Registration Dossier

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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

From the results presented in the report of the available 28 day study a definitive No Observed Adverse Effect Level (NOAEL) for 2,2-dimethylpropane-1,3-diyl dibenzoate of at least 1000 mg/kg bw/day was established due to the lack of general toxicity and due to the lack of affected male and female reproductive organs.

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

Studies dealing specifically with the toxicity to reproduction (fertility assessment) of 2,2-dimethylpropane-1,3-diyl dibenzoate are not identified. However, there is a repeated dose toxicity study in rats over a period of 28 days available in which rats were dosed up to and including 1000 mg/kg bw/day. A test according to OECD TG 414 (Developmental toxicity: rat, oral) was conducted.

As confirmed by literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007, Dent 2007) histopathological examinations in repeated dose toxicity are of high value and of high sensitivity for evaluation of reproductive toxicity. Therefore, at least for fertility assessment, repeated dose toxicity studies showing no adverse effect should be taken into account. Also it is agreed that histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. Therefore, repeated dose toxicity studies should be considered sufficient information to evaluate toxicity on fertility, if histological examination of the reproductive organs is covered by the repeated dose toxicity study and the mode of action of the test substance does not give evidence for a specific toxicity (as would e.g. sex hormone receptor binding activity).

For 2,2-dimethylpropane-1,3-diyl dibenzoate a 28-day study is available (van Otterdijk 2015). During the 28-day treatment period male and female Wistar rats received 0, 100, 300 or 1000 mg/kg bw/day 2,2-dimethylpropane-1,3-diyl dibenzoate dissolved in polyethylene glycol 400 by gavage. The study was performed according to OECD TG 407 and compiled in 2014. Reproductive organs of the rats of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day (limit dose).

In conclusion, due to the lack of any effects on reproductive organs up to and including the limit dose of 1000 mg/kg bw/day over 28 days, there is no indication of a specific toxic potential to the reproductive organs. Therefore, based on the considerations above, no further testing should be required for fertility assessment.

This is in accordance to Annex IX, Section 8.7.3, column 1 of Commission Regulation (EU) 2015/282 amending Annexes VII, IX and X to Regulation (EC ) No 1907/2006.

A screening for reproductive/developmental toxicity (OECD TG 421 or 422) does not need to be conducted, if a prenatal developmental toxicity is available. A test according to OECD TG 414 (Developmental toxicity: rat, oral) is available to fulfil this requirement.

2,2-Dimethylpropane-1,3-diyl dibenzoate is practically non-toxic following acute oral and dermal application (LD50 > 2000 mg/kg bw), is only slightly irritating to the skin and eyes, does not show sensitizing potential and developed no mutagenic activity in the in-vitro tests (MNT, HPRT and Ames test). A NOAEL of 1000 mg/kg bw/day in male and female rats is the result in a subacute oral study.

A developmental toxicity study (rat, oral) according to OECD TG 414 is available. In the study according to OECD TG 414 twenty-two female rats were administered once daily 100, 300 or 1000 mg/kg bw/day 2-dimethylpropane-1,3-diyl dibenzoate by oral gavage 7 days a week from Day 6 to Day 20 post-coitum. No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane- 1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).

Further testing is not necessary. This is in accordance to ANNEX VIII, Section 8.7.1., column 2 of Regulation (EC) No 1907/2006 (REACH).

References

Mangelsdorf et al. 2003:

Some aspects relating to the evaluation of the effects of chemicals on male fertility. Regulatory toxicology and Pharmacology 36, 69-98.

Ulbrich & Palmer 1995:

Detection of effects on male reproduction - a literature survey. J. Am. College of Toxicology 14, 293-327.

Janer et al. 2007:

A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study. Reproductive Toxicology 24, 103-113.

Dent, 2007:

Strength and limitations of using repeated-dose toxicity studies to predict effects on fertility. Regulatory Toxicology and Pharmacology 48, 241-258.


Effects on developmental toxicity

Description of key information

In a study according to OECD guideline 414 twenty-two female rats were administered once daily doses of 100, 300 or 1000 mg/kg bw/day 2-dimethylpropane-1,3-diyl dibenzoate by oral gavage 7 days a week from Day 6 to Day 20 post-coitum.

