Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Feb 2020 to 22 June 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

Premating exposure duration for parental (P0) animals: 10 weeks (7 days/week)
- Basis for dose level selection: The initial concentrations were selected in consultation with the sponsor and were based on the results of a preliminary dose-range finding study in rats OECDE 421 ( Charles river stdy N° 0213169)

- Inclusion/exclusion of extension of Cohort 1A 1B:, 2 A , 2B, and 3 is triggered, because the substance fulfil the requirements under Annex X column 2.

- Termination time for F2: is applicable since the F2 generation is included in this study.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: These Cohorts are included.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: This Cohort is included.
- Route of administration is oral gavage.
-The oral route of exposure was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate

EC Number:

906-170-0

Molecular formula:

CH3CO2(CH2)nCO2CH3 Where n = 2, 3 and 4

IUPAC Name:

Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate

Test material form:

other: colorless liquid

Details on test material:

See information in the field "Confidential details on test material"

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Lyon, France

Number of F0-Males: 100 (25 animals/group).
Number of F0-Females: 100 (25 animals/group; nulliparous and
nonpregnant).
F0-males and females came from different
zones.
Number of F1-Animals After Allocation into Cohorts: 20 pups/sex/group for Cohorts 1A and 1B and 10 pups/sex/group for Cohorts 2A, 2B and 3.
Number of Positive Control Animals for TDAR Assay: 10/sex (nulliparous and nonpregnant females).
Age at the Initiation of Dosing (F0 Animals): Approximately 6 weeks.
Weight at the Initiation of Dosing (F0 Animals): F0 males: 133.1 g to 221.7 g.
F0 females: 112.9 g to 157.8 g.

Animals husbandry :

Environmental conditions:
- Temperature (°C): 19°C to 25°C
- Humidity (%): > 35%
- Air changes : At least 10 air changes per hour.
Lighting cycle : 12 hours light and 12 hours dark (except during
designated procedures).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis as indicated in the following table:

Dose Formulation Sample Collection Schedule
Occasion Formulation Samples Collected a, b
Day 1 Groups 1 and 3 5 x 500 mg (middle)
Groups 2 and 4 5 x 500 mg (top, middle, bottom)

Week 11 Groups 1 to 4 5 x 500 mg (middle)
Week 18 Groups 1 to 4 5 x 500 mg (middle)
Week 33 Groups 1 to 4 5 x 500 mg (middle)
a: Samples were stored in a freezer set to maintain -20°C.
b: 2 samples for analysis and 3 samples for backup.

All samples to be analyzed were shipped (in dry ice) to the Test Site for analysis.

Duration of treatment / exposure:

F0 animals (both sexes): 10 weeks before mating, throughout the mating period and up to the day before necropsy.
F0 animals (females): During gestation (the first day of gestation was designated as GD0) and at least 21 days after parturition, up to and including the day before scheduled necropsy (the day of birth was designated as LD0).
Apparently unmated females for at least 23 days after the last day of the mating period.

F1 animals: During lactation (up to PND21), pups were not dosed directly but could potentially be exposed to the test item in utero,
via maternal milk, from exposure to maternal urine/feces. After weaning (PND21), F1 animals were dosed up to and including the day before scheduled necropsy. The F1 surplus and Cohort 2B animals were not dosed.

Frequency of treatment:

once per day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

110 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

330 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Cohort 1A (20/Sex/Group)
Cohort 1B (20/Sex/Group)
Cohort 2A (10/Sex/Group)
Cohort 2B (10/Sex/Group)
Cohort 3 (10/Sex/Group)

Control animals:

yes, concurrent vehicle

Details on study design:

Group No. Test Material Dose Level (mg/kg/day) Dose Volume a (mL/kg/day) Dose Concentration (mg/mL)
1 Control 0 5 0
2 Test item 110 5 22
3 Test item 330 5 66
4 Test item 1000 5 200
5b Cyclophosphamide 7.5 5 1.5
a::Based on the most recent body weight measurement.
b: For positive control animals from Cohort 3 only.

The Group 1 animals (control) received the vehicle (PEG 400).

