, middle, and bottom of the prepared concentrations and analysed for homogeneity by HPLC.

For stability studies, the doses mixed for the low and high homogeneity study were removed from the refrigerator on the day of sampling (3, 9-10, 21, and 32 days) and brought to ambient conditions. The formulations were stirred for two hours prior to sampling. Triplicate 1-g aliquots were removed and analysed by high performance liquid chromatography. Dose formulations at 2 and 200 mg/mL were stable for 32 days when stored in the refrigerator.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group after 14 days of treatment. All animals were randomly selected for pairing, and remained together for 14 days with no change in mating partners. Positive evidence of mating was confirmed by the presence of a vaginal sperm or copulatory plug. Females were examined daily during cohabitation for the presence of sperm or copulation plug in the vaginal tract. The day when evidence of mating was identified was termed gestation day (GD) 0. On GD 0 the mating pairs were separated and the male and female were individually housed.

Duration of treatment / exposure:

Females received 14 doses prior to pairing (one dose per day) and were dosed through lactation day 3 (the day prior to necropsy).

Female rats of the F0 generation) were administered DMBPC orally by gavage at 0, 50, 200 or 1000 mg/kg/day at a dose volume of 5 mL/kg/day in 0.5 % aqueous methyl cellulose, ten/animals/sex/dose, for two weeks of prebreed exposure, during two weeks of mating, and three weeks of gestation and lactation each (females).

Frequency of treatment:

Once daily

Duration of test:

Through lactation

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50 virgin females were evaluated in this study, 10/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a range-finding study, employing doses of 0, 50, 200, and 1000 mg/kg/day, administered by oral gavage at 5 mL/kg for 14 days.

Maternal examinations:

Observations and examinations: Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the time of onset and degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behaviour pattern.

The body weights were determined and recorded initially and weekly until confirmation of mating. During gestation, F0 females were weighed on gestational day (GD) 0, 7, 14, and 20. Dams producing litters were weighed on PND 0 and 4, and body weight gains were computed.

Feed consumption measurements were recorded weekly for all F0 parental study animals during the prebreed treatment period. During pregnancy of F0 females, feed consumption was recorded for GD 0-7, 7-14, and 14-20. During lactation of the F1 litters, maternal feed consumption was measured for PND 0-4. Feed consumption was not measured during the cohabitation period, since two adult animals (breeding pair) were in the same cage. Feed consumption collection periods corresponded to the collection of the animals' body weight data.

A Functional Observational Battery (FOB), including home cage observations, handling observations, and open field observations, was performed on all animals at least once per week during prebreed, mating (both sexes), gestation and lactation (females) treatment periods.
Five F0 males and five females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy.
In addition, haematology and clinical biochemistry assays were performed at necropsy for five randomly selected parental F0 females per dose group.

- Haematology Parameters Examined: Red blood cell (nucleated RBC), Red cell distribution width, RBC Morphology, Mean platelet volume, White blood cell (Total/differential), Mean corpuscular volume, Mean corpuscular haemoglobin, Platelet counts, Mean corpuscular haemoglobin concentration , Haemoglobin concentration and Haematocrit.
- Clinical Chemistry Parameters Examined: Albumin, Creatinine, Aspartate aminotransferase, Glucose, Alanine aminotransferase, Total protein and sodium, Urea nitrogen, Potassium, Total cholesterol and Chloride.

- Postmortem examinations: A complete necropsy was conducted on all F0 animals at termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. The number of nidation scars and corpora lutea were recorded for all F0 females. The fixed (buffered neutral 10 % formalin) uteri from any females of the F0 generation failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy. This staining procedure did not interfere with subsequent histopathologic evaluation. The ovaries and uterus (with cervix and vagina) were weighed and retained for all F0 animals. The liver, heart, kidneys, adrenals, brain, spleen and thymus were weighed and retained from 5 selected animals/group. In addition, the spinal cord, thyroid, stomach, urinary bladder, sciatic nerve, femur, small and large intestines (including Peyer’s patches), trachea and lungs, lymph nodes and all gross lesions were retained from 5 animals/group. Full histopathology of the retained organs was performed on five high does and control F0 animals.

Ovaries and uterine content:

The number of nidation scars and corpora lutea were recorded for all F0 females. The fixed (buffered neutral 10 % formalin) uteri from any females of the F0 generation failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy.

Fetal examinations:

Beginning on GD 20, each female was observed twice daily (a.m. and p.m.) for evidence of littering. On the day of birth (PND 0), anogenital distance was measured and body weight recorded for all live F1 pups in all litters. Body weight was recorded for all live pups on PND 4 prior to euthanasia. Any female which did not show evidence of successful mating after 14 days of cohabitation was weighed weekly, and treatment continued until GD 26 or delivery occurred. If a female without a confirmed GD 0 date was, in fact, pregnant and delivered a litter, her lactational information was collected as described below.

All F1 pups were sexed and examined as soon as possible after birth (PND 0) to determine the number of viable and stillborn from each litter. Thereafter, litters were evaluated for survival through PND 4. Individual anogenital distance and body weight were recorded on PND 0 for all F1 offspring. All live pups were counted, sexed, weighed individually, and examined grossly at birth and on PND 4. The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified. All pups were examined for physical abnormalities at birth and on PND 4. All pups dying during the early lactation period were necropsied, when possible, to investigate the cause of death. On the day of birth (PND 0), anogenital distance was measured and body weights recorded for all live F1 pups in all litters. F1 litters were weighed and euthanised on PND 4, with all pups examined externally and viscerally after euthanasia.

Statistics:

Frequency data such as reproductive indices (e.g., mating and fertility indices) were not transformed. All indices were analysed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, then a Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between each treatment group and the control group. Anogenital distance was analysed by Analysis of Covariance (ANCOVA; in addition to ANOVA analysis) using body weight at measurement as the covariate. FOB data measured in this study are based on relatively small numbers of animals per group, and the outcomes are likely sparse, skewed, or heavily tied. Therefore, exact versions of standard asymptotic tests were used whenever possible.