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane-1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
In a study according to OECD guideline 414 twenty-two female rats were administered once daily doses of 100, 300 or 1000 mg/kg bw/day 2-dimethylpropane-1,3-diyl dibenzoate by oral gavage 7 days a week from Day 6 to Day 20 post-coitum. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral (including sex) and skeletal malformations and developmental variations.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Additional information:
Test Facility Test Item Number: 05810/B
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Husbandry
Housing
On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding equipped with water bottles. The room in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, sex and animal number.
Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20.9 to 21.3°C with an actual daily mean relative humidity of 43.32 to 51.54%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Food
Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Water
Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 400
Details on exposure:
Vehicle
Identification: Polyethylene glycol 400
Supplier : Merck, Darmstadt, Germany
Specific gravity: 1.125
• Polyethylene glycol Stability for at least 5 hours at room temperature is confirmed over the concentration range 20 to 200 mg/mL, Test Facility Study No. 506379

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility.
Reserve Samples
For each batch (lot) of Test Item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Dose Formulation and Analysis
Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after adding the vehicle to the test item. Formulations were heated to a maximum temperature of 45±5°C for a duration of 25-105 minutes to obtain visual homogeneity (see deviation in Appendix 7). Formulations were released for dosing when they obtained a temperature of 40°C or lower.
Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing, except when the dosing formulations and vehicle were transferred to the animal room. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Formulation and Analysis
Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after adding the vehicle to the test item. Formulations were heated to a maximum temperature of 45±5°C for a duration of 25-105 minutes to obtain visual homogeneity (see deviation in Appendix 7). Formulations were released for dosing when they obtained a temperature of 40°C or lower.
Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing, except when the dosing formulations and vehicle were transferred to the animal room. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Sample Collection and Analysis
Dose formulation samples were collected for analysis.

All samples were stored on dry ice immediately after sampling. The analytical laboratory was notified before shipment of the samples. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70 °C until analysis.
Analytical Method
Analyses were performed by LC-DAD using a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if coefficient of variation (CV) of concentrations was <= 10% for each group.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Additional stability analysis was conducted to determine the stability under the conditions of the deviation. Stability of the prepared formulation was determined at 5 hours at room temperature. Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Details on mating procedure:
Time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating) and were 10 - 14 weeks old and weighed between 164 and 233 g.
Duration of treatment / exposure:
From Day 6 to 20 post-coitum.
Frequency of treatment:
Once daily.
Duration of test:
Scheduled necropsy was conducted on the following days:
Females surviving to planned necropsy: Day 21 post-coitum.
Females with abortion or early delivery: Within 24 hours of abortion or early delivery.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day
Remarks:
2,2-Dimethylpropane-1,3-diyl dibenzoate
Dose / conc.:
300 mg/kg bw/day
Remarks:
2,2-Dimethylpropane-1,3-diyl dibenzoate
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
2,2-Dimethylpropane-1,3-diyl dibenzoate
No. of animals per sex per dose:
22 animals per sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Necropsy – F0-Generation
All animals (including the animal that was found dead) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues (except the uterus) were weighed.
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals found dead).
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
For the animal that was found dead before planned necropsy, these findings were reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

Terminal Procedures – F1-Generation
Fetal Examinations – F1-Generation
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals found dead) was determined.
For late resorptions and recognizable fetuses of females that were found dead, a gross external examination was performed (if possible).
Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar2.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique3. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin.
Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson4.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).
All specimens were archived in glycerin with bronopol as preservative.
Maternal examinations:
Necropsy – F0-Generation
All animals (including the animal that was found dead) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues (except the uterus) were weighed.
Ovaries and uterine content:
Necropsy – F0-Generation
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals found dead).
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
For the animal that was found dead before planned necropsy, these findings were reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.
Fetal examinations:
Terminal Procedures – F1-Generation
Fetal Examinations – F1-Generation
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals found dead) was determined.
For late resorptions and recognizable fetuses of females that were found dead, a gross external examination was performed (if possible).
Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar2.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique3. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin.
Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson4.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).
All specimens were archived in glycerin with bronopol as preservative.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Indices:
Maternal Variables
Body Weight Gains: Calculated between at least each scheduled interval.
Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.