Rationale for the dose selection: Dose levels were based on a simplified OECD 421 study in the Wistar rat (Charles River Study No. 20213169). In this study, 10 rat/sex were dosed at 0, 500 and 1000 mg/kg/day by oral administration (gavage) from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy for females or from 14 days before mating, throughout mating and up to the day before necropsy (i.e., minimum 28 days) for males. There was no sign of systemic parental toxicity or any developmental effect at any dose levels. Doses of 110, 330 and 1000 mg/kg/day were therefore selected to have a ratio of 3 between each dose levels for this OECD 443 study.

Positive control:

Cyclophosphamide monnohydrate for the T-cell Dependent Antibody Response (TDAR) Assay

Examinations

Parental animals: Observations and examinations:

Animals were observed for general health/mortality and moribundity at least twice daily (except on days of receipt and study termination where frequency was at least once daily).
Animals were not removed from cage during observation, unless necessary for confirmation of possible findings.

6.3. Body Weights
Each male was weighed at least weekly.
Each female was weighed as follows:
• At least weekly during pre-mating and mating periods (only pre-mating data are
reported).
• On GD0, 4, 7, 11, 14, 17 and 20.
• On LD1, 4, 7, 10, 14, 17 and 21.
6.4. Food Consumption
Food consumption of males was recorded weekly during the pre-mating period from Day 1.
Food consumption of females was recorded for the following periods:
• Weekly during the pre-mating period from Day 1.
• On GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20.
• On LD1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21.
Food consumption during mating was not recorded for practical reasons.
Clinical Observations
- Cage Side Observations
Cage side observations were performed at least once daily on non-dosing days. During the dosing period, animals were observed before and at least once after dosing. Animals were not
removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Hypersalivation Observations
A specific individual out-of-cage examination was performed at least twice after dosing at least every 3 weeks to detect postdosing hypersalivation and any associated clinical
changes.
- Arena Observations Animals were observed for specific clinical signs in a standard arena once before the first administration of the test item and at least weekly thereafter (except during pairing or
delivering).
- Detailed Clinical Observations
Animals were removed from the cage and a detailed clinical observation was performed in the same time as arena observation.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily and used to determine the stage of estrus for each female from 14 days prior to mating and until identification of mating or separation from the male. On the day of terminal necropsy or for moribund animal, a vaginal lavage was also taken to determine the stage of estrus for each female.

Sperm parameters (parental animals):

Sperm analysis was performed for all surviving males using automated equipment(Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive
motility. Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.

Postmortem examinations (parental animals):

Blood samples were collected acording to tables 9-10-11-12. Urine samples were processed and analyzed for the parameters listed in the table 13

Paired organs were weighed together and if a difference in size was observed, the abnormally sized organs were also weighed separately. The organs were weighed after dissection of fat and other contiguous tissues. Blocks were prepared only for tissues which were evaluated histopathologically. Histopathological examination was performed as follows:
• For all organs/tissues from all adult animals found dead during the study.
• For all macroscopic lesions from all dose group animals.
• For all organs/tissues from all adult animals of Groups 1 (control) and 4 (high dose).
• Reproductive organs for males that failed to sire, females which failed to deliver and females with total litter loss.

Tissues identified in Text Table 16 (except animal identification and bone marrow smears) were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Postmortem examinations (offspring):

See tables 17-18-19-20

Statistics:

Statistical analysis was performed, where appropriate, by the data acquisition software.

Reproductive indices:

Pre-Coital Interval (in Days): Sum of days until successful insemination/Number of inseminated females
Male and Female Copulation Index (in %): Number of inseminated females / Number of paired females X 100
Male and Female Fertility Index (in %): Number of pregnant females / Number of inseminated females X 100
Pre-Birth Loss (in %): Number of implantations - Number of offspring born / Number of implantations X 100
Irregularity Index: Mean standard deviation of length of the oestrous cycle/√ (length of the oestrous cycle)
Days in Estrus: Number of estrus days / Number of smears x 100

Offspring viability indices:

Live Birth Index (in %): Number of pups born alive / Number of pups born x 100
Viability Index (in %): Number of pups alive on PND4/Number of pups alive at birth x100
Lactation Index (in %): Number of pups alive on PND21/Number of pups alive on PND4 x100
(after culling)
Sex Ratio (Proportion of Male Pups in %): Number of males/Number of pups x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related clinical sign in any group for either sex. Transient hypersalivation and/or abnormal foraging and pedalling were noted just after dosing in all groups including control throughout the premating (both sexes), gestation and lactation periods. Signs of habituation were observed since the incidences of hypersalivation and associated clinical signs during the lactation period were lower when compared with the premating period. These findings noted in all groups, including the control, were considered to be a physiological response to the vehicle (PEG 400) and not test item-related in view of the comparable incidence between treated and control groups. Stereotypic behavior was noted on 1 occasion (LD4) for 1 female in the 110 mg/kg/day group (No. 82). In the absence of similar finding in higher dose groups this was considered incidental. Isolated clinical signs were noted for males and/or females including broken or missing tooth, red stained fur, chromodacryorrhea, noisy breathing, exophthalmos or increased eye size,
malocclusion, soft/liquid feces, bleeding, sores, scabs, red/dark vaginal discharge, localized hairloss or bent tail amongst treated and control groups and considered incidental or related to the pregnancy status of the females.
Clinical signs noted during the arena observation were limited to tactile reflex for 1 control male and 1 female in the 110 mg/kg/day group, considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female No. 177 given 1000 mg/kg/day was found dead on Day 5 at the morning mortality check. There was no clinical signs nor macroscopic findings indicative of a test item-related effect, so this isolated unscheduled death was likely incidental.
Male No. 16 in the control group was found dead on Day 5. Before death, this animal was pale, had irregular breathing, was lying down and had purple area on the scrotum. At necropsy, blood was noted in the abdominal cavity and in the scrotum and dark focus
was observed in the stomach. This isolated death in the control group was incidental. As these findings were likely not test item-related and because this occurred during the first week of dosing, both animals were replaced according the Test Facility SOP’s.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a lower mean body weight gain at 1000 mg/kg/day during the lactation period when compared with the concurrent control (-19% from LD1 to LD21) but the mean value was comparable with the Historical Control Data (HCD) mean (-6%). In addition, in the
absence of any impact on the mean terminal body weight on LD21 (-2%), this was considered not toxicologically relevant.
There was no test item-related effect on mean body weight gain in any group for males from Day 1 to Day 84 nor for females during premating or gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Consistent with the lower mean body weight gain noted (see Section 16.4), there was statistically significantly reduced mean food consumption at 1000 mg/kg/day throughout the lactation period when compared with the concurrent control (-7% from LD1 to LD21) but the mean value was comparable with the HCD mean (-3%) and therefore considered not toxicologically significant. There was no test-item related effect on mean food consumption during premating (both sexes) and gestation periods in any groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in hematological parameters in any group for either sex that were considered toxicologically relevant. Minor statistically significant differences arising between control and treated animals, such as decreased red blood cells count and haematocrit concentration along with increased percentage of reticulocyte and decreased lymphocyte and white blood cells count in males treated at 1000 mg/kg/day were considered not toxicologically relevant as they were of low magnitude and not observed for females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in serum clinical chemistry parameters in any group for either sex that were considered toxicologically relevant. Minor statistically significant differences arising between control and treated animals were noted including increased chloride and glucose serum concentration at 1000 mg/kg/day and a decrease in urea serum concentration at 330 and 1000 mg/kg/day for males and an increase in calcium and albumin serum concentration and a decrease in creatinine concentration at 1000 mg/kg/day for females. These changes were considered not toxicologically relevant as they were of low magnitude and/or observed in 1 sex only.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on the mean Total T4 or mean TSH serum level. Although statistical significance and/or slight fluctuations were observed among treated F0 animals, in general, mean Total T4 and TSH serum levels of F0 males and F0 females at all dose levels appeared similar to mean Total T4 and TSH serum levels of controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no test item-related effect on urine analysis parameters in any group for either sex.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no test item-related microscopic finding in any group for either sexes. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the mean estrous cycle length and regularity in any group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no test item-related effect on sperm count, motility or morphology in any group.

Reproductive performance:

no effects observed

Description (incidence and severity):

The majority of mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore comparable in all groups (i.e., 2.7, 2.9, 2.7 and 3.4 days in the control, 110, 330 and 1000 mg/kg/day groups, respectively). There was no test item-related effect on the mean number of implantation sites. Mean values in the treated groups (13.8, 14.3 and 13.0 at 110, 330 and 1000 mg/kg/day, respectively) were above the control mean value (12.9).
There was no test item-related effect on the mating performance in any group. After 2 weeks of mating, all pairs of animals mated in all groups. There was no test item-related effect on fertility. All mated females became pregnant, with the exception of 1 female in each of the control, 330 and 1000 mg/kg/day groups. This was considered incidental.