Indices:

The following indices were calculated: Male Mating Index (%); Male Fertility Index (%); Male Pregnancy Index (%); Female Mating Index (%); Female Fertility Index (%); Female Gestational Index (%); Live birth index (%); Day 4 Survival Index (%, survival between PND 0 and 4); and Mean Live Litter Size.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animals died during the test.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 female body weight for SD 14 and body weight changes for SD 0-14 were significantly reduced at 1000 mg/kg/day. F0 maternal body weights during gestation were significantly reduced at 200 and 1000 mg/kg/day for gestation day (GD) 7 and 14, whereas body weight change showed no effect of treatment among groups. During lactation, F0 maternal body weights were significantly reduced on post natal day (PND) 0 and 4 at 1000 mg/kg/day. Maternal body weight change was not affected during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption expressed as g/day was significantly reduced for the F0 females at 200 and 1000 mg/kg/day for SD 0-7. When expressed as g/kg/day, feed consumption was significantly reduced at 200 mg/kg/day for SD 0-7. During gestation, maternal feed consumption was significantly reduced when expressed as g/day for GD 0-7 and GD 7-14 at 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haemoglobin was significantly reduced at 50, 200, and 1000 mg/kg/day, the haematocrit was reduced at 50 and 200 (but not 1000) mg/kg/day, and there was a reduced red blood cell count at 50 (but not 200 or 1000) mg/kg/day. Red blood cell distribution width (a measure of variation in red blood cell size) was significantly increased at 50, 200, and 1000 mg/kg/day, but there was no change in mean corpuscular volume (the size of the red blood cell) at any dose, and there was no evidence of extramedullary haematopoiesis in the livers or spleens (expected if there were decreases in circulating erythrocytes) of females at 1000 mg/kg/day. Due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters are considered incidental, due to biologic variation, and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry values for F0 females were unaffected, except alanine aminotransferase was significantly increased at 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic findings.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

for risk assessments

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect on offspring survival

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect on sex ratio (% males) per litter

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect on body weights per litter. Reduced body weight per litter at 1000 mg/kg/day on PND 4 was observed for males only. No effects on body weights per litter of female pups or both male and female pups combined were noted.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect on offspring survival

Details on embryotoxic / teratogenic effects:

There was no evidence of F1 offspring toxicity at any dose, either pre- or postnatally, with no effects on offspring survival, anogenital distance, sex ratio (% males) per litter, or body weights per litter. Reduced body weight per litter at 1000 mg/kg/day on PND 4 was observed for males only. No effects on body weights per litter of female pups or both male and female pups combined were noted.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the NOAEL for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.

Executive summary:

The toxicity of the test material was assessed in a combined repeated dose and reproduction / developmental screening study conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day in 0.5 % aqueous methyl cellulose.

Ten/animals/sex/dose were treated for two weeks pre-breeding (males and females), two weeks of mating (males and females), and three weeks of gestation and lactation each (females).

Body weights and feed consumption were recorded at least weekly during the pre-breed period for both sexes and for females during gestation and lactation; clinical signs were recorded at least once daily. A Functional Observational Battery (FOB) was performed on all animals at least once per week during pre-breed, mating (both sexes), gestation, and lactation (females) treatment periods. F0 females were necropsied on lactation day 4 with complete histologic evaluation of five females in the 0 and 1000 mg/kg/day groups. In addition, haematology and clinical biochemistry assays were performed at necropsy for five randomly selected parental animals per dose group.

No parental animals died during the test. Adult F0 parental toxicity was observed at 1000 mg/kg bw/d, as evidenced by reduced body weight and food consumption. There were no treatment-related gross or histopathologic findings in parental animals and no neurobehavioral alterations or significant effects on reproductive success at any dose. Reproductive indices, including Male Mating Index (%), Male Fertility Index (%), Male Pregnancy Index (%), Female Mating Index (%), Female Fertility Index (%), and Female Gestational Index (%), did not differ from controls. Litter parameters, including Live Birth Index (%), Day 4 Survival Index (%, survival between PND 0 and 4), and Mean Live Litter Size, did not differ from controls. There was no evidence of F1 offspring toxicity at any dose.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

neurotoxicity: oral

Remarks:

other: Combined repeated dose toxicity study (including functional observational battery) with reproductive / developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

This OECD 422 study includes most of the end points that are covered in an OECD 424 study (Neurotoxicity Study in Rodents), including body weight and food consumption, detailed clinical observations, functional observational battery, haematology and clinical biochemistry, and histopathology. However, an ophthalmology examination was not performed and the histopathological examination of neurological tissues was not as extensive as for an OECD 424 study (i.e., only brain [cerebrum, cerebellum and pons], spinal cord, and sciatic nerve were examined histologically in the OECD 422 study).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty virgin male and 50 virgin female Crl:CD®(SD) rats were received from the supplier on May 14, 2001. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 63 days old upon receipt and 10-11 weeks old at initiation of test article administration. The range of F0 male body weights at the start of the prebreed exposure period was 315.0 to 361.7 g. The range for F0 females was 213.2 to 248.5 g. During the one-week acclimation the animals were examined by a veterinarian, weighed, and representative animals were subjected to faecal examination and serum viral antibody analysis. In addition, one rat per sex was selected as a sentinel which remained in the study room under the identical conditions of the study animals and was sacrificed and subjected to faecal examination and serum viral antibody analysis and the end of the study. All evaluations were negative and the animals assigned to study were deemed healthy and appropriate for use.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt, all F0 parental test animals were housed individually, except during the mating period when they were house two per cage, in clean, solid-bottom polycarbonate cages with stainless-steel wire lids. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The animal room was on a 12-hour light cycle with a temperature range of 68.0 to 73.6 °F and a relative humidity range of 45.8 to 66.6 % throughout the study. Certified rodent chow and city tap water were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose (CAS No. 9004-67-5; 4000 centipoises, Fisher Scientific)

Details on exposure:

DMBPC was weighed into colour-coded glass beakers for each concentration. The formulations were not corrected for the purity of the test substance. Approximately 400 mL of vehicle was added to 2-litre calibrated beakers to a 1-litre mark. With the vehicle in the 2-litre beakers being stirred on magnetic stir plates, DMBPC was slowly added and the suspensions were stirred for at least 15 minutes. Additional vehicle was added to the calibration line. All test article formulations were then sonicated for 30 minutes, then stirred at least 5 minutes, allowed to rest for 2.5 hours, then stirred for an additional time (30 minutes to 2.5 hours), depending on the formulation, prior to analytical and archival sampling.

