2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2019 to 10 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: Preliminary Study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 May 2019 - 16 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Reproductive performance study in the Sprague-Dawley rat by oral gavage administration for dose selection to a full extended-one generation study (F0 + F1)

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles

Limit test:

no

Justification for study design:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Dose levels of 0, 300, 600 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on findings from an OECD408 study in the Sprague-Dawley rat (RTC Study No. A2297) and an OECD 414 study in the Sprague-Dawley rat (Charles River Study No. 497432)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier)l:
Perstorp Holding AB (Sponsor)
Lot/Batch number: 190100256 (exp. 06 Feb 2022)
- Purity: 99.2%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity and stability was confirmed in corn oil formulations at nominal concentrations of 5 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for 5 days and refrigerated storage (2 to 8ºC) for up to 15 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulation frequency: weekly

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance (ex. 90 days repeated dose study) which provide better results-comparison between studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: (F0) 36 males & 36 females (F1) Where possible, at least one male and one female were selected from each selected litter with up to two males and two females selected from each litter if required
- Source: Charles River (UK) Ltd. Kent, UK
-Duration of acclimatization: Six days before commencement of treatment
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (F0) 69-75 (M+F) days, (F1) Selected animals were microchipped on Day 18-20 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age.
- Weight at study initiation: (F0) Males: 322-388 g; Females: 198-251 g
- Fasting period before study: none
- Housing: cages compromised of polycarbonate body with stainless steel mesh lid; solid bottom cages used except during pairing when grid bottom cages were used; softwood based bark-free fiber bedding; number of animals per cage according to standards
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified pelleted diet, no added antibiotic or other chemotherapeutic or prophylactic agent (restricted diet overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): potable water, ad libitum (except during urine collection)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 2 to 8ºC, or transferred immediately to the animal unit, and dispensed daily.

The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Dose levels of 0, 300, 600 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on earlier findings

Amount of vehicle (if gavage): 4 ml/kg

Due to the viscosity of the high dose level (1000 mg/kg/day) problems were encountered when using the flexible cannula for dose administration to the weanling animals, which resulted in protracted dose administration and dosing omissions. During Days 2-7 of the F1 generation a reduced dose level (600 mg/kg/day) was administered to Group 4 animals to ensure that there were no further omissions. From Week 2 of the F1 generation (nominal Day 35 of age) animals in the high dose resumed treatment at 1000 mg/kg/day

Details on mating procedure:

Pairings commenced after a minimum of two weeks of treatment at a 1:1 male:female ratio for up to two weeks. Ejected copulation plugs were checked daily in cage trays and sperm in the vaginal smear. One detected this was considered Day 0 of gestation and the pair was separated. The pre-coital interval was calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

F0 animals- For a minimum of 15 days before pairing until scheduled termination.

F1 animals- From Day 21 of age up to approximately eight weeks of age following
completion of sexual maturation.

Frequency of treatment:

Daily, approx. the same time each day

Details on study schedule:

F1 generation treated from weaning on Day 21 of age up to approximately eight weeks of
age following completion of sexual maturation.

Dose / conc.:

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

Remarks:

Volume: 4 mL/kg/day
75 mg/mL (nominal concentration)

Dose / conc.:

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

Remarks:

Volume: 4 mL/kg/day
150 mg/mL (nominal concentration)

Dose / conc.:

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

Remarks:

*Selected F1 animals during Week 1 of the F1 generation (Days 2-7)
Volume: 4 mL/kg/day
250 mg/mL (nominal concentration)

No. of animals per sex per dose:

F0: 9
F1: 10

Control animals:

yes, concurrent vehicle

Positive control:

No

Parental animals: Observations and examinations:

Body Weight:
Group mean values and SD were calculated from individual body weight data on each recorded occasion. Group mean weight changes were calculated from the weight changes of individual animals. Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.

Food consumption:
During the pre-pairing periods of the study where animals were gang housed, values are presented for the average amount of food consumed per animal in each cage in each experimental period (g/animal/day). During the gestation and lactation phases, where females were individually housed, group mean values and D were calculated from individual values. After Day 14 of lactation, food consumption is increasingly influenced by the offspring and is no longer an accurate reflection of maternal consumption. The column header day numbers represent the days on which the full feeder and empty feeder were recorded.