Details on results (P0)

See above

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: No adverse effects

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Endocrine findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Reproductive function: sperm measures:

effects observed, non-treatment-related

Reproductive performance:

no effects observed

Details on results (P1)

See above

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Remarks on result:

other: No adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male in the 1000 mg/kg/day (No. 322) group had convulsive episode during dosing on Day 62. This isolated finding is part of the background considered normal for this strain of rat. Transient hypersalivation and/or abnormal foraging and pedalling were noted just after dosing in all groups including control with a higher incidence in the treated groups throughout the dosing period. These findings noted in all groups, including the control, were considered to be a physiological response to the vehicle (PEG 400) and not test item-related in view of the comparable incidence between treated and control groups.
Isolated clinical signs were noted for males and/or females including broken tooth, teeth cut, long teeth, torn claw, liquid feces, chromodacryorrhea, hunched gait, malocclusion, red vaginal discharges, piloerection, sores, scabs, localized or extensive hairloss, incomplete hair growth, noisy breathing, dirty tail, red stained fur or bent tail amongst treated and control groups and considered incidental or related to the pregnancy status of the females. Clinical signs noted during the arena observation were limited to tactile reflex, alertness, abnormal behaviour or posture observed sporadically amongst treated and control groups and therefore considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item-related mortality in any group for any F1 cohort

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on mean body weight gain in any group for either sex. There was slightly lower mean body weight gain in all treated groups for females during the lactation period (Cohort 1B) when compared with the control group considered not toxicologically significant in the absence of dose-related trend and any effect on terminal mean body weight (LD21).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Although there were some variations that occasionally reach statistical significance, there was no test item-related effect on mean food consumption in any group for either sex.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in hematological parameters that were considered toxicologically
significant in any group for either sex. There was lower white blood cells count including lower neutrophil, lymphocyte, monocyte,
eosinophil and basophile counts in all treated groups for females when compared with the concurrent control group. However, mean values were comparable with or higher than that of the HCD means, therefore this was considered not toxicologically significant.
Other minor statistically significant differences arising between control and treated animals, such as decreased red blood cells count and hematocrit concentration in all treated groups for males, increased mean corpuscular hemoglobin concentration for males at 1000 mg/kg/day and decreased monocyte and lymphocyte counts for males in all treated groups were considered not toxicologically relevant as they were not dose-related and/or of low magnitude.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in serum clinical chemistry parameters in any group for either sex that were considered toxicologically relevant. Minor statistically significant differences arising between control and treated animals were noted including decreased phosphorus and urea serum concentration in males at 1000 mg/kg/day and an increase in glucose serum concentration in females at 1000 mg/kg/day and an increase in triglyceride serum concentration at 330 and 1000 mg/kg/day for females. These changes were considered not toxicologically relevant as they were of low magnitude and/or observed in 1 sex only.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross findings in any group and any cohort for either sex. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Histopathological findings:

no effects observed

Description (incidence and severity):