Prior to and during the study, dose formulations encompassing the range of dose concentrations employed in the study (2 and 200 mg/mL) were found to be homogeneous, stable for four hours under room temperature (to simulate daily dosing periods), and stable for at least 32 days in amber bottles under refrigerated conditions (approximately 4 °C). Based on these stability results, formulations were used within the stability limits established and were stored under refrigeration. Dose formulations were originally analysed as 91.3 - 110 % of nominal concentrations, although an error was subsequently detected after the formulations were presented to the animals, which indicated that the top dose formulated on June 28, 2001, was not 91.3 % of nominal but 86.7 % of nominal. The use of this formulation was unavoidable and had no effect on the study. No DMBPC was detected in the vehicle control formulations, with an estimated limit of detection of 0.4 mg/mL.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC (CAS No. 2362-14-3) orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each dose formulation prepared for use on study were analysed by single injection using high performance liquid chromatography (HPLC) with a Model 680 Automated Gradient Controller (Waters Corporation), a Model 510 pump and a Model 710B WISP autosampler. The column was a Zorbax SB-C18 4.6 x 50 mm; 3.5 µm (Agilent), and the guard column was a Zorbax SB-C18 (Agilent) 4.6 x 12.5mm; 5 µm. The detector was a Model 757 absorbance detector (Applied Biosystems). The regression equation for the vehicle standard data was calculated, and the data were plotted for a visual evaluation of linearity. The concentration of DMBPC was calculated in the dose formulation samples (mg/mL) from the peak area ratio for each sample and the linear regression equation.

Standards for acceptable accuracy of formulations were the mean of the analysed samples was within ± 10 % of nominal, and the % RSD (Relative Standard Deviation) for triplicate samples did not exceed 10 %. If one or more of these standards were not met, the dosing formulations were not administered to the animals until the problem was resolved by analysis of the archived sample of the dosing formulations and/or after reformulation and reanalysis. All dose formulations used in the study had analytical values of 91.4 to 110 % of target concentrations with one dose of 86.7 %. Vehicle control formulations contained no DMBPC, with an estimated detection limit of 0.4 mg/mL.

Prior to and during the performance of this study, aliquots of doses, encompassing the range of concentrations that were to be used in the study (2 and 200 mg/mL), were used to assess stability of the dose suspensions. Triplicate one-mL aliquots were collected from the top, middle, and bottom of the prepared concentrations and analysed for homogeneity by HPLC.

For stability studies, the doses mixed for the low and high homogeneity study were removed from the refrigerator on the day of sampling (3, 9-10, 21, and 32 days) and brought to ambient conditions. The formulations were stirred for two hours prior to sampling. Triplicate 1-g aliquots were removed and analysed by high performance liquid chromatography. Dose formulations at 2 and 200 mg/mL were stable for 32 days when stored in the refrigerator.

Duration of treatment / exposure:

Males: Two weeks prior to mating and during the two week mating period (for a total of 28 doses)
Females: Two weeks prior to mating, during the two week mating period, through three weeks of gestation and through lactation day 3 (the day prior to necropsy)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose. Five additional males were assigned to each of the 0 and 1000 mg/kg/day groups and were designated as recovery males.

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a range-finding study, employing doses of 0, 50, 200 and 1000 mg/kg/day, administered by oral gavage at 5 mL/kg for 14 days. There was no effect of treatment in either sex on any parameter examined. Therefore, the doses chosen for this study were 0, 50, 200 and 1000 mg/kg/day. Animals were assigned to the different groups by means of randomisation stratified by body weight, such that the body weights of all groups by sex were homogeneous at study initiation. All animals assigned to each dosage level were exposed to their respective dose concentrations once daily by gavage from the first day of pre-breed to the day prior to necropsy, seven days per week, throughout the study.

Observations and clinical examinations performed and frequency:

Observations and examinations: Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the time of onset and degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behaviour pattern.

The body weights of the F0 male rats were determined and recorded initially and weekly through mating. The body weights of F0 female rats were recorded in the same manner until confirmation of mating. During gestation, F0 females were weighed on gestational day (GD) 0, 7, 14, and 20. Dams producing litters were weighed on post natal day (PND) 0 and 4, and body weight gains were computed.

Feed consumption measurements were recorded weekly for all F0 parental study animals during the pre-breed treatment period. During pregnancy of F0 females, feed consumption was recorded for GD 0-7, 7-14, and 14-20. During lactation of the F1 litters, maternal feed consumption was measured for PND 0-4. Feed consumption was not measured during the cohabitation period, since two adult animals (breeding pair) were in the same cage. Feed consumption collection periods corresponded to the collection of the animals' body weight data.

Once during quarantine, and weekly thereafter until necropsy, all parental animals were evaluated with the Functional Observational Battery (FOB), including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: Not examined
CHOLINESTERASE ACTIVITY: Not examined

Neurobehavioural examinations performed and frequency:

Functional Observational Battery (FOB), including home cage observations, handling observations, and open field observations, was performed on all animals at least once per week during pre-breed, mating (both sexes), gestation, and lactation (females) treatment periods.

Grip Strength (forelimb and hindlimb) was performed on week 4 (F0 males) and during pre-breed and PND 4-6 (F0 females).

Motor Activity and Auditory Startle examinations were performed on week 4 (F0 males) and during pre-breed and PND 4-6 (F0 females).

