Propylidynetrimethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An EOGRTS according to OECD TG 443 was requested by ECHA as a result of a compliance check (CCH-D-2114356723-46-01/F).

Based on the results of this extended one generation reproductive toxicity study (including Cohort 1A, 1B (extended to a second generation), 1C and Surplus), the following no-observed-adverse-effect level (NOAEL) of Trimethylolpropan were established:

General Toxicity:

The decreased body weight gain (F0-generation only) in combination with decreased food efficiency at 6600 ppm (corresponding to mean actual test article intake between 750-1050 mg/kg/day for males and females) in both generations were considered adverse.

The adverse test item-related microscopic findings from 2200 ppm onward consisted of vacuolation of the gray matter of periventricular areas of the brain and in areas adjacent to the central canal of the spinal cord and cytoplasmic vacuolation of skeletal muscle myofibers. These microscopic findings of the F0-Generation were repeated in the F1-Generation.

Based on these results, a NOAEL of 740 ppm (corresponding to an actual test article intake between 74-99 mg/kg/day for males and females) for general toxicity was established.

Reproductive Toxicity:

Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations.

Based on these results, a NOAEL of 2200 ppm (corresponding to an actual test article intake between 225-289 mg/kg/day for males and females) for reproductive toxicity was established.

Developmental Toxicity:

Overall, developmental toxicity following treatment with Trimethylolpropan at 6600 ppm consisted of a decreased post-implantation survival index (resulting in a decreased litter size), decreased mean combined pup weights and decreased pup viability. Furthermore, adverse test item-related clinical signs/macroscopic findings of the brain/skull and/or eye were observed in three and five affected pups at 6600 ppm, in the F1-generation and F2-generation, respectively.

For the observations made with a single incidence (hydrocephaly and small eye; F2-generation) at 740 ppm, followed by a single incidence (partly closed eye; F2-generation) at 2200 ppm, a test item-related effect could not be excluded. Notably, these are singly occurrences at the mid and low dose level which might therefore be considered a chance finding.  However, based on the similar nature and rarity of these findings, a test-item-related effect could not be excluded.

Based on these results, a NOAEL for developmental toxicity could not be established.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The objective of this study is to provide an evaluation of the pre- and postnatal effects of Trimethylolpropan 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, is expected to identify specific target organs in the offspring. The dose levels in this study were selected to be 0, 740, 2200, 6600 ppm (anticipated dose levels: 0, 70-110, 200-330, 600-1000 mg/kg/day). The water concentrations were adjusted according to the rapid increase in body weight and constant water consumption, resulting in remarkable changes in relative water consumption.
In addition, the study will provide and/or confirm information about the effects of Trimethylolpropan on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters are considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

GLP compliance:

yes

Limit test:

no

Justification for study design:

The design of this study was based on the final decision on a compliance check of Trimethylolpropan by ECHA (Decision no. CCH-D-2114356723-46-01/F, date 21 Mar 2017).

In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD Guideline 443. Extended One-Generation Reproductive Toxicity Study, June 2018.
• OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han (Crl: WI(Han) rat was chosen as the animal model for this study as it is an accepted rodent species for reproduction and developmental toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Environmental Acclimation
The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of administration.

Selection, Assignment, Replacement, and Disposition of Animals
F0- animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females will be randomized separately.

On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups will be culled on PND 4.
On PND 21, pups from available litters per group were selected and assigned according to the following schedule.

Cohort Designation Animals/group Age at necropsy
1A 20 M + 20 F PND 89-95
1B Reprotox. 20 M + 20 F PND 140-155
1C 20 M + 20 F After VP/PBS positive*
Surplus Thyroid hormones 10 M + 10 F PND 22
* VP = vaginal patency, BPS = balanopreputial separation

HUSBANDRY
Housing
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm)
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm or type 2000P; 61x43.5x21.5 cm) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 23°C with an actual daily mean relative humidity of 43 to 62%. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Drinking water preparations were freely available to each animal via water bottles. During the acclimatization period, animals had free access to municipal tap water (without the test item) via water bottles/containers. During refreshment of drinking water animals had no access to water for a maximum of 2 hours.
Water bottles were clearly labeled with Test Facility Study No., group, and animal number.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended actions were documented in the study records.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Experimental Design

The actual test item intake is indicated in the tables below. Mean test item intake was within the anticipated dose levels for Groups 1, 2 and 3 for both sexes (i.e. 0, 70-110 and 200-330 mg/kg/day, respectively) and for Group 4 for males only (600-1000 mg/kg/day). For females at the highest concentration, the mean test item intake was slightly above the anticipated dose level. Although the overall test item intake only exceeded the 1000 mg/kg/day with 1-5%, more pronounced exceedance was observed on individual days during the study, with the exception of the lactation phase of the F0-generation.

Test Item Intake – F0-Generation
Mean over means intake [mg test item/kg body weight]
(mean range indicated within brackets)
Group no. 2 3 4
Nominal drinking
water inclusion
level (ppm) 740 2200 6600

Sex Study period
Males Pre-mating 76 (57-104) 233 (160-317) 851 (632-1168)
Post-mating 60 (52-68) 161 (129-202) 724 (627-812)
Mean of means 74 225 838

Females Pre-mating 100 (78-120) 309 (235-373) 1175 (756-1480)
Post-coitum 90 (45-115) 284 (163-381) 1122 (365-1451)
Lactation 84 (55-139) 264 (162-417) 634 (293-894)
Mean of means 93 289 1014


Test Item Intake – F1-Generation
Mean over means intake [mg test item/kg body weight]
(mean range indicated within brackets)
Group no. 2 3 4
Nominal drinking
water inclusion
level (ppm) 740 2200 6600

Sex Study period
Males Post-weaning 92 (52-147) 257 (172-395) 786 (495-1242)
Post-mating 60 (56-66) 168 (150-188) 601 (511-659)
Mean of means 86 240 750

Females Pre-mating 103 (48-145) 292 (216-381) 1009 (580-1350)
Post-coitum 91 (67-107) 2 64 (201-311) 1027 (890-1206)
Lactation 103 (80-157) 270 (210-449) 1153 (845-1668)
Mean of means 99 279 1050


Drinking Water Preparation and Analysis
Preparation of Drinking Water Containing Test Item
The test item was diluted with the required amount of municipal tap water. Drinking water solutions were prepared at least weekly and were stored at room temperature for a maximum of 6 days if not used on the same day. The prepared drinking water solutions were stirred before administration.
No adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Any residual volumes were discarded.

Sample Collection and Analysis
Drinking water preparation samples were collected for analysis.
All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.

Details on mating procedure:

Cohabitation/Mating Procedure – F0-Generation
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating, after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study No. 518618).
Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.
Stability Analysis
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation drinking water containing test item at 9000 ppm was determined at 8 days at room temperature under normal laboratory light conditions.
Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Dose Formulation Analyses
Accuracy
The concentrations analyzed in the drinking water solutions of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the Group 1 drinking water.
Homogeneity
The drinking water solutions of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Residue analysis
No test item was detected in the residue samples.
Stability
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 8 days and in a refrigerator (2-8°C) for at least 8 days.

Duration of treatment / exposure:

Administration of Test Materials
The test item was administered to the appropriate animals by inclusion in the drinking water ad libitum.
F0-males and females were treated up to and including the day before scheduled necropsy.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled water from the water bottle or when they commence drinking for themselves.
From weaning onwards (PND 21), F1-animals of Cohorts 1A were drinking water containing test item up to and including the day before scheduled necropsy. The F1-animals of Cohort 1C, Cohort Surplus and Spares (not assigned to one of the cohorts) were administered drinking water containing test item until the day of necropsy
The F1-animals of Cohort 1B were treated from weaning onwards, including at least 10 weeks prior to the second mating period, the duration of pregnancy and at least 21 days after delivery up to and including the day of scheduled necropsy.
The first day drinking water preparations containing test item were available to the animals was designated as Day 1.
The amount of test item incorporated in the drinking water was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and water consumption values.
The drinking water solutions were refreshed at least every other day at approximately the same time.
F0 generation
Males 11-12 weeks (including 10 weeks pre-mating)
Females 16-19 weeks (including 10 weeks pre-mating)
Females which failed to deliver or had a total litter loss 13-14 weeks

F1 Generation
Cohort 1A 10-12 weeks
Cohort 1B 14-19 weeks
Cohort 1C 3-5 weeks
Cohort Surplus N/A
Females which failed to deliver or had a total litter loss 15-17 weeks

Frequency of treatment:

Daily (drinking water study).

Details on study schedule:

Unscheduled Deaths – F0-Generation
No F0-animals died during the course of the study.
 
Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies are summarized below:
Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
Females which delivered: LD 23-25.
Females which failed to deliver
(Nos. 122-124-140-141-153-160-170-174): With evidence of mating: Post-coitum Days 25 and 27.
Females with total litter loss
(Nos. 107 and 190): W ithin 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Terminal Procedures – F1 and F2-Generation until Weaning
Unscheduled Deaths– F1 and F2-Generation
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

Culled Pups (PND 4) – F1 and F2-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Scheduled Euthanasia – F2-Generation
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. The animals were not deprived of food overnight.
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples were weighed and collected. The selected litters were documented in the study files by the study director in advance.
All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.