Oestrous cyclicity (parental animals):

The number and percentage of F0 females showing the following classifications of estrous cycles before pairing are presented:

Regular: All observed cycles of 4 or 5 days
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Postmortem examinations (offspring):

Findings from examination of offspring are presented on an individual basis for affected litters and offspring.

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analyzed at each timepoint separately:
-Body weight, using absolute weights and gains over appropriate study periods
-Food consumption, over appropriate study periods
-Gestation length
-Sexual maturation, age and body weight at completion
-Litter (implantations, litter size, sex ratio, percentage male, post implantation survival index, live birth index and &viability index), before cull study periods

The following comparisons were performed:
-Group 1 vs 2, 3 and 4

Significant differences between the groups compared were expressed at the 5% (p<0.05) or1% (p<0.01) level.

Parametric/Non-Parametric
Tests include Bartlett's test, Willams' test, t-tests, Dunnett's tests, Kruskal-Wallis' tests, Wilcoxon rank sum tests, Cochran-Armitage tests, Chi-square tests, one- and two-tailed linear-by-linear tests

Reproductive indices:

Individual data was tabulated. Group values were calculated for males and females
separately for the following:
Percentage mating (%) = [(Number of animals mating)/(Animals paired)] x 100
Conception rate (%) =[(Number of animals achieving pregnancy)/(Animals paired)] x 100
Fertility index (%) =[(Number of animals achieving pregnancy)/(Animals paired)] x 100
Gestation index (%) = [Number of live litters born)/(Number pregnant)] x 100

Offspring viability indices:

Post implantation survival, live birth, viability, and lactation indices were calculate for F0.

Post implantation survival index (%) = (Total number of offspring born)/(Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.


Live birth index (%) = (Numer of offspring on Day 1 after littering)/(Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling)/(Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering)/(Number of live offspring on Day 4 (after culling)) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Signs:
No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment with Di-Trimethylolpropane.

Macropathology:
Macroscopic examination of males after nine weeks of treatment and females on Day 21 of lactation did not reveal any abnormality that could be related to treatment with Di-Trimethylolpropane

Dermal irritation (if dermal study):

not examined

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body Weight:
For the first two weeks before pairing body weight gain for males receiving Di-Trimethylolpropane were significantly low when compared with Controls; p<0.05 at 300 and 600 mg/kg/day and p<0.01 at 1000 mg/kg/day. Overall (Days 1-64) the group mean bodyweight gain was slightly low for males receiving Di-Trimethylolpropane when compared with Controls; a dose response was evident however the differences did not attain statistical significance. Body weight change for females before pairing and during gestation was unaffected by administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day. During late lactation (LD18-21) females receiving 300 mg/kg/day showed greater mean weight loss than the current Controls (-27g vs -6 g; p<0.01) and the overall body weight gain (LD1-21) for females receiving Di-Trimethylolpropane was low when compared with Controls at all dose levels (p<0.05) however there was no evidence for a dose response.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption:
Before animals were paired for mating, food consumption for male animals receiving Di-Trimethylolpropane showed no adverse effects of treatment, when compared with Controls. Female animals receiving Di-Trimethylolpropane at 300, 600 and 1000 mg/kg/day, however, showed low food consumption during Days 11 to 14 before pairing at approximately 71-76% of Controls. Food consumption for female animals during gestation and lactation were similar to Controls and considered to be unaffected by treatment.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation indices were unaffected by administration of Di-Trimethylolpropane to parental animals.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation indices were unaffected by administration of Di-Trimethylolpropane to parental animals.

No indication of an effect caused by administration of Di-Trimethylolpropane to parental animals.

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical condition of offspring was unaffected by parental treatment with Di-Trimethylolpropane.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Litter size, offspring survival and sex ratio were unaffected by parental treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter:
On Day 1 of age there was no clear effect on the mean body weight for both male and female offspring. Overall body weight gains for offspring derived from the groups that received Di-Trimethylolpropane were statistically significantly high when compared with Controls (p<0.05) and the mean body weight of offspring from the treated groups was statistically significantly high from Day 11 of age (p<0.05).