Sacrifice and (histo)pathology:

All adult animals were euthanised by CO₂ asphyxiation. F1 pups on PND 4 were sacrificed by decapitation. Records were kept documenting the fate of all animals received for the study.

Postmortem examinations (Parental animals): A complete necropsy was conducted on all F0 animals, including recovery males, at termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. The ovaries, uterus (with cervix and vagina) prostate, epididymides, testes and seminal vesicles (with coagulating glands and their fluids) were weighed and retained for all F0 animals. The liver, heart, kidneys, adrenals, brain (including cerebrum, cerebellum, and pons), spleen and thymus were weighed and retained from 5 selected male and female animals/group. In addition, the spinal cord, thyroid, stomach, urinary bladder, sciatic nerve, femur, small and large intestines (including Peyer’s patches), trachea and lungs, lymph nodes and all gross lesions were retained from 5 F0 adult males and females/group including all recovery males. Full histopathology of the retained organs was performed on five high does and control F0 animals.

Postmortem examinations (Offspring): All F1 pups were examined externally for gross abnormalities, euthanised on PND 4 and examined viscerally.

Other examinations:

Prior to necropsy, five parental males per group were housed in metabolism cages overnight and urine was collected. The urine samples were transferred to the clinical chemistry laboratory where the presence of glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite and WBCs in urine were evaluated by visual observation of colour changes on Multistix® 10 SG Reagent Strips.

Five males and five females per group were fasted overnight prior to necropsy. At necropsy, blood was collected via the tail vein for haematology and clinical biochemistry. Clinical chemistry assays for albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), total cholesterol, creatinine, glucose, and total protein were performed on a Baker Instruments chemistry analyser equipped with Pipettor 2000™ automatic micro-sample robotic pipettor. All assays were performed by standard UV or colorimetric methods. Analysis for the electrolytes sodium, potassium, and chloride was performed on a NOVA Biomedical CRT 5+ electrolyte analyser utilising ion-selective electrode methodology. Haematological analyses were determined on a Serono-Baker Diagnostics System 9010™ haematology analyser, an automated ten-parameter cell counter equipped with variable thresholds to accommodate veterinary samples. The parameters measured included RBC morphology, red blood cell (RBC), white blood cell (WBC), and platelet (PLT) counts; haemoglobin concentration (HGB); haematocrit (HCT); red cell distribution width (RDW); mean platelet volume (MPV); and the red blood cell indices mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).

Statistics:

FOB data measured in this study are based on relatively small numbers of animals per group, and the outcomes are likely sparse, skewed, or heavily tied. Therefore, exact versions of standard asymptotic tests were used whenever possible.

In general, the unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using either parametric ANOVA under the standard assumptions or robust regression method. The homogeneity of variance assumption was examined via Levene's Test. If Levene's Test indicated lack of homogeneity of variance (p<0.05), robust regression methods were used to test all treatment effects. The presence of linear trends was analysed by GLM procedures for homogenous data or by robust regression methods for non-homogenous data. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS 6.12 was used to evaluate the overall effect of treatment and when a significant treatment effect was present, to compare each exposed group to control via Dunnett's Test. For the litter-derived percentage data (e.g., PND 0-4 pup survival indices), the ANOVA was weighted according to litter size. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for parental and pup body weights, parental organ weight parameters and feed consumption, percent F1 males per litter, and F1 anogenital distance. For any endpoints which involved only two groups (e.g., for F0 recovery males, etc.), Student’s t-test was used for the two-group comparisons.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight (males & females) at 1000 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females only at 1000 mg/kg/day

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

NTE and cholinesterase activity were not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):Reproductive indices for the F0 generation, including Male Mating Index (%), Male Fertility Index (%), Male Pregnancy Index (%), Female Mating Index (%), Female Fertility Index (%), and Female Gestational Index (%), did not differ from controls. Litter parameters, including Live Birth Index (%), Day 4 Survival Index (%, survival between PND 0 and 4), and Mean Live Litter Size, did not differ from controls.

There was no evidence of F1 offspring toxicity at any dose, either pre- or postnatally, with no effects on offspring survival, anogenital distance, sex ratio (% males) per litter, or body weights per litter. Reduced body weight per litter at 1000 mg/kg/day on PND 4 was observed for males only. No effects on body weights per litter of female pups or both male and female pups combined were noted. (migrated information)

Details on results:

Mortality: No parental animals died during the test.

Body weight and weight gain: At 1000 mg/kg/day, F0 male body weights were significantly reduced on study day (SD) 14 and 21 and male body weight changes were significantly reduced for SD 7 to 14 and SD 0 to 28. Absolute body weights for recovery males at 1000 mg/kg/day remained lower throughout the two week recovery period; however, body weight gain was comparable to the recovery control males. F0 female body weight for SD 14 and body weight changes for SD 0-14 were significantly reduced at 1000 mg/kg/day. F0 maternal body weights during gestation were significantly reduced at 200 and 1000 mg/kg/day for gestation day (GD) 7 and 14, whereas body weight change showed no effect of treatment among groups. During lactation, F0 maternal body weights were significantly reduced on post natal day (PND) 0 and 4 at 1000 mg/kg/day. Maternal body weight change was not affected during lactation.

Food consumption: Feed consumption expressed as g/day was significantly reduced for the F0 females at 200 and 1000 mg/kg/day for SD 0-7. When expressed as g/kg/day, feed consumption was significantly reduced at 200 mg/kg/day for SD 0-7. During gestation, maternal feed consumption was significantly reduced when expressed as g/day for GD 0-7 and GD 7-14 at 1000 mg/kg/day.

Organ weights: At necropsy, paired testes weight, relative to terminal body weight, was significantly increased at 1000 mg/kg/day; however, organ weights, relative to terminal brain weights, were unaffected across groups.

Clinical chemistry: The F0 males’ sodium level was significantly lower at 200 mg/kg/day, and chloride was significantly higher at 200 and 1000 mg/kg/day. Clinical chemistry values for F0 females were unaffected, except alanine aminotransferase was significantly increased at 1000 mg/kg/day.