Reproduction and Developmental Variables
For each group, the following calculations were performed:
Pre-implantation loss (%): (number of corpora lutea - number of implantation sites) : (number of corpora lutea) x 100
Post-implantation loss (%) (number of implantation sites - number of live fetuses) : (umber of implantation sites) x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): (number of viable fetuses affected/litter) : (number of viable fetuses/litter) x 100
Historical control data:
Historical control data are available.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and body weight gain corrected for the weight of the gravid uterus of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar between treated and control animals over the study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no test item related macroscopic findings were noted in any of the groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: No treatment-related changes were noted in any of the maternal parameters investigated in this study (e.g. clinical appearance, body weight, food consumption and macroscopic examination).
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no treatment related effect on mean fetal body weights.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size for any group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External malformations and variations were not seen in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg.
Two malformations were revealed at skeletal examination in fetuses of which the dams received test item. Due to single occurrence and/or occurrence in control fetuses only, these malformations were considered to be chance findings.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg bw/day.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No treatment-related changes were noted in any of the developmental parameters investigated in this study (e.g. fetal body weights, external, visceral (including sex) and skeletal malformations and developmental variations).
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane-1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

 Dose group     1     2     3     3
   No.  %  No.  %  No.  %  No.  %
 Females on study     22     22     22     22

 Females that aborted

or delivered

 0  0.0  0   0.0  0   0.0  0   0.0
 Females that died  0   0.0  0   0.0  0   0.0  1  4.5
 Females that aborted  0   0.0  0   0.0  0   0.0  0   0.0
 Nongravid  0   0.0  0   0.0  0   0.0  0   0.0
 Gravid  0   0.0  0   0.0  0   0.0  1  100.0
 Females that were euthanized  0   0.0  0   0.0  0   0.0  0   0.0
 Nongravid  0   0.0  0   0.0  0   0.0  0   0.0
 Gravid  0   0.0  0   0.0  0   0.0  0   0.0

 Females examined at

scheduled necropsy-@

 22  100.0  22  100.0  22  100.0  21  95 .5
 Nongravid  0   0.0  2  9.1  0  0.0  0  0.0
 Gravid  22  100.0  20  90.9  22  100.0  21  100.0
 with resorptions only  0  0  1  5.0  0  0.0  0  0
 with viable fetuses  22  100.0  19  95.0  22  100.0  21  100.0
 Total females gravid  22  100.0  20  90.9  22  100.0  22  100.0

1- 0 MG/KG       2- 100 MG/KG        3- 300 MG/KG        4- 1000 MG/KG

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

                           
 Group  

Sex

 M

Sex 

F

 Viable

fetuses

 Dead

fetuses

Resorptions

 Early

 Resorptions

Late

 Post

implantation

loss

 Implantation

sites

 Corpora

lutea

 Pre

implantation

loss

Fetal

weights

in grams 

 No. of

gravid

females

 1  Total  111  129  240  0  9  0  9  249  264  15  NA  22
   Mean  5.0  5.9  10.9  0.0  0.4  0.0  0.4  11.3  12.0  0.7  5.2  
   S.D.  1.70  1.86  1.48  0.00  1.14  0.00  1.14  1.52  1.45  1.21  0.27  
 2  Total  106  104  210  1  6  0  7  217  231  14  NA  20
   Mean  5.3  5.2  10.5  0.1  0.3  0.0  0.4  10.9  11.6  0.7  5.2  
   S.D.  2.23  2.12  2.84  0.22  0.66  0.00  0.67  2.80  2.42  0.86  0.38  
 3  Total  120  126  246  0  7  0  7  253  261  8  NA  22
  Mean   5.5  5.7  11.2  0.0  0.3  0.0  0.3  11.5  11.9  0.4  5.3  
   S.D.  2.30  2.31  2.32  0.00  0.57  0.00  0.57  2.13  1.61  0.95  0.18  
 4  Total  96  130  226  0  8  0  8  234  241  7  NA  21
   Mean  4.6  6.2  10.8  0.0  0.4  0.0  0.4  11.1  11.5  0.3  5.3  
   S.D.  1.89  1.78  1.45  0.00  0.97  0.00  0.97  1.65  1.81  0.48  0.36  

None significantly different from control group

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

-----------------------------------------------------------------------------------------------------------------------------------

1- 0 MG/KG        2- 100 MG/KG        3- 300 MG/KG        4- 1000 MG/KG

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

Group 

 0 MG/KG

 100 MG/KG

 300 MG/KG

 1000 MG/KG

 Corpora lutea

 

 

 

 Mean

 12.0

 11.6

 11.9

 11.5

 S.D.