F1:
Body weight of selected F1 animals on Day 21 of age was significantly high when compared with Controls (p<0.05/0.01) at all dose levels, with no evidence of a dose response. At 1000 mg/kg/day the body weight gain for males from Day 21 to 25 of age was slightly high when compared with Controls (p<0.05); this difference was not apparent for selected F1 female animals. At formal commencement of the F1 generation on Day 28 ±2 days of age, the mean body weight of selected F1 animals from the treated groups was marginally high when compared with Controls, but the differences did not attain statistical significance and a dose response was not apparent. Overall the subsequent body weight gain for selected F1 males receiving Di-Trimethylolpropane up to termination was essentially similar across the groups. Selected F1 females receiving 600 or 1000 mg/kg/day showed slightly high weight gain (p<0.05) resulting in slightly high mean body weight on Day 25 of the F1 generation (p<0.05); these differences were not dose related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for selected F1 animals receiving Di-Trimethylolpropane was slightly high for both males and females at all dose levels, with females showing the greater difference from Controls.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

The age and body weight for animals at attainment of either vaginal opening or balano preputial separation was unaffected by treatment with Di-Trimethylolpropane

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of selected F1 animals did not reveal any findings that could be attributed to treatment.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Examination of selected F1 animals did not reveal any findings that could be attributed to treatment.

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of this preliminary study, and in the absence of any overt toxicity, there is nothing to preclude the use of 1000 mg/kg/day as the high dose in the subsequent OECD443 study; however as 600 mg/kg/day (150 mg/mL administered at 4 ml/kg) was the maximum practicable dose level for weanling animals, selected F1 animals would need to receive this reduced dose level from weaning up to approximately Day 35 of age.

Executive summary:

Oral gavage administration of Di-Trimethylolpropane at dose levels of 300, 600 and 1000 mg/kg/day was generally well tolerated with no effects on clinical condition (F0 and F1), food consumption (F1), reproductive performance (F0), litter size/offspring survival (F1) macropathology (F0 and F1) or sexual maturation (F1). Minor non-adverse effects of treatment were primarily limited to F0 male bodyweight, F0 female lactation bodyweight, F1 offspring bodyweight and F1 selected bodyweight, and a slight effect on F0 female food consumption prior to pairing. Due to the viscosity of the high dose level (1000 mg/kg/day) problems were encountered when using the flexible cannula for dose administration to the weanling animals, which resulted in protracted dose administration and dosing omissions. During Days 2-7 of the F1 generation a reduced dose level (600 mg/kg/day) was administered to Group 4 animals to ensure that there were no further omissions. From Week 2 of the F1 generation (nominal Day 35 of age) animals in the high dose resumed treatment at 1000 mg/kg/day. Based on the results of this preliminary study, and in the absence of any overt toxicity, there is nothing to preclude the use of 1000 mg/kg/day as the high dose in the subsequent OECD443 study; however as 600 mg/kg/day (150 mg/mL administered at 4 ml/kg) was the maximum practicable dose level for weanling animals, selected F1 animals would need to receive this reduced dose level from weaning up to approximately Day 35 of age.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: Based on results derived from a previously performed 90 day toxicity study, a combined repeated dose toxicity screening study and a deveopmental toxicity study
- Exclusion of extension of Cohort 1B: No information indicate the genotoxicity, endocrine disruption related effect of this substance. No information indicate the substance leads to significant exposure or specific metabolism pattern that would require extended exposure. Therefore, extension of cohort 1B to include the F2 generation was not performed.
- Exclusion of developmental neurotoxicity Cohorts 2A/2B and Cohort 3: No indication of the developmental neurotoxicity or immunotoxicity of this substance was seen from existing information. Cohort 2A/2B and cohort 3 are considered not needed at this moment.