Haematology: Haemoglobin was significantly reduced at 50, 200, and 1000 mg/kg/day, the haematocrit was reduced at 50 and 200 (but not 1000) mg/kg/day, and there was a reduced red blood cell count at 50 (but not 200 or 1000) mg/kg/day. Red blood cell distribution width (a measure of variation in red blood cell size) was significantly increased at 50, 200, and 1000 mg/kg/day, but there was no change in mean corpuscular volume (the size of the red blood cell) at any dose, and there was no evidence of extramedullary haematopoiesis in the livers or spleens (expected if there were decreases in circulating erythrocytes) of females (or males) at 1000 mg/kg/day. Due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters are considered incidental, due to biologic variation, and not treatment related.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced body weight (males and females) and food consumption (females only) at 1000 mg/kg/day

Remarks on result:

other: Generation: other: - F0 (migrated information)

Dose descriptor:

NOAEL

Remarks:

for risk assessments

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight and body weight gain reductions lower than 10 %

Remarks on result:

other: Generation: maternal (migrated information)

Conclusions:

Under the conditions of this study, the NOAEL for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day. There were no significant neurobehavioural / neurotoxicological alterations observed following oral exposure of F0 parents to DMBPC up to doses of 1000 mg/kg bw/day.

Executive summary:

The toxicity of the test material was assessed in a combined repeated dose and reproduction / developmental screening study conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day in 0.5 % aqueous methyl cellulose.

Ten/animals/sex/dose were treated for two weeks pre-breeding (males and females), two weeks of mating (males and females), and three weeks of gestation and lactation each (females). Five additional males per group from the control and 1000 mg/kg/day groups were designated as recovery animals and held without dosing for two weeks after the male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high dose group.

Body weights and feed consumption were recorded at least weekly during the pre-breed period for both sexes and for females during gestation and lactation; clinical signs were recorded at least once daily. A Functional Observational Battery (FOB) was performed on all animals at least once per week during pre-breed, mating (both sexes), gestation, and lactation (females) treatment periods. Five F0 males and five females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy. F0 males were necropsied after the mating period with complete histologic evaluation of five males in the 0 and 1000 mg/kg/day groups. F0 females were necropsied on lactation day 4 with complete histologic evaluation of five females in the 0 and 1000 mg/kg/day groups. In addition, haematology, clinical biochemistry, and urinalysis (males only) assays were performed at necropsy for five randomly selected parental F0 males and females per dose group.

No parental animals died during the test. No test substance-related clinical observations were noted throughout the study. In F0 males, a statistically significant decrease in body weight was found at the highest dose on study day 14 (p<0.01) and study day 21 (p<0.05); however, there was not a statistically significant difference in body weight between the controls and the high dose on study day 28. Similarly while the body weight change in F0 males was statistically significant between the control and 1000 mg/kg bw/d dose group from day 7 to day 14 and from day 0 to day 28, there was not a statistically significant difference from day 0 to 7, 14 to 21, or from day 21 to 28. In addition, no statistically significant effects in body weight or body weight change were observed between controls and 1000 mg/kg bw/d dose groups used for recovery animals at any point of the study. Alterations in haematology parameters were observed during the study, but due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters were considered incidental, due to biologic variation, and not treatment related. There were no significant alterations in clinical chemistry parameters. At necropsy, paired testes weight, relative to terminal body weight, was significantly increased at 1000 mg/kg bw/d in parental males; however, organ weights, relative to terminal brain weights, were unaffected across parental groups. All other absolute organ weights and organ weights relative to terminal body weight were not affected by treatment. There were no treatment-related gross or histopathological findings in parental animals and no neurobehavioral alterations or significant effects on reproductive success at any dose.

There was no evidence of F1 offspring toxicity at any dose.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day. There were no significant neurobehavioural / neurotoxicological alterations observed following oral exposure of F0 parents to DMBPC up to doses of 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-cyclohexylidenedi-o-cresol

EC Number:

219-110-7

EC Name:

4,4'-cyclohexylidenedi-o-cresol

Cas Number:

2362-14-3

Molecular formula:

C20-H24-O2

IUPAC Name:

4-[1-(4-hydroxy-3-methylphenyl)cyclohexyl]-2-methylphenol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Dimethyl cyclohexyl bisphenol (DMBPC; CAS No. 2362-14-3)
- Synonyms: Dimethyl bisphenolcyclohexane; 4,4’-cyclohexylidene di-o-cresol; 1,1-Bis(4-hydroxy-3-ethyl)cyclohexane; Bis-OC-Z
- Appearance: fine white powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty virgin male and 50 virgin female Crl:CD®(SD) rats were received from the supplier on May 14, 2001. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 63 days old upon receipt and 10-11 weeks old at initiation of test article administration. The range of F0 male body weights at the start of the prebreed exposure period was 315.0 to 361.7 g. The range for F0 females was 213.2 to 248.5 g. During the one-week acclimation the animals were examined by a veterinarian, weighed, and representative animals were subjected to faecal examination and serum viral antibody analysis. In addition, one rat per sex was selected as a sentinel which remained in the study room under the identical conditions of the study animals and was sacrificed and subjected to faecal examination and serum viral antibody analysis and the end of the study. All evaluations were negative and the animals assigned to study were deemed healthy and appropriate for use.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt, all F0 parental test animals were housed individually, except during the mating period when they were house two per cage, in clean, solid-bottom polycarbonate cages with stainless-steel wire lids. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The animal room was on a 12-hour light cycle with a temperature range of 68.0 to 73.6 °F and a relative humidity range of 45.8 to 66.6 % throughout the study. Certified rodent chow and city tap water were available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose (CAS No. 9004-67-5; 4000 centipoises, Fisher Scientific)

Details on exposure:

DMBPC was weighed into colour-coded glass beakers for each concentration. The formulations were not corrected for the purity of the test substance. Approximately 400 mL of vehicle was added to 2-litre calibrated beakers to a 1-litre mark. With the vehicle in the 2-litre beakers being stirred on magnetic stir plates, DMBPC was slowly added and the suspensions were stirred for at least 15 minutes. Additional vehicle was added to the calibration line. All test article formulations were then sonicated for 30 minutes, then stirred at least 5 minutes, allowed to rest for 2.5 hours, then stirred for an additional time (30 minutes to 2.5 hours), depending on the formulation, prior to analytical and archival sampling.