 1.45

 2.42

 1.61

 1.81

 N

 22

 20

 22

 21

 Implantation sites

 

 

 

 

 Mean

 11.3

 10.9

 11.5

 11.1

 S.D.

 1.52

 2.80

 2.13

 1.65

 N

 22

 20

 22

 21

 Viable fetuses (%)

 

 

 Mean

 96.8

 92.7

 96.7

 97.1

 S.D.

 8.45

 22.4

 6.29

 7.17

 N

 22

 20

 22

 21

 Dead fetuses (%)

 

 

 

 Mean

 0.0

 5.0

 0.0

 0.0

 S.D.

 0.00

 22.36

 0.00

 0.00

 N

 22

 20

 22

 21

 Early resorptions (%)

 

 

 

 

 Mean

 3.2

 2.4

 3.3

 2.9

 S.D.

 8.45

 5.14

 6.29

 7.17

 N

 22

 20

 22

 21

 Late resorptions (%)

 

 

 

 Mean

 0.0

 0

 0

 0

 S.D.

 0.00

 0.00

 0.00

 0.00

 N

 22

 20

 22

 21

 Total resorptions (%)

 

 

 

 

 Mean

 3.2

 2.4

 3.3

 2.9

 S.D.

 8.45

 5.14

 6.29

 7.17

 N

 22

 20

 22

 21

 Pre-implantation loss (%)

 

 

 

 

 Mean

 5.3

 8.2

 3.6

 2.7

 S.D.

 9.31

 16.64

 10.00

 3.90

 N  22  20  22  21
 Post-implantation loss (%)        
 Mean  3.2  7.4  3.3  2.9
 S.D.  8.45  22.40  6.29  7.17
 N  22  20  22  21
 Males (%)        
 Mean  46.4  50.3  48.4  42.3
 S.D.  14.66  16.25  17.74  15.19
 N  22  19  22  21
 Females (%)        
 Mean  53.6  49.7  51.6  57.7
 S.D.  14.66  16.25  17.74  15.19
 N  22  19  22  21
 Male fetal weights (g)        
 Mean  5.4  5.4  5.5  5.4
 S.D.  0.32  0.39  0.17  0.43
 N  22  19  22  21
 Female fetal weights (g)        
 Mean  5.1  5.0  5.2  5.1
 S.D.  0.28  0.42  0.21  0.31
 N  22  19  22  21
 Combined fetal weights (g)        
 Mean  5.2  5.2  5.3  5.3
 S.D.  0.27  0.38  0.18  0.36
 N  22  19  22  21

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA AND IMPLANTATION SITES COMPARED USING DUNNETT'S TEST

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

            Fetuses           Litters
 Dose group:  1  2  3  4  1  2  3  4
 Number examined externally  240  210  246  226  22  19  22  21
 Number with findings  0  0  0  0  0  0  0  0
 Number examined viscerally  121  108  124  114 22  19  22  21
 Lung abnormal lobation  1  0  0  0  1  0  0  0
 Situs inversus  1  0  0  0  1  0  0  0
 Number examined skeletally  120  102  122  112  22  19  22  21
 Bent limb bone(s)  1  0  0  0  1  0  0  0
 Rib anomaly  1  0  0  0  1  0  0  0
 Vertebral centra anomaly  0  1  0  0  0  1  0  0
 Vertebral anomaly with or without associated rib anomaly  1  0  0  1  1  0  0  1
 Total number with malformations                
 External:  0  0  0  0  0  0  0  0
 Soft tissue:  1  0  0  0  1  0  0  0
 Skeletal:  3  1  0  1  3  1  0  1
 Combined:  3  1  0  1  3  1  0  1

1- 0 MG/KG        2- 100 MG/KG        3- 300 MG/KG        4- 1000 MG/KG

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

 Dose group:  1  2  3  4
 Number of litters examined externally    22  19  22  21
 Number of litters with findings    0  0  0  0
                
 Number of litters examined viscerally    22  19  22  21
 Lung-abnormal lobation  Mean  0.8  0.0  0.0  0.0
 S.D.  3.55  0.00  0.00  0.00
 Situs inversus  Mean  0.8  0.0  0.0  0.0
   S.D.  3.55  0.00  0.00  0.00
                