- Route of administration
Based on the information provided in the technical dossier and/or in the chemical safety report, ECHA agreed that the oral route - which is the preferred one as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) Chapter R.7a, section R.7.5.4.3 - was the most appropriate route of administration. More specifically, even though the information indicated that human exposure to the registered substance by the inhalation route is likely, the exposure concentrations reported in the chemical safety report for the inhalation route are low. Hence, the test was performed by the oral route.
- Other considerations
The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

EC Number:

245-509-0

EC Name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Cas Number:

23235-61-2

Molecular formula:

C12H26O5

IUPAC Name:

2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Details on test material:

- Name of test material (as cited in study report): DI-TMP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier)l: Perstorp Holding AB (Sponsor)
Lot/Batch number: 190100256 (exp. 06 Feb 2022)
- Purity: 99.2%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity and stability was confirmed in corn oil formulations at nominal concentrations of 5 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for 5 days and refrigerated storage (2 to 8ºC) for up to 15 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulation rfequency: weekly
The test item formulations were ‘gritty’ consistency and the following procedure was implemented as a precautionary measure to reduce the risk of any potential irritation to the mouth and/or oesophagus during gavage administration:
-The dose was drawn up via the catheter and the external surface of the catheter wiped on paper tissue.
-The catheter surface was rinsed in a container of tap water (container 1) and wiped dry on clean paper tissue.
-Then the outside of the catheter was rinsed further in a second container of tap water (container 2) prior to intubation.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance (ex. 90 days repeated dose study) which provide better results-comparison between studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: 27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships
- Source: Charles River (UK) Ltd. Kent, UK
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: (F0) 28-34 days, (F1) 21 days selection; 28 days allocation/formal start
- Weight at study initiation: (F0) Males: 72-132 g; Females: 52-93 g
- Fasting period before study: none
- Housing: cages compromised of polycarbonate body with stainless steel mesh lid; solid bottom cages used except during pairing when grid bottom cages were used; softwood based bark-free fiber bedding; number of animals per cage according to standards
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified pelleted diet, no added antibiotic or other chemotherapeutic or prophylactic agent (restricted diet overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): potable water, ad libitum (except during urine collection)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

In-Life Dates:
16 Oct 2019 to 5 May 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 2 to 8ºC, or transferred immediately to the animal unit, and dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water):TG recomends "where possible, the use of an aqueous solution/suspension is considered first,followed by consideration of a solution/suspension in oil". Corn oil was deemed most appropriate for species selection and substance properties by the Sponsor.
- Concentration in vehicle (nominal): 0, 25, 75, 250 mg/ml (150mg/mL instead of 250 mg/mL day 21 of age to nominal day 35 of age)
- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1/1(sibling pairing was not permitted)
- Length of cohabitation: max. 2 weeks (separation at detection of mating)
- Proof of pregnancy: The day of detection of an ejected copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M/062/19). The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 25 μg/mL to 250 μg/mL.The formulations for Week 1 (F0 generation), Week 1 (F1 generation) and Last Week of F1 generation were sampled. For all groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel. Two samples from all groups were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. The mean concentration were within 10% of the nominal concentration, confirming the accuracy of formulation, with the exception of one formulation (Group 4 in the last week of the F1 generation) that was 10.8% of the nominal concentration. The difference from mean remained within 3%, confirming precise analysis, with the exception of Group 4, Last Week of F1 generation which was ±7.37%. The results for the Group 4 Last Week of F1 generation was not investigated, however the procedural recovery samples prepared at the same concentration as these samples was 99.9% of nominal concentration, confirming accur cy of dilution.

The original analysis for Week 1 (F0 generation) was diluted to below the quantifiable concentration range in error. The samples were rediluted from extracts to bring them within the calibration range but the system suitability failed on that occasion. Therefore, the original results are reported. The individual procedural recovery prepared at the concentration of the samples was 103.3% of nominal concentration, confirming that the calculated concentration of this group is acceptable.

Stability analyses performed previously 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.

Duration of treatment / exposure:

The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)

Frequency of treatment:

Daily, approx. the same time each day

Details on study schedule:

- Age at mating of the mated animals in the study (F0): 14-15 weeks (after 10 weeks dosing)
-The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Remarks:

F1: Day 21 of age to nominal Day 35 of age - dose reduced to the maximum practicable for the dosing equipment required for weanling animals (600 mg/kg/day)

No. of animals per sex per dose:

25 males + 25 females (F0), 20 males + 20 females (F1 cohort 1A), 20 males + 20 females (F1 cohort 1B)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on results derived from a previously performed 90 day toxicity study, a combined repeated dose toxicity screening study, a deveopmental toxicity study, and a preliminary reproductive study. All dose levels of 300, 600 and 1000 mg/kg/day were generally well tolerated with minor non-adverse effects of treatment primarily limited to F0 male bodyweight, F0 female lactation body weight, F1 offspring body weight and F1 selected bodyweight, and a slight effect on F0 female food consumption prior to pairing. Low and intermediate dose levels of 100 and 300 mg/kg/day were selected to provide an approximate 3-fold dose interval. Since 600 mg/kg/day (150 mg/mL administered at 4 ml/kg) was the maximum practicable dose level for weanling animals, selected F1 animals received this reduced dose level from weaning up to approximately Day 35 of age.

- Fasting period before blood sampling for clinical biochemistry:
yes

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination Once each week
After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: :
F0 males Day that treatment commenced.
Each week.
Before necropsy.

F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post partum.
Before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females Weekly, from the day that treatment commenced until paired formating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-20 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (ad libitum)

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
- Dry smears: For 15 days before pairing, using cotton swabs.

- Wet smears: After pairing until evidence of mating confirmed.For four days before scheduled termination (nominally Days 25 to 28 post partum). Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

Sperm parameters (parental animals):

Motility:
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA). F0 Group 2 Male 36 unable to assess 200 sperm at motility analysis.

Sperm morphology: Groups 1 and 4
A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

Sperm morphology: Groups 2 and 3
Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4
The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3
Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4
After removal of the tunica, the left testis of each male was then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3
Samples frozen for possible future assessment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes , litters culled to 10 (where possible 5 males and 5 females), When the total number of pups in a litter on PND 4 was ≤ 10 pups, no litter size adjustment occurred.


PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function.

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on LD 0. The live pups were counted, sexed, weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including PND 4. Additionally, on Day 1 of age, all offspring received a qualitative assessmentof body temperature, state of activity and reaction to handling and the ano-genital distance was mesured.

After being sexed dircetly before and after culling (PND 4), the pups were not sexed again until day 21 of age. From PND 5, the total live pups were counted daily and examined for ill-health, evidence of reaction to treatment again, and weighed on PND 7, 14 and 21. On day 13, male offsprings nipple/aerolae were counted.

- Selection and weaning of F1 animals for Cohort 1A and 1B
Two males and two females were selected from each selected litter and one male and one female from each litter, where available, were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (28 +/-2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

- Assessment of F1 Sexual Maturation (Cohorts 1A and 1B)
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, along with the body weight on that day. For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected. Commencing at PND 38, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, along with the body weight on that day.

In-life procedures. observations and measurements F1 (Cohorts 1A and 1B)
- Mortality/Moribundity Checks
Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

- Detailed Clinical Observations
Animals were subjected to detailed clinical observations weekly from weaning on PND 21, starting on a suitable day within one week of weaning of the majority of litters. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also examined. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as
appropriate.

- Pre and Postdose Observations
Detailed observations were performed to establish and confirm a pattern of signs in
association with dosing according to the following schedule:
Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week.
Detailed observations were recorded at the following times in relation to dose administration:
- Prior to dosing
- One to two hours after completion of dosing
- As late as possible in the working day

- Body weights
Recorded on Days 1, 4, 7, 14 and 21 of age; Selected F1 generation: Days 23, 25, 27* and 29* of age. (* Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days)). Plus before necropsy.


- Food consumption
From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

- Oestrus Cycle Monitoring
Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.
For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

- Haematology (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Activated partial thromboplastin time, Fibrinogen and prothrombin time

- Clinical Chemistry (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides
Sodium, Potassium, Chloride, Sample quality

- Urinalysis (Cohort 1A; 10 rats/sex/group):
Last week of dosing
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood, sodum, potassium, and chloride. microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
-Epithelial cells (Epi)
-Leucocytes (WBC)
- Erythrocytes (RBC)
- Casts
- Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals


- Thyroid stimulating hormone, TSH and T4 (Conditions, blood sample site, and anaesthetic caried based on age)
F1 - culled offspring Ten litters per group - pooled litter sample on Day 4 of age.* Plus ten male and ten female animals per group on Day 22 of age from as many litters as possible)
F1 adults- Cohort 1A - Ten male and 14# female animals per group at termination

Postmortem examinations (parental animals):

SACRIFICE
F0 animals:
- Male animals: After weaning of the F1 animals, after conifmration that no further mating was required
- Maternal animals: Day 28 post partum
- Females failing to mate: Following completion of pairing females terminated after showing an estrus smear
- Method: Carbon dioxide asphyxiation with subsequent exsanguination

GROSS NECROPSY
- F0
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues

ORGAN WEIGHTS
F0:
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), optic nerves, pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.