Unscheduled Deaths – F0-Generation
If an animal dies on study, a necropsy will be conducted and specified tissues will be saved, but not weighed. If necessary, the animal will be refrigerated to minimize autolysis.
Animals may be euthanized for humane reasons as per Test Facility SOPs. These animals will be deeply anaesthetized using isoflurane and subsequently exsanguinated. They will undergo necropsy, and specified tissues will be retained, but not weighed.

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia will have a terminal body weight recorded and will be deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies are summarized below:
Males (which sire and fail to sire): After successful mating and a minimum of 10 weeks of treatment.
Spare females: LD 22.
Females which deliver: LD 23-25.
Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27. Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
Females with total litter loss: Dams with no surviving pups will be euthanized within 24 hours after the last pup is found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy will be fasted overnight with a maximum of 24 hours before necropsy. Water will be available.

Unscheduled Deaths– F1-Generation and F2-Generation
Pups and fetuses may be euthanized for humane reasons.
Recognizable fetuses of females that die spontaneously or are euthanized in extremis will be examined externally, sexed (both externally and internally, if possible).
Stillborn pups and pups found dead between birth and PND13 will be sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy will be performed including sex determination (both externally and internally, if possible).

Culled Pups (PND 4) – F1-Generation and F2-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) will be euthanized by decapitation. From two extra pups per litter, blood will be collected (F1-generation only), if possible.
Sex will be determined both externally and internally (if possible). Pups will be externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities will be recorded.

Scheduled Euthanasia – F2-Generation
Scheduled necropsy of the F2-animals of Cohort 1B will be conducted on PND 21-23. The animals will not be deprived of food overnight.
From 10 selected litters/group, terminal body weight will be determined for one male and one female pup. Subsequently, these pups will be deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals will be subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded.

Dose / conc.:

0 ppm

Remarks:

F0-animals: Group 1 (Control)
See 'Details on exposure'

Dose / conc.:

740 ppm

Remarks:

F0-animals: Group 2;
See 'Details on exposure'

Dose / conc.:

2 200 ppm

Remarks:

F0-animals: Group 3;
See 'Details on exposure'

Dose / conc.:

6 600 ppm

Remarks:

F0-animals: Group 4;
See 'Details on exposure'

Dose / conc.:

0 ppm

Remarks:

F1-animals: Group 1 (Control); Cohort 1A, 1B, 1C, Surplus

Dose / conc.:

740 ppm

Remarks:

F1-animals: Group 2; Cohort 1A, 1B, 1C, Surplus

Dose / conc.:

2 200 ppm

Remarks:

F1-animals: Group 3; Cohort 1A, 1B, 1C, Surplus

Dose / conc.:

6 600 ppm

Remarks:

F1-animals: Group 4; Cohort 1A, 1B, 1C, Surplus

No. of animals per sex per dose:

F0-animals: Group 1, 2, 4 and 4: 25 males and 25 females/group.
F1-animals: Group 1, 2, 3, and 4: each cohort 20 males and 20 females in cohort 1A, 1B and 1 C; and each group (surplus cohort) 10 males and 10 females.
Remark: Each F1 group (1, 2, 3, and 4) had 4 cohorts (Cohort 1A, Cohort 1B, Cohort 1C and a surplus cohort).

Control animals:

yes, concurrent vehicle

Details on study design:

Justification of Route and Dose Levels
The design of this study was based on the final decision on a compliance check of Trimethylolpropan by ECHA (Decision no. CCH-D-2114356723-46-01/F, date 21 Mar 2017).
The oral route of administration via drinking water inclusion was selected because this was specified in the final decision by ECHA (Decision no. CCH-D-2114356723-46-01/F, date 21 Mar 2017).

The administration of Trimethylolpropan via inclusion in the drinking water was considered to be the most suitable route based on analytical results and the results of the 14-day dose range finder (DRF), in which palatability of Trimethylolpropan via diet and drinking water was investigated.

The dose levels were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with drinking water exposure of Trimethylolpropan in rats, and in an attempt to produce graded responses to the test item. In this preliminary study, dose concentrations of 1500, 3000 and 6000 ppm were tested. Toxicologically relevant body weight effects were observed at 6000 ppm in males and females; a reduced body weight gain noted for males during the entire treatment period (mean body weights about 8% lower than controls) and in females, a body weight loss of -6% was noted during premating resulting in 10% lower mean body weights during entire treatment period. No relevant effects were noted in any of the remaining parental or reproductive and developmental parameters investigated in this study. A dose concentration of 6000 ppm correspond with an actual test item intake of 579 mg/kg in males during premating and mating, and in females 728 mg/kg, 960 mg/kg and 617 mg/kg during the premating, post-coitum and lactation period, respectively. As the observed toxic effects in this study was 10% lower mean body weights in females, the highest dose concentration was increased with 10% in aim to achieve an actual test item intake up to 1000 mg/kg without leading to severe toxicity in females.

The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

None.

Parental animals: Observations and examinations:

In-life Procedures, Observations, and Measurements – F0-Generation
Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F0-Generation
Clinical observations were performed at least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – F0-Generation
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – F0-Generation
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

Water Consumption – F0-Generation
Water consumption was quantitatively measured at least over every two days, except for males and females which were housed together for mating and for females without evidence of mating. Water consumption was measured daily during post-coitum and lactation.
Water consumption was also measured on the days the concentration of test item in de drinking water was changed.

Cohabitation/Mating Procedure – F0-Generation
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating, after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
For one couple (Male No. 5, Female No. 105), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated 11 days after the actual mating date. The actual mating date was designated Day 0 post-coitum.
Detection of mating was not confirmed in first instance for Female Nos. 107 and 118. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

General Reproduction Data – F0-Generation
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.


Laboratory Evaluations
Clinical Pathology
Sample Collection
F0-animals and Cohort 1A animals
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room.
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals2 housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water (without test item) was available.
Culled pups of the F1-generation and F2-generation (PND 4) pups
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
Samples were collected according to the table below. After collection, samples were transferred to the appropriate laboratory for processing.

Hematology
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the following parameters:
White blood cells (WBC)
Neutrophils (absolute)
Lymphocytes (absolute)
Monocytes (absolute)
Eosinophils (absolute)
Basophils (absolute)
Red blood cells
Reticulocytes (absolute)
Red Blood Cell Distribution Width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets
A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. These smears were not examined.

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for the following parameters:
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)

Clinical Chemistry
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-Heparin as anticoagulant. Samples at a target volume of 1.0 mL were collected in tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids), which was analyzed for thefollowing parameters:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile Acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

Thyroid hormone
Blood samples at a target volume of 1.0 mL (F0-animals and F1-animals of Cohort 1A and F2-animals), 0.5 mL (pooled PND 4 pups) and 1.0 mL (Cohort Surplus PND 22 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum of F0-animals, Cohort 1A animals and Cohort Surplus animals (i.e. PND 22 pups) was used for measurement of both T4 and TSH. Pooled serum of culled PND 4 pups was used for measurement of T4 only. Any remaining sample was discarded.
Thyroxine (T4)
Thyroid Stimulating Hormone (TSH)

Urinalysis
Urine samples were analyzed for the following parameters:
Volume
Specific gravity
Clarity
Colour
pH
Blood
White blood cells (WBC)
Bilirubin
Urobilinogen
Protein
Ketones
Glucose
Nitrite
Sediment:
White blood cells (WBC-sed.)
Red blood cells (RBC-sed.)
Casts
Epithelial cells
Crystals
Bacteria
Other

Oestrous cyclicity (parental animals):

Estrous cycle determination – F0-Generation
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.

Sperm parameters (parental animals):

Sperm Analysis – F0-Generation
For all surviving males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
As for animal no. 96 (Group 4), abnormalities were noted in both epididymis, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.

Litter observations:

In-life Procedures, Observations, and Measurements – F1-Generation and F2-Generation until Weaning (PND 21)
The in-life procedures, observations, and measurements listed below were performed for the pups until weaning (PND 21).

Mortality/Moribundity Checks – F1 and F2-Generation
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F1 and F2-Generation
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Body Weights – F1 and F2-Generation
Live pups were weighed individually on PND 1, 4, 7, 13 and 21.
For animals of Cohorts and Surplus and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy.

Sex – F1 and F2-Generation
Sex was externally determined for all pups on PND 1 and 4.

Anogenital Distance – F1 and F2-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1 and F2-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Culling – F1 and F2-Generation
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done.

Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

In-life Procedures, Observations, and Measurements – F1-Generation and F2-Generation from Weaning (PND 21) onwards:
Cohorts 1A, 1B and 1C (60 animals/sex/group)
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort Surplus and spare F1-animals as these were terminated on PND 22 and 22-24, respectively.