Prior to and during the study, dose formulations encompassing the range of dose concentrations employed in the study (2 and 200 mg/mL) were found to be homogeneous, stable for four hours under room temperature (to simulate daily dosing periods), and stable for at least 32 days in amber bottles under refrigerated conditions (approximately 4 °C). Based on these stability results, formulations were used within the stability limits established and were stored under refrigeration. Dose formulations were originally analysed as 91.3 - 110 % of nominal concentrations, although an error was subsequently detected after the formulations were presented to the animals, which indicated that the top dose formulated on June 28, 2001, was not 91.3 % of nominal but 86.7 % of nominal. The use of this formulation was unavoidable and had no effect on the study. No DMBPC was detected in the vehicle control formulations, with an estimated limit of detection of 0.4 mg/mL.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC (CAS No. 2362-14-3) orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group after 14 days of treatment. All animals were randomly selected for pairing, and remained together for 14 days with no change in mating partners. Positive evidence of mating was confirmed by the presence of a vaginal sperm or copulatory plug. Females were examined daily during cohabitation for the presence of sperm or copulation plug in the vaginal tract. The day when evidence of mating was identified was termed gestation day (GD) 0. On GD 0 the mating pairs were separated and the male and female were individually housed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each dose formulation prepared for use on study were analysed by single injection using high performance liquid chromatography (HPLC) with a Model 680 Automated Gradient Controller (Waters Corporation), a Model 510 pump and a Model 710B WISP autosampler. The column was a Zorbax SB-C18 4.6 x 50 mm; 3.5 µm (Agilent), and the guard column was a Zorbax SB-C18 (Agilent) 4.6 x 12.5mm; 5 µm. The detector was a Model 757 absorbance detector (Applied Biosystems). The regression equation for the vehicle standard data was calculated, and the data were plotted for a visual evaluation of linearity. The concentration of DMBPC was calculated in the dose formulation samples (mg/mL) from the peak area ratio for each sample and the linear regression equation.

Standards for acceptable accuracy of formulations were the mean of the analysed samples was within ± 10 % of nominal, and the % RSD (Relative Standard Deviation) for triplicate samples did not exceed 10 %. If one or more of these standards were not met, the dosing formulations were not administered to the animals until the problem was resolved by analysis of the archived sample of the dosing formulations and/or after reformulation and reanalysis. All dose formulations used in the study had analytical values of 91.4 to 110 % of target concentrations with one dose of 86.7 %. Vehicle control formulations contained no DMBPC, with an estimated detection limit of 0.4 mg/mL.

Prior to and during the performance of this study, aliquots of doses, encompassing the range of concentrations that were to be used in the study (2 and 200 mg/mL), were used to assess stability of the dose suspensions. Triplicate one-mL aliquots were collected from the top, middle, and bottom of the prepared concentrations and analysed for homogeneity by HPLC.

For stability studies, the doses mixed for the low and high homogeneity study were removed from the refrigerator on the day of sampling (3, 9-10, 21, and 32 days) and brought to ambient conditions. The formulations were stirred for two hours prior to sampling. Triplicate 1-g aliquots were removed and analysed by high performance liquid chromatography. Dose formulations at 2 and 200 mg/mL were stable for 32 days when stored in the refrigerator.

Duration of treatment / exposure:

Males: Two weeks prior to mating, during the two week mating period (for a total of 28 doses)
Females: Two weeks prior to mating, during the two week mating period and through lactation day 3 (the day prior to necropsy)

Frequency of treatment:

once daily

Details on study schedule:

- Details on study schedule: Males received a total of 14 or 15 doses prior to mating (one dose per day). Males were dosed throughout the mating period through the day prior to euthanasia for a total of 28 doses. Females received 14 doses prior to pairing (one dose per day) and were dosed through lactation day 3 (the day prior to necropsy). After the two-week prebreed exposure period, animals were randomly mated within treatment groups for a two-week mating period to produce the F1 generation, with exposure continuing. Five additional males per group in the control and high dose (1000 mg/kg/day) groups were retained as recovery animals for two weeks after the end of the dosing period to evaluate recovery from any possible treatment-related effects identified in the high dose group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/group; 5 additional males in the control and high concentration (recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a range-finding study, employing doses of 0, 50, 200, and 1000 mg/kg/day, administered by oral gavage at 5 mL/kg for 14 days.

Examinations

Parental animals: Observations and examinations:

Observations and examinations: Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the time of onset and degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behaviour pattern.

The body weights of the F0 male rats were determined and recorded initially and weekly through mating. The body weights of F0 female rats were recorded in the same manner until confirmation of mating. During gestation, F0 females were weighed on gestational day (GD) 0, 7, 14, and 20. Dams producing litters were weighed on PND 0 and 4, and body weight gains were computed.

Feed consumption measurements were recorded weekly for all F0 parental study animals during the prebreed treatment period. During pregnancy of F0 females, feed consumption was recorded for GD 0-7, 7-14, and 14-20. During lactation of the F1 litters, maternal feed consumption was measured for PND 0-4. Feed consumption was not measured during the cohabitation period, since two adult animals (breeding pair) were in the same cage. Feed consumption collection periods corresponded to the collection of the animals' body weight data.