 Number of litters examined skeletally    22  19  22  21
 Bent limb bone(s)  Mean  0.9  0.0  0.0  0.0
   S.D.  4.26  0.00  0.00  0.00
 Rib anomaly  Mean  0.8  0.0  0.0  0.0
   S.D.  3.55  0.00  0.00  0.00
 Vertebral centra anomaly  Mean  0.0  1.1  0.0  0.0
   S.D.  0.00  4.59  0.00  0.00
 Vertebral anomaly with or without associated rib anomaly  Mean  0.9  0.0  0.0  1.0
   S.D.  4.26  0.00  0.00  4.36
           
 Number of litters examined    22  19  22  21
 Total malformations          
 Percent per litter with external malformations  Mean  0.0  0.0  0.0  0.0
 S.D.  0.00  0.00  0.00  0.00
 Percent per litter with soft tissue malformations  Mean  0.8  0.0  0.0  0.0
   S.D.  3.55  0.00  0.00  0.00
 Percent per litter with skeletal malformations  Mean  2.6  1.1  0.0  1.0
   S.D.  6.66  4.59  0.00  4.36
 Total percent per litter with malformations  Mean  2.4  1.1  0.0  1.0
   S.D.  6.27  4.59  0.00  4.36

1- 0 MG/KG        2- 100 MG/KG        3- 300 MG/KG        4- 1000 MG/KG

SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO.]

            Fetuses           Litters
 Dose group:  1  2  3  4  1  2  3  4
 Number examined externally  240  210  246  226  22  19  22  21
 Number with findings  0  0  0  0  0  0  0  0
                 
 Number examined viscerally  121  108  124  114  22  19  22  21
 Liver - small supernumerary lobe(s)  3  5  5  1  3  5  3  1
                 
 Number examined skeletally  120  102  122  112  22  19  22  21
 14th rudimentary rib(s)  53  55  66  76  19  18  20  21
 14th full rib(s)  5  6  13  10  5  4  9  8
 Pelvic gridle- caudal shift  6  5  13  9  5  4  9  8
 Bent rib(s)  9  8  12  7  8  6  8  4
 Sternebra(e) malaligned (slight or moderate)  12  19  16  9  9  13  11  7
 reduced ossification of the skull  7  13  15  11  5  9  8  7
 7th cervical ossification site(s)  10  6  6  9  6  5  5  5
 Metacarpal(s) and/or metatarsal(s) unossified  4  4  0  1  3  1  0  1
 7th cervical full rib(s)  0  2  0  0  0  2  0  0
 Sternebra(e) #5 and/or #6 unossified  0  1  1  0  0  1  1  0

1- 0 MG/KG        2- 100 MG/KG        3- 300 MG/KG        4- 1000 MG/KG

Conclusions:
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane-1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).
Executive summary:

In a study according to OECD guideline 414 twenty-two female rats were administered once daily doses of 100, 300 or 1000 mg/kg bw/day 2-dimethylpropane-1,3-diyl dibenzoate by oral gavage 7 days a week from Day 6 to Day 20 post-coitum. The following parameters and end points were evaluated in this study for the F0-generation:  mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.  In addition, the following parameters were determined for the F1-generation:  the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral (including sex) and skeletal malformations and developmental variations.

In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels were selected based on the results of the repeated dose 28 day oral toxicity study with 2,2-Dimethylpropane-1,3-diyl dibenzoate by daily gavage in the rat (Test Facility Study No. 506379), and in an attempt to produce graded responses to the test item.  Treatment up to 1000 mg/kg bw/day was well tolerated.  No toxicologically significant changes were noted in any of the parameters investigated in the study.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation:  mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.  In addition, the following parameters were determined for the F1-generation:  the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral (including sex) and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane-1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