Mortality/Moribundity Checks – Cohorts 1A, 1B and 1C
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations– Cohorts 1A, 1B and 1C
Clinical observations were performed at least once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – Cohorts 1A, 1B and 1C
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

Body Weights – Cohorts 1A, 1B and 1C
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation.
For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – Cohorts 1A, 1B and 1C
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

Water Consumption – Cohorts 1A, 1B and 1C
Water consumption was quantitatively measured at least over every two days, from weaning onwards up to the day prior to scheduled necropsy.
Water consumption was also measured on the days the concentration of test item in de drinking water was changed.

Vaginal Patency – Cohorts 1A, 1B and 1C
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency is present, by visual inspection of the vaginal area.
Body weight was recorded on the day of acquisition of vaginal patency.

Balanopreputial Separation – Cohorts 1A, 1B and 1C
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards Ref. 2 until balanopreputial separation was present, by visual inspection of the genital area.
Body weight was recorded on the day of acquisition of balanopreputial separation.

Stage of Estrus Determination – Cohorts 1A, 1B and 1C
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Cohort 1A (20 animals/sex/group)
The in-life procedures, observations, and measurements listed below were performed for the F1-females of Cohort 1A only.

Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods.
During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. The estrus cycle data of the first period is not reported.
During the second period, daily vaginal lavage was performed from PND 75 to 88.

Cohort 1B (20 animals/sex/group)
The in-life procedures, observations, and measurements listed below were performed for the F1-animals of Cohort 1B only.

Body Weights – Cohort 1B
Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohort 1B
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

Estrous Cycle Determination – Cohort 1B
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed from start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

Cohabitation/Mating Procedure – Cohort 1B
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
Detection of mating was not confirmed in first instance for Female Nos. 501 and 520. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

General Reproduction Data – Cohort 1B
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Postmortem examinations (parental animals):

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.


Histology – F0 and F1-Generation
Tissues mentioned below from a selection of the F0 and F1-animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).

Bone marrow, Bone, sternum, Brain, Cervix, Epididymis, Eye, Gland, adrenal, Gland, coagulation, Gland, harderian, Gland, mammary, Gland, parathyroid, Gland, pituitary, Gland, preputial, Gland, prostate, Gland, seminal vesicle, Gland, thyroid, Heart, Kidney, Large intestine, Cecum, Large intestine, colon, Large intestine, colon, Large intestine, rectum, Liver, Lung, Muscle, skeletal, Nerve, optic, Nerve sciatic, Ovaries, Oviducts, Skin, Smal intestine, duodenum + ileum + jejunum, Spincal cord, Spleen, Stomach, Testes, thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

Postmortem examinations (offspring):

Terminal Procedures – F1 and F2-Generation until Weaning
Unscheduled Deaths– F1 and F2-Generation
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. At the discretion of the Study Director a limited necropsy was performed for pups sacrificed in extremis between birth and PND13. Additionally, livers were collected of pups sacrificed in extremis, at the discretion of the Study Director.

Culled Pups (PND 4) – F1 and F2-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Scheduled Euthanasia – F2-Generation
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. The animals were not deprived of food overnight.
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues were weighed and collected. The selected litters were documented in the study files by the study director in advance.
All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.
Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.
 
For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues were weighed and collected.

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated.
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Sperm Analysis – Cohort 1A
For all surviving males of Cohort 1A, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One pup (male or female) was selected per litter (20 litters in total).
One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:

Parameter (Abbreviation) Unit Markers for identification
T-cells % Lymphoid cells CD3+/CD45RA-
T-helper cells % Lymphoid cells CD3+/CD4+/CD8-
T-cytotoxic cells % Lymphoid cells CD3+/CD4-/CD8+
B-cells % Lymphoid cells CD3-/CD45RA+
NK-cells % Lymphoid cells CD3-/CD161a+
Ratio T-helper cells/
T-cytotoxic cells (Th/Tc) - -

The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Cohort 1B
Scheduled necropsy of the F1-Generation of Cohort 1B was conducted on the following days:
F1-Males (which sired and failed to sire): Following completion of the mating period.
F1-Females which delivered: LD 21-23
F1-Females which failed to deliver: With evidence of mating: Post-coitum Days 25-27 (nos. 641, 716,717 and719).
Without evidence of mating: Approximately 24-26 days after the last day of the mating period (Nos. 508, 571, 713and 723).
F1-Females with total litter loss (No. 509): Within 24 hours after the last pup was found dead or missing.

The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
On PND 22, blood samples (1.0 mL) were collected between 7.00 and 10.30 a.m. from all animals by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
 
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Reproductive indices:

Mating index males and females (%), Precoital time, Fertility index males and females (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%), Percentage live males and females at first litter check (%).

Offspring viability indices:

Viability index (%), Weaning index (%), Percentage live males and females at weaning(%).

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related effects on body weight and body weight gain were observed up to 2200 ppm.

In 6600 ppm-treated females, body weight and body weight gain were statistically significantly reduced from Week 4 of premating onwards. Mean body weight gain ranged between 0.81 to 0.89x of controls during the premating period, resulting in a mean body weight that was 0.93x of controls at the initiation of mating. Although body weight gains during gestation and lactation were considered unaffected by treatment up to 6600 ppm, body weights remained significantly lower throughout gestation and lactation and at termination, mean body weight of 6600 ppm-treated females was 0.93x of controls. The statistically significantly increased body weight gain in females at 6600 ppm on Day 21 of lactation was considered the result of smaller litter sizes in this group and consequently a lower burden on the dams.
For males treated at 6600 ppm, a similar trend was observed for body weight and body weight gain from Week 4 of premating onwards, statistically significant in Week 2 of mating only. Mean body weight gain was 0.91-0.98x of controls, resulting in statistically significantly reduced terminal body weights (0.94x) at the end of the treatment period.
Any other changes between treated animals and control, reaching statistical significance, were considered not to be test-item related in the absence of a dose-related effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 2200 ppm.
Relative food consumption in males and females treated at 6600 ppm was statistically significantly increased from Week 4 of premating onwards (except for lactating females, see below). This increase in both sexes was the result of decreased body weights in these animals and therefore considered not directly related to treatment. Additionally, mean food intake during the lactation phase was statistically significantly lower (0.80x) in 6600 ppm-treated females than in concurrent controls. This was considered to be the result of the smaller litter sizes at this high dose and therefore secondary to treatment.
Further statistically significant differences in food consumption for males at 6600 ppm were considered not to represent a change of toxicological significance, due to the minimal magnitude of the change.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Throughout the premating and mating period, water consumption (both absolute and relative to body weight) were significantly increased at the high dose animals (6600 ppm) compared to concurrent control. A similar trend, was noted at 740 and 2200 ppm, showing a clear dose response.
During gestation, this increase in water consumption was less pronounced and mainly observed at 6600 ppm (reaching statistically significance for absolute values only). For 2200 ppm-treated females, a slight increase in relative water consumption (not statistically significant) was observed between Day 1 to 11, subsequently returning to normal values. The slight decrease in water consumption observed during lactation in high dose females was attributed to the smaller litter size at this dose level

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in males or females treated at 6600 ppm. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
• Increased lymphocyte count (1.47x; within internal control range) and white blood cell counts (WBC, 1.39x; within internal control range) in males.
• Increased red blood cell distribution width (RDW; 1.10x; within internal control range) in males.
• Increased mean corpuscular volume (MCV; 1.04x; within internal control range) and mean corpuscular haemoglobin (MCH; 1.06x; within internal control range) in females.
An apparent dose-related increase in platelet counts was noted in all treated males compared to controls (1.06x, 1.12x and 1.16x at 740, 2200 and 6600 ppm, respectively). As these changes were not statistically significant and remained within the normal range, this was considered of no biological relevance.
The statistically significantly decreased monocyte counts at 6600 ppm (0.67x) were considered not to represent a change of biological relevance based on the minimal magnitude of change.
Other statistically significant differences in haematology parameters were considered not to represent a change of biological relevance as values remained within the normal range and/or occurred in the absence of a dose-related response.
Coagulation parameters of treated rats were considered not to have been affected by treatment up to 6600 ppm. Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
• Increased urea levels in treated males (1.23x, 1.25x and 1.23x at 740, 2200 and 6600 ppm, respectively; within internal control range).
• Decreased creatinine level in males at 6600 ppm (0.93x; within internal control range).
• Increased sodium levels in all treated males (1.01x in all treatment groups; within internal control range).
• Increased inorganic phosphate level in males at 6600 ppm (1.11x; within internal control range).
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum levels of TSH (thyroid stimulating hormone) and T4 (thyroxine) in were considered unaffected by treatment with the test item.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in treated females at 2200 and 6600 ppm. Relative differences in mean values as compared to the control group are indicated between parentheses.
• Decreased urinary volume (0.67x and 0.61x at 2200 and 6600 ppm, respectively; within internal control range).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Trimethylolpropan were noted in the brain, spinal cord and skeletal muscle of the 2200 and 6600 ppm group males and females.
Brain: Vacuolation of the gray matter, primarily located periaqueductal in the midbrain, and at a lesser amount subventricular (3rd and 4th ventricle) were noted in a few males and in most of the females of the 2200 ppm group and in all rats of the 6600 ppm group (up to moderate).
Spinal cord: Vacuolation of the gray matter, primarily located around the central canal was noted in a few females of the 2200 ppm group and all rats of the 6600 ppm group (up to slight).
Skeletal muscle: Vacuolation, (with or without eosinophilic inclusion) was observed in a few males and females of the 2200 ppm group, and in most rats of the 6600 ppm group (up to moderate). Furthermore, there was an increased incidence and/or severity of animals showing myofiber degeneration/regeneration (minimal) and/or mononuclear cell infiltrate (up to slight) incidence in 2200 and 6600 group males and in 2200 ppm group females.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. 