MICROSCOPIC EVALUATION
F0:
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

Postmortem examinations (offspring):

SACRIFICE
F1 animals:
Unselected offspring On Day 4 and Day 22 of age.
Cohort 1A animals At approximately 13 weeks of age.
Cohort 1B animals At approximately 14 weeks of age.
- Method:
F1 adults: Carbon dioxide asphyxiation with subsequent exsanguination.
F1 offspring 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination
F1 offspring less than 14 days of age: Subcutaneous injection of sodium pentobarbitone.

GROSS NECROPSY
F1 animals:
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues

ORGAN WEIGHTS
- F1 Cohort 1A
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), lymph nodes (mesenteric, left axillary), oovaries (L+R), pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.
- F1 Cohort 1B
epididymides, ovaries (L+R), pituitary, prostate (dorsolateral and ventral combined), seminal vesciles (with coagulation gland), testes, uterus with cervix and oviducts
- Unselected F1 offspring on Day 22 of age
brain (cerebellum, cerebrum, midbrain), spleen, thymus


MICROSCOPIC EVALUATION
- F1 Cohort 1A
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

- F1 Cohort 1B
Abnormalities

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analyzed at each timepoint separately:
-Body weight, using absolute weights and gains over appropriate study periods
-Food consumption, over appropriate study periods
-Hematology
-Blood chemistry
-Urinalysis
-Estrous cycles
-Pre-coital interval
-Gestation length and gestation index
-Stage of estrous cycle at termination
-Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
-Ano-genital distance, adjusted for Day 1 offspring body weight
-Sexual maturation, age and body weight at completion
-Organ weights, absolute and relative to body weight
-Corpora lutea and ovarian primordial follicle count
The following comparisons were performed:
-Group 1 vs 2, 3 and 4
-Group 1 vs 4 for ovarian follicle counts and corpora lutea data

Significant differences between the groups compared were expressed at the 5% (p<0.05) or1% (p<0.01) level.

Parametric/Non-Parametric
Tests include Bartlett's test, Willams' test, t-tests, Dunnett's tests, Kruskal-Wallis' tests, Wilcoxon rank sum tests, Cochran-Armitage tests, Chi-square tests, one- and two-tailed linear-by-linear tests

Reproductive indices:

Post implantation survival, live birth, viability, and lactation indices were calculate for F0.

Post implantation survival index (%) = (Total number of offspring born)/(Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.


Live birth index (%) = (Numer of offspring on Day 1 after littering)/(Total number of offspring born) x 100

Offspring viability indices:

Viability index (%) = (Number of live offspring on Day 4 before culling)/(Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering)/(Number of live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of F0 adult animals was unaffected by treatment. There were no signs at either routine physical examination or in association with dose administration that could be attributed to Di-Trimethylolpropane

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of ten animals were either found dead or were killed for reasons of animal welfare. Of these, five animals had macroscopic findings that were consistent with dosing trauma and as such these deaths were unrelated to administration of Di-Trimethylolpropane (Group 3 male no.53, Group 2 females no. 228 and 229, Group 3 female no. 271 and Group 4 female no. 276). Three Control animals also died prematurely. The remaining two decedent animals (Group 2 male nos 29 and 41) either died or were killed prematurely following convulsions/seizures. No significant macroscopic or microscopic finding were observed that explained the cause of death in these two animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall the bodyweight gain for males during treatment and for females before pairing and during gestation was unaffected by administration of Di-Trimethylolpropane. On Day 1 of lactation the mean body weight for females receiving 1000 mg/kg/day was slightly but statistically significantly low at approximately 95 % of Controls (p<0.05); however the overall body weight gain for females at this dose level was significantly high when compared with Controls (p<0.01). Body weight gain during lactation for females receiving 100 or 300 mg/kg/day was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