- Cohort 1A:
Following quantitative evaluation of ovaries, there was a test item-related statistically significantly lower mean count of primordial/small growing follicles at 1000 mg/kg/day when compared with control females. There were no differences in the mean numbers of
corpora lutea. Following standard histopathologic examination, no test item-related findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item.
- Cohorts 2A and 2B:
No test item-related microscopic findings were noted in the nervous system, following detailed examination of the following subregions of the brain (Cohorts 2A and 2B) and spinal cord, dorsal root ganglia and ventral root fibers, skeletal muscle, sciatic and tibial nerves, eye and optic nerves (Cohort 2A). The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore,
were considered unrelated to administration of test item.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on the mean Total T4 or mean TSH serum level. Mean Total T4 serum levels appeared slightly decreased in F1 Cohort 1A males at 330 mg/kg/day (0.78x of controls, statistically significant) and slightly increased in F1 Cohort 1A females at 110 and 330 mg/kg/day (both 1.28x of controls). These slight changes occurred in the absence of a dose-related trend, because mean Total T4 serum levels of F1 Cohort 1A males and F1 Cohort 1A males at the other dose levels appeared similar to mean Total T4 serum levels of controls. Mean TSH serum levels appeared increased in F1 Cohort 1A females at 110 mg/kg/day (2.61x of controls) and 330 mg/kg/day (3.89x of controls, statistical significant), but these changes occurred in the absence of a dose-related trend, because mean TSH levels of F1 Cohort 1A females at 1000 mg/kg/day appeared similar to mean TSH serum levels of controls. Mean TSH serum levels appeared slightly increased in F1 Cohort 1A males at 1000 mg/kg/day (1.57x of controls), but this change did not achieve statistical significance. Mean TSH serum levels appeared similar to mean Total TSH serum levels of controls for F1 Cohort 1A males at 110 and 330 mg/kg/day.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Auditory Startle Reflex - Cohort 2A
There was no test item-related effect on the auditory startle reflex test in any group for either sex. The percentage of habituation to repeated acoustic stimuli was comparable between treated and control groups.
- Functional Observation Battery - Cohort 2A
There was no test item-related effect on the neurobehavioral examinations performed between PND63 and PND75 in Cohort 2A male and female rats. The statistically significantly lower number of fecal bolus noted in Groups 2 and 4 male rats was not dose-related and was not associated with any changes in other functional observation battery parameters. Therefore, this effect was considered incidental and not test item-related.
- Motor Activity - Cohort 2A
There was no test item-related effect on habituation to a novel environment or on motor activity in any group for either sex.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

- Splenic Lymphocyte Subpopulation Analysis
Cohort 1A: There was no test item-related effect on the immune system in any group for either sex.
- Humoral Response Following KLH Administration (TDAR) - Cohort 3
There was no test item-related effect on TDAR results in any group for either sex, while treatment of control animals with Cyclophosphamide resulted in immunosuppression, as evidenced by higher incidences of non-responders in both genders.

Details on results (F1)

See above

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: No adverse effects

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be test item-related.
Incidental clinical signs of pups consisted of occasional hematoma, sores, hairloss, swollen, weakness, incomplete hair growth, scabs or teeth marks. In view of their nature and/or sporadic occurrence including in controls, these findings were considered of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings:

no effects observed

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not specified

Details on results (F2)

See results above

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2 (cohort 1B)

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: No adverse effects observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Any other information on results incl. tables

See attached document for Tables regarding P0, P1, F1 and F2.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this extended 1 generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Effect Levels (NOELs) of Reaction Mass of Dimethyl Adipate, Dimethyl Glutarate and Dimethyl Succinate (EC 906-170-0) were established:
General Toxicity (F0): At least 1000 mg/kg/day.
Reproductive Toxicity (F0): At least 1000 mg/kg/day.
General Toxicity (F1): At least 1000 mg/kg/day.
Developmental Toxicity (F1): At least 1000 mg/kg/day.

Executive summary:

The objective of this study was to provide an evaluation of the pre and postnatal effects of the test item, Reaction Mass of Dimethyl Adipate, Dimethyl Glutarate and Dimethyl Succinate (EC 906 -170 -0), on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: Gonadal function, the estrous cycle, spermatogenesis, mating behaviour, fertility, pregnancy, parturition, and lactation. Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems. 1.2. Procedures The test item, Reaction Mass of Dimethyl Adipate, Dimethyl Glutarate and Dimethyl Succinate (EC 906-170-0), was administered by daily oral gavage at dose levels of 110, 330 and 1000 mg/kg/day to groups of 25 male and 25 female Wistar rats (F0 generation). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group of 25 rats/sex received the same dose volume (5 mL/kg/day) of the control item (PEG 400). The inseminated females were allowed to litter, and development of the offspring was observed up to weaning on Postnatal Day (PND) 21. The following parameters and endpoints were evaluated in all F0 animals: Mortality, clinical signs (including arena observations), body weights and body weight gains, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic observations, organ weights and histology findings. F0 males were euthanised after at least 12 weeks of exposure and F0 females after at least 16 weeks of dosing. From weaning, the selected F1-pups were allocated to 5 cohorts (1A, 1B, 2A, 2B and 3) of 20 males and 20 females for the first 2 and 10 males and 10 females for the last 3. All F1 animals, except Cohort 2B, were administered by daily oral gavage from PND21 up to the day before the day of scheduled necropsy at the same dose levels of 110, 330 and 1000 mg/kg/day. A fourth group received the same dose volume (5 mL/kg/day) of the control item (PEG 400). Except for Cohort 2B, F1 animals were observed for mortality, clinical signs (including arena observations), body weights and body weight gains, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups and Cohort 1A), pup anogenital distance, pup number of aerola/nipples, estrous cycles (Cohort 1A), sexual maturation, sperm quality (Cohort 1A), neurobehavioral tests (Cohort 2A), macroscopic observations, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. Humoral response was also evaluated following KLH administration (TDAR assay) for Cohort 3 animals. F1 animals from Cohort 2B were not dosed and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis. F1 animals from Cohort 3 were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls. F1 animals from Cohort 2A were sacrificed after neurobehavioral tests between PND76 and PND90. F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99. Surplus offspring (10/sex/group) not included in any of the F1 cohorts, were selected for thyroid hormone analysis and organ weights on PND21. F1 females from Cohort 1B were mated and allowed to litter and development of the offspring was observed up to weaning on PND21. The following reproduction/developmental parameters were determined: Mating performance and fertility, and delivery and litter data. For Cohort 1B, F1 males were sacrificed after at least 17 weeks of dosing and F1 females after at least 16 weeks of dosing. F2 offspring were observed for early postnatal development, mortality, clinical signs, body weights, sex, anogenital distance, number of aerola/nipples, organ weights and macroscopic findings. F2 offspring were sacrificed on PND21-23. 1.3. Results Samples collected from formulations of Group 2, Group 3 and Group 4 were accurate and homogeneous. A minimal response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 11.