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, concentration and morphology were considered unaffected by treatment with the test item.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating Index – F0-Generation
Mating index was considered not to be affected by treatment with the test item.

Precoital Time – F0-Generation
Precoital time was considered not to be affected by treatment with the test item.

Number of Implantation Sites – F0-Generation
Number of implantation sites was considered unaffected by treatment with the test item.

Fertility Index – F0-Generation
Fertility index was considered not to be affected by treatment.

Histopathological evaluation of reproductive performance - F0-Generation
There were 3/25 couples of the control group, 4/25 couples of the 740 ppm group, 4/25 couples of the 2200 ppm group and 2/25 couples of the 6600 ppm group without healthy offspring. No histopathologically identified abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Male No. 96 at 6600 ppm showed unilateral lack of sperm in the epididymis and vacuolation and germ cell depletion of the testis. Since these findings occurred unilateral in a single male, with offspring, they were regarded to be unrelated to treatment. The testes of all other examined males revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Gestation Index and Duration – F0-Generation
The duration of gestation was approximately one day longer in 6600 ppm-treated females compared with control mean (22.0 vs. 21.1 days). As this difference was statistically significant and the value at 6600 ppm was at the upper limit of the internal control range , a relation with treatment with the test item could not be excluded. Duration of gestation remained within the normal range at 740 and 2200 ppm.
Gestation index was considered not to be affected by treatment up to 6600 ppm.

Parturition/Maternal Care – F0-Generation
No signs of difficult or prolonged parturition were noted among the pregnant females.

Post-Implantation Survival Index – F0-Generation
At 6600 ppm, post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was lower; 59% compared with 95% for the controls.
The total number of offspring born compared to the total number of uterine implantations was considered unaffected by treatment up to 2200 ppm.

Litter Size – F0-Generation
Up to 2200 ppm, litter size remained unaffected by treatment with the test item.
Litter size was statistically significantly reduced at 6600 ppm (mean number of pups was 7.9 compared to 10.8 in the controls).

Live Birth Index – F0-Generation
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 99, 100, 97 and 98% for the control, 740, 2200 and 6600 ppm groups, respectively.

Viability Index – F0-Generation
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered unaffected by treatment up to 2200 ppm.

Weaning Index –F0-Generation
The weaning index was considered unaffected by treatment up to 2200 ppm.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental results - F0 generation

Effect level:

740 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remarks on result'

Remarks on result:

other: see 'Remarks'

Remarks:

Treatment with Trimethylolpropan, resulted in a decrease in body weight gain in high dose animals (6600 ppm) from Week 4 (females) or Week 5 (males) of premating onwards. Throughout the premating and mating period, a dose-related increase in water consumption was observed in all treatment groups, statistically significant for high dose animals (6600 ppm) only. Following treatment at 2200 and 6600 ppm, adverse test item-related microscopic findings were observed in the brain, spinal cord and skeletal muscle myofibers in males and females. Eosinophilic irregular inclusions were also present in the cytoplasmic vacuoles of skeletal muscle, which were suggestive of degenerated myofibrils and/or a storage myopathy and considered adverse. Additional test item-related changes in organ weights were observed, comprising a higher mean liver weight (absolute and relative to body weight; not statistically significant for absolute values at 2200 ppm) in 2200 ppm and 6600 ppm-treated males and higher mean ovary to body weight ratio in 6600 ppm-treated females.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive results - F0 generation

Effect level:

6 600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks on results'

Remarks on result:

other:

Remarks:

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs) up to the highest dose level tested (6600 ppm).

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental results – F0 generation / F1 generation (pre-weaning)

Effect level:

2 200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks on result'

Remarks on result:

other:

Remarks:

No developmental toxicity was observed up to 2200 ppm. The duration of gestation was approximately one day longer in 6600 ppm-treated females compared with control mean (22.0 vs. 21.1 days). Developmental toxicity following treatment with Trimethylolpropan at 6600 ppm consisted of a decreased post-implantation survival index (resulting in a decreased litter size), decreased viability and weaning indices, decreased mean combined pup weights and increased spleen weights in male pups at PND 22. The total number of offspring born compared to the total number of uterine implantations (post-implantation survival index) was lower following treatment with Trimethylolpropan at 6600 ppm).

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 200 ppm

System:

other:

Organ:

other: Following treatment at 2200 and 6600 ppm, adverse test item-related microscopic findings were observed in the brain, spinal cord and skeletal muscle myofibers in males and females.

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical Observations – F1-Generation
No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations in males and females up to 6600 ppm.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

Mortality – F1-Generation
No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body Weights and Body Weight Gains – F1-Generation
No test item-related effects on body weight and body weight gain were observed up to 2200 ppm.
From weaning onwards, body weights for both sexes were statistically significantly lower at 6600 ppm compared to concurrent controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food Consumption – F1-Generation
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 2200 ppm.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water Consumption – F1-Generation
For females, water consumption (both absolute and relative to body weight) were increased, in all test-item treated groups from Week 4-5 after weaning onwards, reaching statistical significance on most occasions during treatment. For males, the effect was less pronounced and without showing a clear dose-response, although statistically significant on several occasions.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Retinal gliosis was observed in a few 6600 ppm group males and females (up to moderate). This finding consisted of an increased cellularity around the ganglion layer of the retina, most likely representing an increased number of glial cells.

Haematological findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Urinalysis findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Behaviour (functional findings):

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Immunological findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Gross pathological findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Neuropathological findings:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Histopathological evaluation of reproductive performance - F1-Generation (Cohort 1B)
There were 2/20 couples of the control group, 1/20 couple of the 740 ppm group and 5/20 couples of the 6600 ppm group with lack of healthy offspring.
Only for 6600 ppm group in one male microscopic evaluation revealed an explanation for the infertility in the form of marked oligospermia (i.e. reduced sperm) of the epididymides and moderate degeneration/depletion of the round spermatids of the testes. The findings in this male were regarded as unrelated to the test item, since these occurred in a single male of Cohort 1B and these findings were absent in the males of Cohort 1A and males of the F0-Generation. For the remaining rats of the infertile couples, no histopathologically identified abnormalities were seen in the reproductive organs, which could account for their lack of offspring.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Reproduction Data – F1-Generation (Cohort 1B)
Mating Index – F1-Generation (Cohort 1B)
Mating index was considered unaffected by treatment with the test item. The mating indices were 95% for the control, 740 and 6600 ppm groups, and 100% for the 2200 ppm group.
Precoital Time – F1-Generation (Cohort 1B)
Precoital time was unaffected by treatment up to 6600 ppm.
Number of Implantation Sites – F1-Generation (Cohort 1B)
Number of implantation sites was unaffected by treatment up to 2200 ppm.
At 6600 ppm the number of implantation sites was significantly reduced compared to the controls (i.e., mean number of 10.4 at 6600 ppm, compared with 12.2 in controls).
Fertility Index – F1-Generation (Cohort 1B)
Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 100%, 100%, 95% and 89% for controls and females treated at 740, 2200 and 6600 ppm, respectively.
The number of non-pregnant females versus mated females was 1/20 and 2/19 in the 2200 and 6600 ppm groups, respectively. As these cases of non-pregnancy remained within the normal biological variation, these were considered not to be related to treatment with the test item.

Developmental Data – F1-Generation (Cohort 1B)
Gestation Index and Duration – F1-Generation (Cohort 1B)
Gestation index was unaffected by treatment up to 2200 ppm and duration of gestation was unaffected by treatment up to 6600 ppm.
Parturation/Maternal Care – F1-Generation (Cohort 1B)
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-Implantation Survival Index – F1-Generation (Cohort 1B)
The total number of offspring born compared to the total number of uterine implantations (post-implantation survival index) was considered not to be affected by treatment up to 2200 ppm.
At 6600 ppm, post-implantation survival index was lower; 65% compared to 93% for the controls.
Litter Size – F1-Generation Cohort 1B
Up to 2200 ppm, litter size remained unaffected by treatment.
Litter size was statistically significantly reduced at 6600 ppm (mean number of pups was 7.5 compared to 11.4 in the controls).
Sex Ratio – F2-Pups
Sex ratio was considered not to be affected by treatment with the test item.
Live Birth Index – F1-Generation (Cohort 1B)
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered to be unaffected by treatment up to 2200 ppm. The live birth indices were 100% for the control, 740 and 2200 groups. At 6600 ppm, the live birth index was slightly reduced (97%).