From Week 6 of treatment up to pairing males receiving 1000 mg/kg/day showed food consumption values that were slightly but significantly high when compared with Controls (p<0.05 - Week 6; p<0.01 - Weeks 7 to 10); food consumption for males receiving 100 or 300 mg/kg/day was similar to Controls throughout the pre-pairing treatment period. Food consumption for females before pairing, during gestation and lactation was unaffected by administration of Di-Trimethylolpropane.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological examination of males and females at scheduled termination did not reveal any changes that could be attributed to Di-Trimethylolpropane; differences were either minor, lacked a dose response or were inconsistent between the sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Females at 1000 mg/kg/day had high plasma phosphorous levels when compared with Controls (p<0.01). Other differences in the blood chemistry at scheduled termination were minor, lacked a dose response or were inconsistent between the sexes.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The individual TSH concentrations in each group, at all-time points and within sexes, showed high variation. There were no observed treatment related effects on serum TSH concentrations in F0 adult animals or in the F1 offspring. There was a suggestion of low TSH levels for the F1 adult animals that received Di-Trimethylolpropane; however, as stated above, due to the high variation it cannot be stated that this was due to the test article or biological variation.

Mean T4 concentrations for F0 adult animals, F1 offspring and selected F1 adult animals were similar to Controls, showing no effects of treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examination of F0 animals at scheduled termination did not reveal any findings that could be related to administration of Di-Trimethylolpropane up to and including 1000 mg/kg/day. Ten control females, five females treated with Di-Trimethylolpropane at 100 mg/kg/day, two females treated with Di-Trimethylolpropane at 300 mg/kg/day and eight females treated with Di-Trimethylolpropane at 1000 mg/kg/day exhibited minimal to marked mineralization in the aorta, other blood vessels at various locations, kidneys and/or stomach. Based on its anatomical distribution and the absence of necrosis, these histopathological findings were consistent with metastatic mineralization/calcification, likely due to pseudohyperparathyroidism related with pregnancy and lactation. Presence in both females from control and treated groups, metastatic mineralization is considered very unlikely to have compromised evaluation or interpretation of any test item related effects.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Pre-coital interval: The majority of animals mated within four days of pairing and the distribution of outliers between the groups showed no relationship to treatment.

Gestation Length and Gustation Index: The duration of gestation was unaffected by treatment with animals littering within the expected range of 22 to 23.5 days. The gestation index was slightly low at 1000 mg/kg/day but this was solely due to a single female (no.276) that was killed for welfare reasons following dosing trauma during gestation and as such the gestation index is deemed unaffected by treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes in the estrous cycles that could be attributed to Di-Trimethylolpropane.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or morphology following treatment with Di-Trimethylolpropane.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility were unaffected by administration of Di-Trimethylolpropane.

Effect levels (P0)

Key result

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:

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general condition of selected F1 animals was unaffected by treatment. There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Four animals from Cohort 1A died prior to scheduled termination. For the two Controls animals (nos. 404 and 601) macroscopic examination revealed dosing trauma as the cause of death. Two males receiving 300 mg/kg/day (nos. 456 and 458) were found dead on Days 19 and 30 respectively. Terminal signs were limited to no. 456 which was observed with abnormal breathing prior to death however macroscopic examination did not reveal any abnormality. The lungs for male No. 456 showed slight multifocal lymphocytic and mononuclear cell infiltrate in a perivascular and peribronchial distribution; the lung lesions were considered to be the major factor contributing to death. For male No. 458 there were no significant microscopic findings to explain the cause of death. Most tissues examined were considered autolytic and readable. The major factor contributing to death was undetermined.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of selected F1 animals on Day 21 of age and at formal start of the F1 generation on nominal Day 28±2 of age were unaffected by administration of Di-Trimethylolpropane and subsequent gain up to scheduled termination showed no adverse effects of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for both male and female animals did not show any adverse effects of treatment.