Male and female Wistar rats received daily 0, 100, 300 or 1000 mg/kg bw/day dissolved in polyethylene glycol 400 by gavage over a period of 28 days. The examinations were done according to OECD TG 407 under GLP conditions. Considering male and female reproductive organs no adverse effects were reported. Therefore a NOAEL (reproductive organs) at least 1000 mg/kg bw/day was established.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Subacute repeated dose toxicicity study according to respective guideline under GLP conditions.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD TG 407
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: groups of 5 animals per sex
- Diet ad libitum
- Water ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
The test substance formulated in polyethylene glycol 400 and administered daily for 28 days by oral gavage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations preparations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Duration of test:
After 28 days of treatment animals were sacrificed.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance formulated in polyethylene glycol 400 and administered daily for 28 days by oral gavage.
The following parameters were evaluated:
- clinical signs
- functional observation test
- body weight and food consumption
- clinical pathology and macroscopy at termination
- organ weights and histopathology on a selection of tissues as required by the guideline

GROSS PATHOLOGY: Yes
- organ weights:
adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus including cervix, prostate, seminal vesicles, thyroid including parathyroid

HISTOPATHOLOGY: Yes
adrenal glands, aorta, brain, caecum, Cervix, clitoral gland, colon, duodenum, epididymides, eyes, female mammary gland, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland exorbital, liver, lung infused with formalin, lymph nodes mandibular and mesenteric, nasopharynx, oesophgus, ovaries, pancreas, Peyer's patches, pituitary gland, preputal gland, prostate gland, rectum, salivary gland, sciatic nerve, seminal vesicles including coagulating glands, skeletal muscles, skin spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid gland including parathyroid, trachea, urinary bladder, uterus, vagina
Statistics:
Dunnett's test, the Steel test, Fisher Exact test, Kruskal Walis test
Dose descriptor:
other: NOAEL (reproductive organs)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Based on the results from organ weights and macroscopic and microscopic examination
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Referring to the parameters examined. The test item was tolerated up to 1000 mg/kg bw/day without toxicologically relevant effects.
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
Salivation was observed in all females and most males at 1000 mg/kg bw/day and most males at 300 mg/kg bw/day on several days after dosing.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

ORGAN WEIGHTS
Organ weights or organ to body weight ratios of treated animals were similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded that could be attributed to treatment with the test substance.
Conclusions:
From the results presented in the report a definitive No Observed Adverse Effect Level (NOAEL) for 2,2-dimethylpropane-1,3-diyl dibenzoate of at least 1000 mg/kg bw/day was established due to the lack of general toxicity and due to the lack of affected male and female reproductive organs.
Executive summary:

Male and female Wistar rats received daily 0, 100, 300 or 1000 mg/kg bw/day dissolved in polyethylene glycol 400 by gavage over a period of 28 days. The examinations were done according to OECD TG 407 under GLP conditions. Considering male and female reproductive organs no adverse effects were reported. Therefore a NOAEL (reproductive organs) at least 1000 mg/kg bw/day was established.

Overall, from the results presented in the report a definitive No Observed Adverse Effect Level (NOAEL) for 2,2-dimethylpropane-1,3-diyl dibenzoate of at least 1000 mg/kg bw/day was established due to the lack of general toxicity and due to the lack of affected male and female reproductive organs.

Additional information

Male and female Wistar rats received daily 0, 100, 300 or 1000 mg/kg bw/day dissolved in polyethylene glycol 400 by gavage over a period of 28 days. The examinations were done according to OECD TG 407 under GLP conditions. Considering male and female reproductive organs no adverse effects were reported. Therefore a NOAEL (reproductive organs) at least 1000 mg/kg bw/day was established.

Overall, from the results presented in the report of the available 28 day study a definitive No Observed Adverse Effect Level (NOAEL) for 2,2-dimethylpropane-1,3-diyl dibenzoate of at least 1000 mg/kg bw/day was established due to the lack of general toxicity and due to the lack of affected male and female reproductive organs.

Justification for classification or non-classification

In a study according to OECD guideline 407 male and female Wistar rats received daily 0, 100, 300 or 1000 mg/kg bw/day dissolved in polyethylene glycol 400 by gavage over a period of 28 days. Considering male and female reproductive organs no adverse effects were reported. Therefore a NOAEL (reproductive organs) at least 1000 mg/kg bw/day was established.

In a study according to OECD guideline 414 twenty-two female rats were administered once daily doses of 100, 300 or 1000 mg/kg bw/day 2-dimethylpropane-1,3-diyl dibenzoate by oral gavage 7 days a week from Day 6 to Day 20 post-coitum. No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2-Dimethylpropane-1,3-diyl dibenzoate was established as being at least 1000 mg/kg bw/day (limit dose; highest dose tested).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Additional information