Viability Index – F1-Generation Cohort 1B
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered unaffected by treatment up to 2200 ppm.
At 6600 ppm, postnatal loss was statistically significantly increased resulting in a slightly decreased viability index (96% compared to 100% in the controls).
Weaning Index – F1-Generation Cohort 1B
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered unaffected by treatment with the test item.

Examination was performed on Cohort 1A (See 'Results: F1 generation')

Key result

Dose descriptor:

NOAEL

Effect level:

2 200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations. Based on these results, a NOAEL of 2200 ppm (corresponding to an actual test article intake between 225-289 mg/kg/day for males and females) for reproductive toxicity was established.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 200 ppm

System:

female reproductive system

Organ:

other: At 6600 ppm the number of implantation sites was significantly reduced compared to the controls (i.e. mean number of 10.4 at 6600 ppm, compared with 12.2 in controls).

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical Observations – F1-Generation
No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations in males and females up to 6600 ppm.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

Mortality – F1-Generation
No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body Weights and Body Weight Gains – F1-Generation
No test item-related effects on body weight and body weight gain were observed up to 2200 ppm.
From weaning onwards, body weights for both sexes were statistically significantly lower at 6600 ppm compared to concurrent controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food Consumption – F1-Generation
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 2200 ppm.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water Consumption – F1-Generation
For females, water consumption (both absolute and relative to body weight) were increased, in all test-item treated groups from Week 4-5 after weaning onwards, reaching statistical significance on most occasions during treatment. For males, the effect was less pronounced and without showing a clear dose-response, although statistically significant on several occasions.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Retinal gliosis was observed in a few 6600 ppm group males and females (up to moderate). This finding consisted of an increased cellularity around the ganglion layer of the retina, most likely representing an increased number of glial cells.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology – F1-Generation (Cohort 1A)
No changes in haematology parameters were noted in males and females at 740 ppm.
The following statistically significant changes were noted in males or females treated at 2200 and/or 6600 ppm. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
• Increased red blood cell distribution width (RDW) in males at 6600 ppm (1.27x; above internal control range) and females (1.07x; within internal control range). Values of 6/10 high dose males were above the highest control value (ranging between 1.08-2.02x of mean control value).
• Increased mean corpuscular volume (MCV) in females at 6600 ppm (1.03x; within internal control range) and mean corpuscular haemoglobin (MCH) at 2200 and 6600 ppm (1.03 and 1.05x, respectively; above internal control range at 6600 ppm).
Other statistically significant differences in haematology parameters were considered not to represent a change of toxicological relevance based on the minimal magnitude of change, as values remained within the normal range and/or occurred in the absence of a dose-related response.

Coagulation – F1-Generation (Cohort 1A)
Coagulation parameters of treated rats were considered not to have been affected by treatment up to 6600 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical Chemistry – F1-Generation (Cohort 1A)
The following statistically significant (unless indicated otherwise) changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses. Relevant historical control data are provided by footnote .
• Decreased total bilirubin levels in males at 740, 2200 and 6600 ppm (0.85x for all dose levels; not statistically significant; below internal control range) and females at 740, 2200 and 6600 ppm (0.86, 0.77 and 0.82x, respectively; all within internal control range).
• Increased urea levels in males at 2200 and 6600 ppm (1.17x and 1.20x, respectively; statistically significant at 6600 ppm only; within internal control range) and females at 2200 and 6600 ppm (1.14x and 1.24x, respectively; statistically significant at 6600 ppm only; within internal control range).
• Decreased creatinine level in females at 6600 ppm (0.87x; below internal control range).
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical Biochemistry (T4 and TSH levels) on PND 4 and 22-24 – F1-Generation
Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered to be unaffected by treatment.
At 6600 ppm, serum TSH levels in male pups at PND 22 were slightly increased compared with controls, whereas serum T4 levels were slightly reduced (mean values 1.58x and 0.88x of controls, respectively). As these values were not statistically significant and remained well within the internal control range , these effects were considered unrelated to treatment with the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis – F1-Generation (Cohort 1A)
Urinary parameters of treated rats were considered to be unaffected by treatment with the test item.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sexual Maturation – F1-Generation
Sexual maturation (both sexes) was considered to be unaffected by treatment with the test item up to 6600 ppm.

Estrous Cycle – F1-Generation (Cohort 1A)
Length and regularity of the estrous cycle were not affected by treatment up to 6600 ppm. Most females had a regular cycle of 4-5 days between PND 75 and 88.
One control female had an irregular cycle, which was unrelated to treatment with the test item.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ Weights until Weaning – F1-Generation (Surplus Cohort)
No test item-related effects on organ weight were observed up to 2200 ppm.
Test item-related higher spleen weights (relative to body weights) were noted in the 6600 ppm Surplus males and females on PND 22.
An increase in spleen weights (absolute and relative to body weight) was noted in all treatment groups, except for the 740 ppm males. As the values at 740 ppm (females), 2200 ppm (both sexes) and 6600 ppm (females) remained within the internal control range, the increased weights at the low and mid dose and high dose females were considered not toxicologically relevant. For males at 6600 ppm, however, relative spleen weights exceeded the internal control range.
The statistically significantly decreased brain weights at 6600 ppm in both sexes were attributed to the decreased body weights, as relative to body weight mean values remained within the same range as concurrent controls.


Organ Weights – F1-Generation
Cohort 1A
Test item-related organ weight differences (absolute and/or relative to body weights) were noted in the 2200 ppm and/or 6600 ppm group males and females.
Principal statistically significant test item-related organ weight changes were noted in the:
Brain: Lower absolute brain weights were noted in 6600 ppm group rats of both sexes. In females, the brain to body weight ratio was statistically significant increased.
Liver: Higher liver weights (organ to body weight ratio) were observed in 2200 and 6600 ppm group rats of both sexes (at 6600 ppm, mean values were out of internal control range)
Ovaries: Significantly higher ovary weights (body to organ weight ratio) were observed in 6600 ppm group females.
There were no organ weight changes in the 740 ppm group of the F1-Generation Cohort 1A.

Cohort 1B
For the females, only the organ weights of rats that delivered healthy offspring were used for the intergroup comparison (i.e. not mated or non-pregnant females and females with total litter loss or implantation sites only were excluded).
Organ to body weight ratios of ovaries, were statistically significant higher in 6600 ppm group females compared to the control group. Since this change was in line with changes in other cohorts and/or generation, it was regarded as test item-related.
Adrenal glands were most likely affected by a test item-related effect on final body weight (females -18% and -15% when excluding some females based on the conditions given in the paragraph above).
Any other differences, including those that reached statistical significance were considered not to be Trimethylolpropan-related due to the lack of dose-related pattern (prostate gland and epididymides of males), and/or general overlap and variability (thyroid gland of females) in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopy until Weaning – F1-Generation from weaning onwards
No macroscopic findings were noted among pups sacrificed before weaning, spare pups and pups of Cohort Surplus (PND 22) that were considered to be related to treatment up to 2200 ppm.
At 6600 ppm, abnormalities of the skull and/or brain were found in three pups. For pup No. 178-5 an enlarged cranium was observed between Days 18 and 20, and at necropsy the brain was found to contain fluid. Dilated brain ventricles were noted at necropsy for pup No. 184-2 and for pup No. 194-5 the skull and brain were found misshapen (calvaria contained watery-clear fluid) and agenesis of the eyes was observed. At the incidence observed, these findings were contributed to treatment with the test item.

Macroscopic Findings – F1-Generation from weaning onwards
Test item-related gross observations were noted in the brain of two 6600 ppm group males of Cohort 1A, consisting of ventricle dilation (Male No. 417) and a flaccid brain containing fluid and a misshapen cranium (Male No. 418). The microscopic correlates of the brain were ventricular dilation (at moderate or marked degree).
There were no test item-related macroscopic findings in F1-females of Cohort 1A.
Possible test item-related findings were present in the spleen and liver of 6600 ppm group males of Cohort 1B, in the form of thickened spleen (two males) and thickened or enlarged liver (9 males). The thickened liver of a single 740 ppm group male was considered as an incidental finding unrelated to treatment with the test item.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathology – F1-Generation
Test item-related microscopic findings were noted in the brain of pups of both sexes of the 6600 ppm group
Brain:
Ventricular dilation was observed at minimal degree in one male and three female pups, at moderate degree in one female pup and at marked degree (correlated with fluid in the skull and flaccid brain) in one male pup. There was no evidence of vacuolation of the gray matter of the periaqueductal or subventricular areas like what was seen in the parental generations.

Ovarian Follicle Counts (Cohort 1A)
There were no test item-related effects on the ovarian follicle counts in the F1 6600 ppm group females (Cohort 1A) and in the Corpora lutea counts of all test item treated F1-groups when compared to control group females (Cohort 1A). Any variation between group mean counts represented biological variability.

Sperm Analysis – F1-Generation (Cohort 1A)
There were no toxicologically relevant changes in sperm motility, concentration and morphology in Cohort 1A males treated up to 6600 ppm.

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Splenic Lymphocyte Subpopulation – F1-Generation (Cohort 1A)
There were no test item-related effects on splenic lymphocyte subpopulations observed.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Generation:

F1

Effect level:

2 200 ppm

Based on:

test mat.