A Functional Observational Battery (FOB), including home cage observations, handling observations, and open field observations, was performed on all animals at least once per week during prebreed, mating (both sexes), gestation and lactation (females) treatment periods.
Five F0 males and five females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy.
In addition, haematology, clinical biochemistry, and urinalysis (males only) assays were performed at necropsy for five randomly selected parental F0 males and females per dose group.
- Haematology Parameters Examined: Red blood cell (nucleated RBC), Red cell distribution width, RBC Morphology, Mean platelet volume, White blood cell (Total/differential), Mean corpuscular volume, Mean corpuscular haemoglobin, Platelet counts, Mean corpuscular haemoglobin concentration , Haemoglobin concentration and Haematocrit.
- Clinical Chemistry Parameters Examined: Albumin, Creatinine, Aspartate aminotransferase, Glucose, Alanine aminotransferase, Total protein and sodium, Urea nitrogen, Potassium, Total cholesterol and Chloride.

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

Parameters examined in P males: The epididymides, testes and seminal vesicles (with coagulating glands and their fluids) were weighed and retained for all F0 animals.

Litter observations:

Beginning on GD 20, each female was observed twice daily (a.m. and p.m.) for evidence of littering. On the day of birth (PND 0), anogenital distance was measured and body weight recorded for all live F1 pups in all litters. Body weight was recorded for all live pups on PND 4 prior to euthanasia. Any female which did not show evidence of successful mating after 14 days of cohabitation was weighed weekly, and treatment continued until GD 26 or delivery occurred. If a female without a confirmed GD 0 date was, in fact, pregnant and delivered a litter, her lactational information was collected as described below.

All F1 pups were sexed and examined as soon as possible after birth (PND 0) to determine the number of viable and stillborn from each litter. Thereafter, litters were evaluated for survival through PND 4. Individual anogenital distance and body weight were recorded on PND 0 for all F1 offspring. All live pups were counted, sexed, weighed individually, and examined grossly at birth and on PND 4. The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified. All pups were examined for physical abnormalities at birth and on PND 4. All pups dying during the early lactation period were necropsied, when possible, to investigate the cause of death.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all F0 animals, including recovery males, at termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. The number of nidation scars and corpora lutea were recorded for all F0 females. The fixed (buffered neutral 10 % formalin) uteri from any females of the F0 generation failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy. This staining procedure did not interfere with subsequent histopathologic evaluation. The ovaries, uterus (with cervix and vagina), prostate, epididymides, testes and seminal vesicles (with coagulating glands and their fluids) were weighed and retained for all F0 animals. The liver, heart, kidneys, adrenals, brain, spleen and thymus were weighed and retained from 5 selected male and female animals/group. In addition, the spinal cord, thyroid, stomach, urinary bladder, sciatic nerve, femur, small and large intestines (including Peyer’s patches), trachea and lungs, lymph nodes and all gross lesions were retained from 5 F0 adult males and females/group including all recovery males. Full histopathology of the retained organs was performed on five high dose and control F0 animals.

Postmortem examinations (offspring):

All F1 pups were examined externally for gross abnormalities, euthanised on PND 4 and examined viscerally.

Statistics:

The unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using either parametric ANOVA under the standard assumptions or robust regression method. The homogeneity of variance assumption was examined via Levene's Test. If Levene's Test indicated lack of homogeneity of variance (p<0.05), robust regression methods were used to test all treatment effects. The presence of linear trends was analysed by GLM procedures for homogenous data or by robust regression methods for nonhomogenous data. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS 6.12 was used to evaluate the overall effect of treatment and when a significant treatment effect was present, to compare each exposed group to control via Dunnett's Test. For the litter-derived percentage data (e.g., PND 0-4 pup survival indices), the ANOVA was weighted according to litter size. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for parental and pup body weights, parental organ weight parameters and feed consumption, percent F1 males per litter, and F1 anogenital distance. For any endpoints which involved only two groups (e.g., for F0 recovery males, etc.), Student’s t-test was used for the two-group comparisons.

Reproductive indices:

The following reproductive indices were calculated: Male Mating Index (%); Male Fertility Index (%); Male Pregnancy Index (%); Female Mating Index (%); Female Fertility Index (%): and Female Gestational Index (%).

Offspring viability indices:

The following litter parameters were calculated: Live birth index (%); Day 4 Survival Index (%, survival between PND 0 and 4); and Mean Live Litter Size.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animals died during the test.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, F0 male body weights were significantly reduced on study day (SD) 14 and 21 and male body weight changes were significantly reduced for SD 7 to 14 and SD 0 to 28. Absolute body weights for recovery males at 1000 mg/kg/day remained lower throughout the two week recovery period; however, body weight gain was comparable to the recovery control males. F0 female body weight for SD 14 and body weight changes for SD 0-14 were significantly reduced at 1000 mg/kg/day. F0 maternal body weights during gestation were significantly reduced at 200 and 1000 mg/kg/day for gestation day (GD) 7 and 14, whereas body weight change showed no effect of treatment among groups. During lactation, F0 maternal body weights were significantly reduced on post natal day (PND) 0 and 4 at 1000 mg/kg/day. Maternal body weight change was not affected during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption expressed as g/day was significantly reduced for the F0 females at 200 and 1000 mg/kg/day for SD 0-7. When expressed as g/kg/day, feed consumption was significantly reduced at 200 mg/kg/day for SD 0-7. During gestation, maternal feed consumption was significantly reduced when expressed as g/day for GD 0-7 and GD 7-14 at 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haemoglobin was significantly reduced at 50, 200, and 1000 mg/kg/day, the haematocrit was reduced at 50 and 200 (but not 1000) mg/kg/day, and there was a reduced red blood cell count at 50 (but not 200 or 1000) mg/kg/day. Red blood cell distribution width (a measure of variation in red blood cell size) was significantly increased at 50, 200, and 1000 mg/kg/day, but there was no change in mean corpuscular volume (the size of the red blood cell) at any dose, and there was no evidence of extramedullary haematopoiesis in the livers or spleens (expected if there were decreases in circulating erythrocytes) of females (or males) at 1000 mg/kg/day. Due to the lack of dose-related, meaningful, or consistent findings in the haematologic assessments in either sex, the observed changes in these parameters are considered incidental, due to biologic variation, and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The F0 males’ sodium level was significantly lower at 200 mg/kg/day, and chloride was significantly higher at 200 and 1000 mg/kg/day. Clinical chemistry values for F0 females were unaffected, except alanine aminotransferase was significantly increased at 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related gross or histopathologic findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced body weight (males and females) and food consumption (females only) at 1000 mg/kg/day

2

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity for risk assessments)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight and body weight gain reductions lower than 10 %

3

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect on offspring survival.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect on body weights per litter. Reduced body weight per litter at 1000 mg/kg/day on PND 4 was observed for males only. No effects on body weights per litter of female pups or both male and female pups combined were noted.