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):

Hematological examination of males and females at scheduled termination did not reveal any changes that could be attributed to Di-Trimethylolpropane; differences were either minor, lacked a dose response or were inconsistent between the sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
Females receiving Di-Trimethylolpropane had high plasma potassium levels when compared with Controls (p<0.01); a dose response was not apparent. Other differences in the blood chemistry at scheduled termination were minor, lacked a dose response or were inconsistent between the sexes.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Males and females receiving 1000 mg/kg/day and females receiving 300 mg/kg/day showed low urinary pH and high specific gravity when compared with Controls; specific gravity for females at 100 mg/kg/day was also high. No other differences were apparent.

Sexual maturation:

no effects observed

Description (incidence and severity):

The age and body weight of females on completion of vaginal opening and males on completion of balano-preputial separation were unaffected by administration

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

On Day 1 of age the ano-genital distance for both male and female offspring from the treated groups were similar to Controls and showed no adverse effect of treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On Day 13 of age the distribution of litters with male offspring that showed nipples did not show any relationship to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females that received 300 or 1000 mg/kg/day had slightly but significantly high absolute and high body weight relative thyroid weight (p<005); this was not evident in the male animals. Females at 1000 mg/kg/day also showed low absolute and body weight relative mesenteric lymph node weight (p<0.05); this was not evident in the male animals. No other differences were apparent.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination performed after at least 10 weeks of treatment revealed no test item related lesions.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings following treatment with Di-Trimethylolpropane from weaning to approximately 13 weeks of age (F1A cohort) or from weaning to approximately 14 weeks of age (F1B). All histological changes were considered to be unrelated to treatment and to be consistent with the pathology background observed in Sprague Dawley rats in these laboratories.

Other effects:

no effects observed

Description (incidence and severity):

Ovarian Follicle Cuonts and Corpora Lutea (Cohort 1A):
Ovarian follicle and corporalutea counts were unaffected by administration of Di-Trimethylolpropane.

Sperm Assessment (Cohort 1A):
There were no adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or morphology following treatment with Di-Trimethylolpropane.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Minor differences were observed in the immunophenotyping parameters between groups for both males and females. These differences were not related to the oral gavage administration of Di-Trimethylolpropane as they were within the ranges observed in Control animals.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

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 treatment related effects were seen.

2

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

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 treatment related effects were seen.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

Executive summary:

In an extended one-generation reproductive toxicity study (OECD 443) in rats, including Cohorts 1A and 1B, Di-Trimethylolpropane (Di-TMP) was administered to groups of 25 animals per sex and dose level by gavage at dose levels of 100, 300, or 1000 mg/kg/day (F0) at a volume dose of 4 mL/kg/day. Additionally, Di-TMP was administered to 40 animals per sex and dose level by gavage at dose levels 100, 300, or 1000mg/kg/day (F1, Cohorts 1A and 1B) at a volume dose of 4 mL/kg/day, except from Day 21 of age to nominal Day 35 of age when the high dose was reduced to 600 mg/kg/day which was maximum practicable for the dosing equipment required for weaning animals. A similarly constituted Control group received the vehicle (F0 & F1), corn oil, at the same volume dose as the treated groups throughout the same periods.

 

Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. All F1 animals were then dosed directly from postnatal day (PND) 21 to at least PND 90. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function (Cohorts 1A and 1B). 

 

Throughout the study, 10 animals were either found dead or were killed for reasons of animal welfare, none were deemed treatment-related. Various effects were seen throughout the study, but differences and/or effects were concluded to be non-treatment-related since they were either minor, lacked a dose response or were inconsistent between the sexes.

 

Overall, administration of Di-Trimethylolpropane in F0 was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters or pathology. Estrous cycles, mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment. Regarding F1 litter responses, the general condition of offspring, litter size, offspring survival, offspring development, thyroid hormones and macropathology were unaffected by treatment. Cohorts 1A and 1B showed that the administration of Di-Trimethylolpropane was well tolerated with no treatment-related mortality and no adverse effects on general condition, body weight, food consumption, sexual maturation, estrous cycles, thyroid hormones, clinical pathology, immunophenotyping parameters, organ weights, ovarian follicle counts, sperm parameters or pathology.

 

Ultimately, oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition, the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.

 

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

 

This study is acceptable and satisfies the guideline requirements for an extended one-generation reproductive study (OECD 443) in the rat.