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Effects on fertility

Description of key information
In a vaginal cornification/uterine weight bioassay, DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose (Bioqual Inc 1997, Vel 001-97).
Effect on fertility: via oral route
Dose descriptor:
NOAEL
500 mg/kg bw/day
Additional information

A two generation study in rats was conducted to assess the effects on reproductive performance of the test material DPGDB. The study was conducted according to OECD and EPA test guidelines, and in compliance with GLP (HLS 2001, VCL 316/004080). According to preliminary results obtained in rats in a dose range-finding study (HLS 2006, VCL315/990088), concentrations of DPGDB in diet at 25000 ppm during two weeks before mating, during gestation and lactation gave evidence of toxicity in dams (mortality, clinical signs). This preliminary study suggested that an upper dietary concentration of DPGDB between 7500 and 15000 ppm would be appropriate in a main two generation study of reproductive performance.

 

In the main study, dietary administration of DPGDB at concentrations of 1000, 3300 or 10000 ppm was generally well tolerated by the P (F0) and subsequent F1 parental animals and their respective progeny. Bodyweight change of F1 females before paring and F1 males were slightly but significantly lower than in Controls.

No adverse effects were seen on overall parental food consumption; food conversion efficiency calculated during the 10 week pre-mating phase was considered similar to controls for both generations.

 Oestrous cycle, mating performance, fertility and fecundity were similar in all groups. Gestation lengths and the parturition process were unaffected by treatment. Assessment of the terminal vaginal smears taken from F0 females revealed a higher incidence of females in oestrus in groups treated with DPGDB compared with controls. This finding was not apparent among F1 females and is considered to be of doubtful biological significance.

Litter parameters at birth of the F1 and F2 progeny and their survival to weaning showed no apparent detrimental effects of treatment with DPGDB. However, in both F1 and F2 offspring at 10000ppm there was a slight reduction on weight gain during days 14-21 of age and this finding may be linked to the transition to direct exposure to the test material as the offspring weaned on to solid diet at the same dietary inclusion levels as their parents.

 No treatment related findings were seen at microscopic examination of the F1 offspring not selected to form the next generation or the F2 offspring killed after weaning. Macropathology, histopathology assessment and sperm analysis for the F0 and F1 adults showed no adverse effects of treatment.

The only possible effect of treatment detected at assessment of organ weights from F1 and F2 offspring was significantly lower absolute and relative spleen weight among F2 males and females compared to controls. The toxicological significance if this finding is uncertain since it was not detected among F1 offspring or among F0/F1 adult animals.

The evidence from this study suggested that a dietary concentration of DPGDB at 10000 ppm should be considered as the No-Observed-Effect-Level (NOEL) for F0 and F1 parent animals. The No-Observed-Adverse-Effect-Level (NOAEL) for survival and growth of the offspring is considered to be 10000 ppm (equivalent to a minimum estimated daily achieved dosage of 500 mg/kg/d).


Short description of key information:
In a two generation study, conducted according to OECD and EPA test guidelines, and in compliance with GLP, realised in rats to assess the effects on reproductive performance of the test material DPGDB (HLS 2001, VCL 316/004080), the NOAEL for F0 and F1 parent animals and the NOAEL for survival and growth of the offspring were both considered to be 10000 ppm in diet.

Effects on developmental toxicity

Description of key information
 A Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Importantly, a 10.5% decrease in fetal body weight in the 500 mg/kg/day dosage group reflects the 17% decrease in feed consumption in the dams during the fetal period and therefore this reduced fetal body weight is related to the maternal toxicity that was observed at that dose level
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
500 mg/kg bw/day
Additional information

A pre-natal development study in rats was conducted to determine the effect of the test material DPGDB when administered during and beyond the organogenesis phase of gestation. The study was conducted according to Japanese, US EPA and OECD test guidelines, and in compliance with GLP (HLS 2000, VCL314/993005).