Remarks:

Based on these results, a NOAEL of 2200 ppm (corresponding to an actual test article intake between 225-289 mg/kg/day for males and females) for reproductive toxicity was established.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

6 600 ppm

System:

other: Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations.

Organ:

other: Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations.

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical Signs and Macroscopic Findings – F2-Pups
No clinical signs occurred and no macroscopic findings were noted among pups that were considered to be related to treatment up to 2200 ppm.
At 6600 ppm, for 5 pups brain abnormalities were noted, consisting of hydrocephaly, a dome-shaped skull, flaccid brain containing fluid and cavity formation in the cerebrum and agenesis of both eyes or fluid in the skull and flaccid brain. At the incidence observed, these finding were considered related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment up to 2200 ppm.
At 6600 ppm, body weights were reduced from PND 7 onwards, reaching statistical significance on PND 21. Mean values for combined pup weight ranging between 0.92-0.94x of controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital Distance – F2-Pups
A dose related decrease in normalized anogenital distance for male pups was observed in all treatment groups (not statistically significant). A similar trend was noted for the normalized anogenital distance in female pups at 6600 ppm.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areola/Nipple Retention – F2-Pups
Treatment up to 6600 ppm had no effect on areola/nipple retention.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ Weights –F2-Pups
Test item-related organ weights differences (absolute and/or relative to body weights) were noted in the 6600 ppm group males and females.
Brain: Lower brain weights were present in the 6600 ppm group in both sexes. (Absolute in males and absolute and relative to body weight ratio in females). The organ weight changes in females at 740 ppm were accompanied by a higher final body weight and not in line with the changes noted in other groups and therefore considered as not test item related.
Spleen: Statistical significant higher spleen weights were present in 6600 ppm group females (absolute and relative to body weight).
Thymus weights were not affected.

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For the observations made with a single incidence (hydrocephaly and small eye; F2-generation) at 740 ppm, followed by a single incidence (partly closed eye; F2-generation) at 2200 ppm, a test item-related effect could not be excluded.
Based on these results, a NOAEL for developmental toxicity could not be established.

Key result

Dose descriptor:

other: Notably, these are singly occurrences at the mid and low dose level which might therefore be considered a chance finding. However, based on the similar nature and rarity of these findings, a test-item-related effect could not be excluded.

Generation:

F2

Effect level:

740 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

740 ppm

System:

other: Single incidence (hydrocephaly and small eye; F2-generation) at 740 ppm, followed by a single incidence (partly closed eye; F2-generation) at 2200 ppm.

Treatment related:

yes

Dose response relationship:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

6 600 ppm

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

An EOGRTS according to OECD TG 443 was requested by ECHA as a result of a compliance check (CCH-D-2114356723-46-01/F).

Conclusions:

An EOGRTS according to OECD TG 443 was requested by ECHA as a result of a compliance check (CCH-D-2114356723-46-01/F).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohort 1A, 1B (extended to a second generation), 1C and Surplus), the following no-observed-adverse-effect level (NOAEL) of Trimethylolpropan were established:

General Toxicity:
The decreased body weight gain (F0-generation only) in combination with decreased food efficiency at 6600 ppm (corresponding to mean actual test article intake between 750-1050 mg/kg/day for males and females) in both generations were considered adverse.
The adverse test item-related microscopic findings from 2200 ppm onward consisted of vacuolation of the gray matter of periventricular areas of the brain and in areas adjacent to the central canal of the spinal cord and cytoplasmic vacuolation of skeletal muscle myofibers. These microscopic findings of the F0-Generation were repeated in the F1-Generation.
Based on these results, a NOAEL of 740 ppm (corresponding to an actual test article intake between 74-99 mg/kg/day for males and females) for general toxicity was established.

Reproductive Toxicity:
Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations.
Based on these results, a NOAEL of 2200 ppm (corresponding to an actual test article intake between 225-289 mg/kg/day for males and females) for reproductive toxicity was established.

Developmental Toxicity:
Overall, developmental toxicity following treatment with Trimethylolpropan at 6600 ppm consisted of a decreased post-implantation survival index (resulting in a decreased litter size), decreased mean combined pup weights and decreased pup viability. Furthermore, adverse test item-related clinical signs/macroscopic findings of the brain/skull and/or eye were observed in three and five affected pups at 6600 ppm, in the F1-generation and F2-generation, respectively.
For the observations made with a single incidence (hydrocephaly and small eye; F2-generation) at 740 ppm, followed by a single incidence (partly closed eye; F2-generation) at 2200 ppm, a test item-related effect could not be excluded.
Notably, these are singly occurrences at the mid and low dose level which might therefore be considered a chance finding. However, based on the similar nature and rarity of these findings, a test-item-related effect could not be excluded.
Based on these results, a NOAEL for developmental toxicity could not be established.

Executive summary:

An EOGRTS according to OECD TG 443 was requested by ECHA as a result of a compliance check (CCH-D-2114356723-46-01/F).

In this OECD TG 443 study the pre- and postnatal effects of Trimethylolpropan 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 were evaluated. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, is expected to identify specific target organs in the offspring. The dose levels in this study were selected to be 0, 740, 2200, 6600 ppm (anticipated dose levels: 0, 70-110, 200-330, 600-1000 mg/kg/day). The water concentrations were adjusted according to the rapid increase in body weight and constant water consumption, resulting in remarkable changes in relative water consumption.

In addition, the study will provide and/or confirm information about the effects of Trimethylolpropan on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters are considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

Due to observations in the F1 generation the study was extended to the F2 generation to finally conclude on the initial observations.

Based on the results of this extended one generation reproductive toxicity study (including Cohort 1A, 1B (extended to a second generation), 1C and Surplus), the following no-observed-adverse-effect level (NOAEL) of Trimethylolpropan were established:

General Toxicity:

The decreased body weight gain (F0-generation only) in combination with decreased food efficiency at 6600 ppm (corresponding to mean actual test article intake between 750-1050 mg/kg/day for males and females) in both generations were considered adverse.

The adverse test item-related microscopic findings from 2200 ppm onward consisted of vacuolation of the gray matter of periventricular areas of the brain and in areas adjacent to the central canal of the spinal cord and cytoplasmic vacuolation of skeletal muscle myofibers. These microscopic findings of the F0-Generation were repeated in the F1-Generation.

Based on these results, a NOAEL of 740 ppm (corresponding to an actual test article intake between 74-99 mg/kg/day for males and females) for general toxicity was established.

Reproductive Toxicity:

Adverse reproductive effects were observed in Cohort 1B animals, in which treatment at 6600 ppm resulted in a significantly reduced number of implantations.

Based on these results, a NOAEL of 2200 ppm (corresponding to an actual test article intake between 225-289 mg/kg/day for males and females) for reproductive toxicity was established.

Developmental Toxicity:

Overall, developmental toxicity following treatment with Trimethylolpropan at 6600 ppm consisted of a decreased post-implantation survival index (resulting in a decreased litter size), decreased mean combined pup weights and decreased pup viability. Furthermore, adverse test item-related clinical signs/macroscopic findings of the brain/skull and/or eye were observed in three and five affected pups at 6600 ppm, in the F1-generation and F2-generation, respectively.

For the observations made with a single incidence (hydrocephaly and small eye; F2-generation) at 740 ppm, followed by a single incidence (partly closed eye; F2-generation) at 2200 ppm, a test item-related effect could not be excluded. Notably, these are singly occurrences at the mid and low dose level which might therefore be considered a chance finding.  However, based on the similar nature and rarity of these findings, a test-item-related effect could not be excluded.

Based on these results, a NOAEL for developmental toxicity could not be established.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study according to OECD TG 443.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An EOGRTS according to OECD TG 443 was requested by ECHA as a result of a compliance check (CCH-D-2114356723-46-01/F).

Effects on developmental toxicity

Description of key information

Two developmental studies according OECD TG 414 - one in rats and another in rabbits – are available. In the rat study the NOAEL of maternal and developmental toxicity was 100 mg/kg/day.
In the rabbit study the maternal and developmental NOAEL for Trimethylolpropan was established as being 450 mg/kg/day, the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Eighty-eight mated female New Zealand White rabbits were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 50, 150 and 450 mg/kg/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Mating procedures:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From Days 6 to 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week.

Duration of test:

28 days

No. of animals per sex per dose:

22 female rabbits per dose

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 450 mg/kg/day was the highest applied dose

Key result

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal findings

A dose-related effect on food consumption and body weight gain was seen at 150 and 450 mg/kg/day. When compared to the control group, reduced mean values for food intake and body weight gain were seen during the first 10 days of treatment (i.e. from Days 6-16 post-coitum). Changes were relatively slight and did not always reach statistical significance. Furthermore, as recovery towards control values was seen during the remainder of the treatment period, the decreases in food intake and body weight gain were not considered to be adverse. Terminal body weights corrected for (gravid) uterine weight were unaffected by treatment.

No effects on food consumption and body weight were seen after treatment at 50 mg/kg/day.