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect on anogenital distance.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross or histopathologic findings.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross or histopathologic findings.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

There was no evidence of F1 offspring toxicity at any dose, either pre- or postnatally, with no effects on offspring survival, anogenital distance, sex ratio (% males) per litter, or body weights per litter. Reduced body weight per litter at 1000 mg/kg/day on PND 4 was observed for males only. No effects on body weights per litter of female pups or both male and female pups combined were noted.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at the maximum dose tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Body Weights/Body Weight Changes - Males (g)

Dose (mg/kg/day)	0	50	200	1000
Body Weights
Day 14
Mean	419.2	411.2	401.9	393.7**
S.D.	6.1	6.6	5.1	5.0
N	10	10	10	10
Day 21
Mean	439.2	431.0	417.7	412.6*
S.D.	6.8	8.6	5.6	5.8
N	10	10	10	10
Body Weight Changes
Days 7 to 14
Mean	36.2	31.1	28.2	20.6**
S.D.	2.5	2.7	1.8	4.4
N	10	10	10	10
Days 0 to 28
Mean	125.9	117.1	104.1	97.4*
S.D.	6.1	9.6	5.2	4.8
N	10	10	10	10
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

 

Body Weights and Body Weight Changes – Females (g)

Dose (mg/kg/day)	0	50	200	1000
Body Weights (Prebreed)
Day 14
Mean	257.4	257.8	247.3	239.4*
S.D.	4.1	6.4	4.8	4.4
N	10	10	10	10
Body Weight Changes (prebreed)
Days 0-14
Mean	25.4	23.5	16.4	10.1*
S.D.	2.1	5.0	3.1	4.0
N	10	10	10	10
Body Weights (Gestation)
GD 7
Mean	297.4	297.2	275.0*	273.7*
S.D.	3.1	7.6	7.1	5.0
N	10	9	7	10
GD 14
Mean	333.4	334.6	307.3*	307.1*
S.D.	3.5	8.8	9.9	4.4
N	10	9	7	10
Body Weights (Lactation)
Lactation day 0
Mean	320.3	317.3	297.9	297.1*
S.D.	5.5	5.8	8.8	5.9
N	10	9	7	10
Lactation day 4
Mean	334.5	339.5	316.3	309.1*
S.D.	4.9	9.0	9.5	5.8
N	10	9	7	10
* Significantly different from the control group at 0.05 using Dunnett’s test

 

Food Consumption – Females

Group (mg/kg/day)	0	50	200	1000
Prebreed
Days 0-7
Mean	21.1	21.4	18.9*	19.1*
S.D.	0.5	0.4	0.7	0.6
N	10	10	10	10
Gestation
GD 0 to 7
Mean	23.6	25.7	21.4	21.0*
S.D.	0.6	0.7	1.1	0.7
N	10	9	7	10
GD 7 to 14
Mean	25.3	27.5	23.8	21.9*
S.D.	0.7	1.0	1.2	0.8
N	10	9	7	10
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.

Executive summary:

The toxicity of the test material was assessed in a combined repeated dose and reproduction / developmental screening study conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

Male and female CD (Sprague-Dawley) rats (the F0 generation) were administered DMBPC orally by gavage at 0, 50, 200, or 1000 mg/kg/day at a dose volume of 5 mL/kg/day in 0.5 % aqueous methyl cellulose.

Ten/animals/sex/dose were treated for two weeks pre-breeding (males and females), two weeks of mating (males and females), and three weeks of gestation and lactation each (females). Five additional males per group from the control and 1000 mg/kg/day groups were designated as recovery animals and held without dosing for two weeks after the male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high dose group.

Body weights and feed consumption were recorded at least weekly during the pre-breed period for both sexes and for females during gestation and lactation; clinical signs were recorded at least once daily. A Functional Observational Battery (FOB) was performed on all animals at least once per week during pre-breed, mating (both sexes), gestation, and lactation (females) treatment periods. F0 males were necropsied after the mating period with complete histologic evaluation of five males in the 0 and 1000 mg/kg/day groups. F0 females were necropsied on lactation day 4 with complete histologic evaluation of five females in the 0 and 1000 mg/kg/day groups. In addition, haematology, clinical biochemistry, and urinalysis (males only) assays were performed at necropsy for five randomly selected parental F0 males and females per dose group.

No parental animals died during the test. Adult F0 parental toxicity was observed at 1000 mg/kg bw/d, as evidenced by reduced body weight (males and females) and food consumption (females only). At necropsy, paired testes weight, relative to terminal body weight, was significantly increased at 1000 mg/kg bw/day in parental males; however, organ weights, relative to terminal brain weights, were unaffected across parental groups. There were no treatment-related gross or histopathologic findings in parental animals and no neurobehavioral alterations or significant effects on reproductive success at any dose. Reproductive indices, including Male Mating Index (%), Male Fertility Index (%), Male Pregnancy Index (%), Female Mating Index (%), Female Fertility Index (%), and Female Gestational Index (%), did not differ from controls. Litter parameters, including Live Birth Index (%), Day 4 Survival Index (%, survival between PND 0 and 4), and Mean Live Litter Size, did not differ from controls. There was no evidence of F1 offspring toxicity at any dose.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic parental toxicity was 200 mg/kg/day; for reproductive and offspring toxicity, the NOAEL was at least 1000 mg/kg/day.