Groups of 22 female rats were selected after mating, and were dosed by oral gavage with corn oil fortified with the test material between day 6 and day 19 of gestation. Dose levels examined were 0 (vehicle control), 250, 500, and 1000 mg/kg bw/day. According to preliminary results obtained in rats in a dose range-finding study (HLS 2000, VCL313/980305), doses up to 1500mg/kg/d during gestation days 6 to 19 gave no adverse effect on dams or foetuses, but maternal toxicity was observed at the highest dose. The highest dose used in the main study was therefore 1000mg/kg/d.

In the rat study, an association between treatment at 1000 and 500 mg/kg bw/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

The increase in cervical ribs at 1000 mg/kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kg bw/day and there was no concomitant change in vertebral configuration.

Salivation after dosing was observed at all dosages of benzoflex, the incidence was dose related but this finding was not considered to be of toxicological importance. At 1000 mg/kg/d, there were no detectable signs of maternal toxicity, there were no maternal deaths and all females had a live litter scarifice. It was concluded that the 1000 mg/kg/d is the NOAEL for maternal toxicity.There were no treatment related effects observed at prenatal survival or growth. At 1000 mg/kg/d, treatment related small but definite increase in the number of fetus with cervical ribs were observed. The no-observed adverse effect level for all aspects of pre-natal development is concluded to be 500 mg/kg bw/day.

Toxicity to reproduction: other studies

Description of key information

A Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. The test substance, dipropyleneglycol dibenzoate (DPGDB), in the vehicle (0.5% carboxymethylcellulose in deionized water) was administered orally by gavage to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from Gestation Days 7–28. Dosage levels were 100, 250, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. No fetal malformations were attributed to the test substance. Other fetal developmental variations occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges, and therefore were not attributed to the test substance.Adverse effects on maternal survival, mean body weight changes, and food consumption were noted in the 500 mg/kg/day group; therefore, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Importantly, a 10.5% decrease in fetal body weight in the 500 mg/kg/day dosage group reflects the 17% decrease in feed consumption in the dams during the fetal period and therefore this reduced fetal body weight is related to the maternal toxicity that was observed at that dose level.

Additional information

A study was performed to investigate whether the test substance, dipropylene glycol dibenzoate, had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay. The study was conducted to GLP but no formal international test guidelines were applicable.

Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control Diethylstilbestrol (DES)) were orally dosed for 7 days. All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.

DES, the positive control, resulted in a dose dependent induction of vaginal cornification. In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/kg/day x 7 days. Hence by this endpoint the test compound showed no estrogenic activity.

DES also induced a dose dependent increase in uterine weight and uterine weight to final body weight ratio. Conversely, DPGDB did not stimulate a uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/ kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.

Justification for classification or non-classification

Based on the usual criteria for classification for developmental toxicity such as treatment related increases in embryofetal mortality with increased resorptions, increase in congenital malformations, decreased fetal bodyweight etc., it is clear that no classification is justified. The only effects observed that require further consideration are the small increase in anomalies, viz. cervical ribs, and a small increase in retarded ossification of the sternebrae. 

 

With respect to retarded ossification, this effect is proceeding at a high rate during the last few days of gestation and a wide range of degree of ossification of fetuses is expected when the fetuses are delivered one or two days before expected parturition. In the control fetuses in the present study incomplete ossification of 5th and/or 6th sternebrae is present in 77/158 (49%) of fetuses. The mid and high dose groups show the same effect in 104/165 (64%) and 104/155 (67%) of fetuses respectively. This is a very small increase in a very common variation which would almost certainly disappear by term and is considered to be of no lasting toxicological significance.