Remaining maternal parameters (i.e. mortality, clinical signs, macroscopic appearance at necropsy and maternal pregnancy data) were unaffected in the 50, 150 and 450 mg/kg/day groups.

Developmental findings

No developmental toxicity was observed after treatment up to 450 mg/kg/day.

The trend towards slightly lower male and female fetal body weights noted at 150 and 450 mg/kg/day was most likely secondary to the lower body weight gains observed in maternal animals from Days 6-16 post-coitum. As changes were only slight (not reaching statistical significance) and all values remained within normal limits, it was not considered to be toxicologically relevant.

Litter size and sex ratio were unaffected by treatment up to 450 mg/kg/day.

There were no treatment-related external, visceral and skeletal variations or malformations.

Executive summary:

Principles of method:

Eighty-eight mated female New Zealand White rabbits were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 50, 150 and 450 mg/kg/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

Maternal findings

A dose-related effect on food consumption and body weight gain was seen at 150 and 450 mg/kg/day. When compared to the control group, reduced mean values for food intake and body weight gain were seen during the first 10 days of treatment (i.e. from Days 6-16 post-coitum). Changes were relatively slight and did not always reach statistical significance. Furthermore, as recovery towards control values was seen during the remainder of the treatment period, the decreases in food intake and body weight gain were not considered to be adverse. Terminal body weights corrected for (gravid) uterine weight were unaffected by treatment.

No effects on food consumption and body weight were seen after treatment at 50 mg/kg/day.

Remaining maternal parameters (i.e. mortality, clinical signs, macroscopic appearance at necropsy and maternal pregnancy data) were unaffected in the 50, 150 and 450 mg/kg/day groups.

Developmental findings

No developmental toxicity was observed after treatment up to 450 mg/kg/day.

The trend towards slightly lower male and female fetal body weights noted at 150 and 450 mg/kg/day was most likely secondary to the lower body weight gains observed in maternal animals from Days 6-16 post-coitum. As changes were only slight (not reaching statistical significance) and all values remained within normal limits, it was not considered to be toxicologically relevant.

Litter size and sex ratio were unaffected by treatment up to 450 mg/kg/day.

There were no treatment-related external, visceral and skeletal variations or malformations.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Trimethylolpropan was established as being 450 mg/kg/day, the highest dose tested.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

This study was conducted in compliance with the following guidelines:
- OECD guidelines: Guidelines for Testing of Chemicals, Section 4: Health Effects, No. 414 “Prenatal Developmental Toxicity Study“, adopted January 22, 2001.
- US-EPA: Health Effects Test Guidelines OPPTS 870.3700: “Prenatal Developmental Toxicity Study“, EPA 712-C-98-207, dated August 1998.
- Japanese MAFF guidelines: Guideline on the Compiling of Test Results on Toxicity “Teratology Study”, 12-Nousan No. 8147 of November 24, 2000, amended June 26, 2001.
- EEC guidelines: Commission Directive 2004/73/EEC, Official Journal of the European Communities L 152, dated April 29, 2004.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Strain: Wistar (Hsd Cpb:WU), SPF-bred

During acclimatization in groups; starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings supplied by Ssniff GmbH, 59494 Soest, Germany. One new wooden block for environmental enrichment (aspen bricks supplied by TAPVEI OY, 73620 Kortteinen, Finland) was added to each animal at start of study and renewed as needed. Wood shavings were spot-checked for possible contaminant levels.

Feeding: Ad libitum; Provimi Kliba Maus/Ratte-Haltung-GLP, Art.- No.: 3883 PM S15, producer: Provimi Kliba SA, 4303 Kaiseraugst, Switzerland
Specification of ingredients and contaminants: According to recommendations of GV SOLAS, Aug. 2001.

Water: Ad libitum; Tap water in polycarbonate bottles.
Specification of contaminants: According to actual German drinking water standards.
Cleaning of Animal Room and Pest Control: Cleaned daily and disinfected at least once a month using a disinfectant (aqueous preparation of Tego 2000 by Th. Goldschmidt AG, 45127 Essen, Germany). Continuous pest control using sticky cockroach traps on pheromone basis (purchased from Killgerm GmbH, 41460 Neuss, Germany).

Cleaning of Cage Material: Routine exchange of cages (with litter) and drinking bottles (at least two times a week); routine exchange of cage lids and racks.
Room Temperature: 22 ± 2°C
Relative Humidity: Approximately 55 %.
Temperature and relative humidity recorded continuously.
Air Change: > 10 per hour
Light/Dark Regimen: 12h/12h
Radio program playing during the light period.

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the formulation was confirmed by the analysis of concentration and stability of dosage forms prepared in the same way as it were done in the study. Data on homogeneity of the test substance in the administration formulations were assessed in study no. 2012/0074/05.
Content checks of the active ingredient in samples with concentrations of 10, 30, and 100 mg/mL were carried out twice for the formulation preparation days of 08-APR- 2013 and 14-APR-2013

Details on mating procedure:

The animals were mated by placing two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

Day 6 – 20 of gestation

Frequency of treatment:

Once daily

Duration of test:

02-APR-12013 – 13-JUN-2013

No. of animals per sex per dose:

25 / 25 / 30 / 25 females per dose
0 / 100 / 300/ 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Maternal examinations:

Evaluation of the general tolerance of the test substance by the females was based on appearance and behavior, food and water intakes, appearance of excretory products, body weight development, and mortality of the animals as well as on gross pathological findings.

Ovaries and uterine content:

uterine weights, individual weight and appearance of the placentas, number of early resorptions (only implantation site visible), late resorptions (fetal or placental remnant visible), and dead fetuses (fetuses without signs of life, but without maceration).

Fetal examinations:

The following parameters were determined and assessed at cesarean section and during the subsequent fetal evaluation without knowledge of treatment groups: number of corpora lutea, number of implantations (in females without visible implantation sites after staining of the uterus with a solution of 10 % ammonium sulfide), number of live fetuses, sex of live fetuses, individual weights of live fetuses, external malformations and other findings deviating from normal, visceral malformations and other findings deviating from normal [evaluation of about half of the fetuses by scalpel and/or razor blade sectioning according to the modified WILSON technique (processing and evaluation after cesarean section without knowledge of treatment group)], findings in abdominal, pelvic, and thoracic organs as well as skeletal findings by the modified DAWSON technique [Every other fetus within a litter was prepared for either skeletal or visceral evaluation with generally the first fetus of each litter assigned to skeletal evaluation. The visceral findings observed during the evisceration of fetuses assigned for skeletal evaluation are listed together with the external findings]

Statistics:

Differences between the control and Trimethylolpropan-treated groups were considered to be significant when p < 0.05.

1 Analysis of Variance (ANOVA); in case of significance Dunnett's test for: feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female

2 N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for, fertility rate, gestation rate, number of implantations per group, number of pre-implantation losses per group, number of post-implantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group
Skeletal localizations with mechanical damage in single fetuses were excluded from the calculation of percentages of affected localizations.

3 Kruskal-Wallis test and in case of significant differences Dunn's test for: number of pre-implantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.

Indices:

included in the report

Historical control data:

included in the report as an Annex.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: body weight decrease

Details on maternal toxic effects:
Statistically significant mean body weight loss occurred at the 1000 mg/kg group between days 8-21 p.c. This resulted in statistically significantly reduced final body weights at 1000 mg/kg bw/day.
Absolute body weight gain (days 6 – 20 p.c. and days 0 – 21 p.c.) and corrected body weight gain (days 0 – 21 p.c.) were statistically significantly decreased by treatment at dose levels of 300 mg/kg bw/day and above. Final mean body weight at 300 mg/kg bw/day was lower but not statistically significantly reduced when compared to the final mean body weight of the control and the low dose group.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: 300 mg/kg/day: body weight decrease; 1000 mg/kg/day anophthalmia, misshapen eyes, dilation of brain ventricles, shortened right hind leg in one litter, placental weight

Details on embryotoxic / teratogenic effects:
Twenty-five, respectively thirty (300 mg/kg group, only) inseminated female Wistar rats each were treated daily orally by gavage with Trimethylolpropan (purity 99.0 %) in tap water from day 6 to day 20 p.c. in the following doses: 0, 100, 300, and 1000 mg/kg body weight (bw)/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

At 1000 mg/kg bw/day, animals were seen with sunken flanks, hypoactive and with decreased water consumption. Mean body weight loss occurred transiently at the 1000 mg/kg dose level. Absolute and corrected body weight gains and carcass weight were also decreased by treatment at dose levels of 300 mg/kg bw/day and above, and resulted in statistically significantly reduced final body weights at 1000 mg/kg bw/day.

Food intake was decreased at 1000 mg/kg, whereas water intake and fecal and urinary excretions were mostly unaffected.
Necropsy revealed no treatment related findings up to 1000 mg/kg.

The gestation rate was unaffected by treatment at dose levels up to 1000 mg/kg.

At 1000 mg/kg, placentas were light discolored or greenish yellowish placental borders were noted and placental weight was minimally reduced.