F0 Generation:

There was no test item-related death in any group for F0 animals. There was no test item-related clinical sign in any group for F0 animals. There was no test item-related effect in any group in the mean F0 body weight gain or food consumption for either sex. There was no test item-related effect in any group on F0 male and female mating and fertility, male copulation, mean number of implantations, mean estrous cycle lengths, mean pre-coital intervals, gestation lengths and sperm parameters. There were no test item-related effects on hematology, coagulation, serum clinical chemistry, urinary parameters or thyroid hormone serum levels for F0 animals at any dose for either sex. There was no test item-related macroscopic or microscopic finding and no test item-related effect in organ weights in any group for either sex.

F1 Generation:

There was no test item-related effect on F1 postnatal survival in any group. The mean number of pups delivered was comparable with that of the control group and the historical control data mean in all treated groups. There were no test item-related effects on F1-pups clinical condition, body weight, anogenital distance, or areolae/nipple at any dose level. There were no test item-related effects on T4 and TSH levels in F1-pups on PND4 and PND21. There was no test item-related effect on the mean ages at attainment of balanopreputial cleavage and vaginal opening for F1 animals in any group. The duration from vaginal opening to first estrous in the treated groups was comparable to the control group. There was no test item-related clinical sign in any group for either sex. There was no test item-related effect on mean body weight gain or mean food consumption in any group for either sex. There were no test item-related effects on hematology, coagulation, serum clinical chemistry, urinary parameters or thyroid hormone serum level for the F1 males and females after approximately 13 weeks of dosing (Cohort 1A) at any dose. There was a test item-related lower fertility index in the 1000 mg/kg/day group when compared with other groups and the HCD range (Cohort 1B) considered adverse as it was also associated with test item-related statistically significantly lower mean count of primordial/small growing follicles at 1000 mg/kg/day when compared with control females (Cohort 1A). There was no test item-related effect in any group on F1 males and females mating, mean number of implantations, mean pre-coital intervals, gestation lengths (Cohort 1B). There were no test item-related effects on mean F1 estrous cycle lengths or spermatogenic parameters noted in any group (Cohort 1A). There was no test item-related immunotoxicity (Cohorts 1A and 3), as evidenced by lymphocyte subpopulation analysis or KLH-IgM responses in a TDAR assay. There was no test item-related neurotoxicity (Cohorts 2A and 2B), as indicated by auditory startle responsiveness at weaning or FOB and motor activity at adulthood, or brain morphometry, brain weight and neurohistopathology. There were no test item-related pathology findings in any groups.

F2 Generation:

There was no test item-related effect on F2 postnatal survival in any group and no test item-related effect on the mean number of pups delivered. There were no test item-related effects on F2-pup clinical condition, body weight, anogenital distance, or areolae/nipple at any dose level. There were no test item-related organ weight changes in any group.