 

With respect to the presence of cervical ribs, this effect was observed in 10/155 (6.5%) fetuses from 6/22 litters in the top dose group. In the mid and low dose groups, 1 fetus from 2 litters were affected in each dose group. The incidence in the top dose group is greater than in the concurrent controls and greater than has been seen in historical control groups. In historical control groups, data are available for 13 studies between 1997-1999, and in 7 of the 13 studies control litters contained fetuses with cervical ribs. The number of affected fetuses per litter ranged from 0-4 per litter. Because of the higher number of affected fetuses in the top dose group in the developmental toxicology study, that also exceed the historical control data from this laboratory, and also above the historical control data for the same strain of rat published by Charles River who showed that in Crl CD SD rats the range for cervical ribs in 2800 control litters was zero to 16.7% of litters (0- 3.7% of fetuses) (Barbeau et al. 2008), this finding is considered to be related to treatment.

 

However, cervical ribs are not regarded as a malformation, but as an anomaly that occurs relatively frequently in rats of this strain. The most common type of this anomaly is reversible, disappearing postnatally, though in some cases can persist postnatally (Chernoff and Rogers, 2004). It is a common response to stress in the dams. The presence of a low incidence as in the present study at toxic dose levels of 1000 mg/kg bw is not normally regarded as any great toxicological significance. There is no evidence reported of gross toxicity in the dams in this study, but examination of the data available from the range-finding study showedthe dose of 1500 mg/kg for 13 days was near to the LD50 for the substance with half the animals becoming moribund within the final two days of exposure. This is further supported by a study quoted in the report of an earlier study in the same strain of female rats dosed with 2000 mg/kg for up to 7 days which resulted in the death of 3 of 8 rats. The onset of toxic signs in these studies did not appear until after several days of intense salivation followed by sudden onset of bodyweight loss and then the animals became moribund within a further two days or so. At PM changes in the consistency of the gut contents was noted as the only finding. Thus, although there were no deaths at 1000 mg/kg after 13 days of dosing in the present study, there were clear signs of marked salivation by that time and no detailed PM examination of gut contents is reported. It is not unreasonable to assume that there was in fact maternal stress at this high dose level.

 

In deciding whether the presence of a small number of minor anomalies at a high dose level of 1000mg/kg bw would qualify for a substance to be classified under the CLP Regulation it is useful to look at the Guidance published by ECHA (ECHA, 2009) in relation to minor effects observed in studies.  In paragraph 3.7.2.4.1., it is stated that “Development of the offspring throughout gestation and during the early postnatal stages can be influenced by toxic effects in the mother either through non-specific mechanisms related to stress and the disruption of maternal homeostasis, or by specific maternally-mediated mechanisms. In the interpretation of the developmental outcome to decide classification for developmental effects it is important to consider the possible influence of maternal toxicity. This is a complex issue because of uncertainties surrounding the relationship between maternal toxicity and developmental outcome. Expert judgement and a weight of evidence approach, using all available studies, shall be used to determine the degree of influence that shall be attributed to maternal toxicity when interpreting the criteria for classification for developmental effects.” In paragraph 3.7.2.4.3., it is further stated that “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity.” 

 

There is considerable discussion in the Guidance in relation to maternal toxicity and it states that even in the presence of considerable maternal toxicity classification (perhaps in Category 2) should be considered where there is “a significant toxic effect in the offspring, e.g. irreversible effects such as structural malformations, embryo/foetal lethality, significant post-natal functional deficiencies.”  

Since no treatment related malformations or embryo-fetal lethality or any other severe irreversible effects were observed in the developmental toxicology study even at the highest dose level of 1000 mg/kg bw, but only a small increase in minor anomalies, it is concluded that a classification for developmental toxicity for dipropylene glycol dibenzoate under the CLP Regulations is not warranted.

A recently conducted Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. No fetal malformations were attributed to the test substance. Other fetal developmental variations occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges, and therefore were not attributed to the test substance.Importantly, decrease in fetal body weight in the 500 mg/kg/day dosage group reflects the decrease in feed consumption in the dams during the fetal period and therefore this reduced fetal body weight is related to the maternal toxicity that was observed at that dose level. There were no severe developmental effects or fetal toxicity was observed at the any dose levels therefore the substance was not classified under GHS classification.