Post-implantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 300 mg/kg. At 1000 mg/kg, the mean post-implantation loss was statistically significantly increased, yet could not be confirmed to a decreased number of living fetuses. The number of fetuses was unaffected by treatment up to 1000 mg/kg bw/day.

Fetal sex distribution was unaffected by treatment at dose levels up to 1000 mg/kg.

Fetal weights were dose-dependently decreased at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw /day.

A treatment related effect on malformations (incidence or type) was not evident at dose levels up to 300 mg/kg.

At the distinctly maternally toxic 1000 mg/kg dose level, a treatment related effect on fetal malformations (mainly dysplastic, fore- and hind limb bones, malformation of eyes and dilation of brain ventricles) is assumed.

A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg. At 1000 mg/kg bw/day, increased fetal deviations were noted as pale skin and slight edema. These findings were assumed as sign of fetal retardation in relation to the decreased maternal weight gain and the decreased fetal weight. A treatment related effect is assumed for the number of slight dilations of the different brain ventricles, taking into account the according malformations and the distinctly reduced fetal weights.

A treatment related effect on skeletal deviations (retardations and variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) and incidence of common skeletal variations (wavy and 14th ribs) at the maternally toxic 1000 mg/kg level, and borderline effects of a few localizations (digits, toes, sternebrae, wavy ribs) at the still maternally toxic 300 mg/kg level in relation to dose-dependently decreased fetal weights at 300 mg/kg and 1000 mg/kg dose levels.

Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined:
Maternal toxicity: 100 mg/kg bw/day
Developmental toxicity: 100 mg/kg bw/day

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Fetal weights

Remarks on result:

other: see 'Remarks'

Remarks:

The fetal weights were dose-dependently decreased at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw /day. A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg. A treatment related effect on skeletal deviations (retardations and variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) and incidence of common skeletal variations (wavy and 14th ribs) at the maternally toxic 1000 mg/kg level, and borderline effects of a few localizations (digits, toes, sternebrae, wavy ribs) at the still maternally toxic 300 mg/kg level in relation to dose-dependently decreased fetal weights at 300 mg/kg and 1000 mg/kg dose levels.

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Fetal weights

Description (incidence and severity):

The fetal weights were dose-dependently decreased at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw /day. A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg. A treatment related effect on skeletal deviations (retardations and variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) and incidence of common skeletal variations (wavy and 14th ribs) at the maternally toxic 1000 mg/kg level, and borderline effects of a few localizations (digits, toes, sternebrae, wavy ribs) at the still maternally toxic 300 mg/kg level in relation to dose-dependently decreased fetal weights at 300 mg/kg and 1000 mg/kg dose levels.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Body Weights

Treatment-related impairment of body weight gain was observed in relation to treatment at 300 mg/kg and above. This was characterized by statistically significant mean body weight loss in the 1000 mg/kg group between days 8-12 p.c. followed by significantly reduced body weight gain from day 12 – 14 p.c. , as well as statistically significantly reduced absolute body weight gain during treatment (day 6-20 p.c.) and gestation (days 0-21 p.c) and distinctly to severely decreased corrected body weight gain and carcass weight at 300 mg/kg and above. At 1000 mg/kg statistically significantly reduced final body weights were seen.

Additionally mean gravid uterus weight was statistically significantly reduced at 1000 mg/kg in relation to statistically significantly reduced fetal weights

Food and Water Intake, Excretory Products

Food intake was statistically significantly decreased between days 6-15 p.c. at the high dose level of 1000 mg/kg. Correspondingly, mean body weight of the animals was statistically significantly decreased starting on day 8 p.c. until necropsy..

Besides findings regarding water intake and urination at 300 mg/kg and above already mentioned in Section 5.2.1, animals nos. 101, 102, 103, 104, and 105 of the 300 mg/kg group showed increased water consumption on a single day (day 15 p.c.). At 1000 mg/kg only animal no. 90 showed increased water consumption together with increased urination starting on day 18 p.c. until necropsy on day 21 p.c. Treatment relation was excluded for these sporadic findings without dose relation.

At 1000 mg/kg light colored feces were seen in animals nos. 9, 14, 26, and 36 on various days between day 11 – 17 p.c. As this observation was most probably related to the high amount of light colored test substance given, toxicological relevance was not attributed to this finding.

Thus, while feed intake was impaired at 1000 mg/kg, mean water intake, and excretions of the females with viable fetuses at cesarean section were unaffected by test item treatment up to 1000 mg/kg.

Necropsy

One female (no. 74) of the 1000 mg/kg group which was sacrificed on day 7 p.c. revealed ulceration at the genital (vulva) region.

Bilateral or unilateral dilation of the renal pelvis occurred at all dose levels in three, respectively two animals at 1000 mg/kg, worms in the intestinal tract were seen in 2 animals at 100 mg/kg, and one animal with the uterus filled with clear fluid was found at 300 mg/kg (the female had no implantation sites). These findings were considered incidental, a treatment related effect is excluded due to a lacking dose dependency, and because these findings are known as spontaneous findings in the rat strain used.

All remaining females showed no remarkable findings.

General Reproduction Data

The fertility rate was incidentally slightly lower at the 300 mg/kg dose level. However, as implantation generally occurs before start of treatment on day 6 p.c. treatment relation was excluded for this finding. The mean number of corpora lutea and preimplantation loss did not differ to a statistically significant extent from the control group values and values were comparable with the range of normal scattering for the rat strain used. Therefore, an impact on the outcome of the study due to unequal distribution of females in the different groups with respect to the parameters mentioned above is excluded.

Effect of Test Compound on Intrauterine Development

Gestation Rate

The gestation rate / gestation index was unaffected by treatment at dose levels up to 1000 mg/kg.

Weight and Appearance of Placentas

At all dose levels mean placental weights were statistically significantly reduced in the low and medium dose and slightly more at 1000 mg/kg

As the placental weights were within the normal range of historical control data (cf. historical control data in the Annex 8.8.9) this findings was assumed to be incidental up to 300 mg/kg. Of note, litter size was incidentally as well higher at 100 and 300 mg/kg.

At 1000 mg/kg placental weight was slightly below the range of historical controls so that treatment relation could not totally be excluded.

Appearance of placentas was unaffected by treatment at dose levels up to 300 mg/kg. At 1000 mg/kg, 11 placentas of 6 litters were light discolored. The incidence for this finding was statistically significantly different to controls. A greenish yellowish placental border was noted in 5 placentas of 2 litters. The overall number of placental findings at 1000 mg/kg (16 placentas within 7 litters) showed statistical significance on an individual fetus and litter basis.

Post-implantation Loss, Number of Fetuses

Post-implantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 300 mg/kg.

At 1000 mg/kg, the mean post-implantation loss was statistically significantly increased. Although the mean number of fetuses was identical to the control group value, treatment relation cannot be excluded as incidence of postimplantation loss was above the range of historical control data

Sex of Fetuses

Fetal sex distribution was unaffected by treatment at dose levels up to 1000 mg/kg.

Fetal Weight

Mean fetal weights were statistically significantly decreased at dose levels of 300 mg/kg bw/day and above. When the mean fetal weight was separated in male and female fetuses, the corresponding fetal male and female weights were also statistically significantly decreased at dose levels of 300 mg/kg bw/day and above.

Eighty-four fetuses at 1000 mg/kg were noted with 3.0 g or less. The smallest fetus weighed 1.93 g. Seventy-nine fetuses weighed between 3 g and 4 g, and 75 fetuses weighed more.

Treatment relation was assumed for these observations at 1000 mg/kg.

Although average litter size at 300 mg/kg – which may have affected fetal weights - was considerably higher than the current control value (mean litter size 13.8 versus 11.9 in the control), treatment relation can neither be excluded for slightly reduced fetal weight at 300 mg/kg since values lay below the range of historical control data

The observation of reduced fetal weights was related to distinctly to severely impaired maternal body weight gain (e.g. corrected body weight gain) at dose levels of 300 mg/kg bw and above.

Fetal Malformations

The overall incidences of fetuses or litters with malformations lay within the range of historical control data and revealed no statistical significance, and were thus unaffected by treatment at dose levels up to 300 mg/kg.

At 1000 mg/kg bw/day, statistically significantly increased individual malformations were noted mainly as dysplastic forelimb bones (humerus, ulna, radius, scapula, and clavicle at the right or left site, or bilateral) and hind limb bones (dysplastic femurs), a combined skeletal finding (1st sacral vertebral arch has the shape of lumbar vertebral arch, left; pelvis shift to caudal, left), and alterations of the eyes at the head as eyehole reduced in size (skeletal finding), eye rudiment flat (external finding), eye ball missing or reduced in size (visceral finding), eye hole reduced in size (visceral finding)], and dilation of brain ventricles (visceral finding). The incidence of these findings was not covered by historical data with necropsy on day 20 p.c. or day 21 p.c .

The other skeletal findings were assumed incidental and a sign of retardation as a lot of fetuses were noted with very low weight between 2.55 g (fetus no. 346, dam no. 29; fetus no. 430, dam no. 36) and 3.82 g (fetus no. 451, dam no. 39).

During gross external examination, five fetuses (nos. 683, 687, 692, 695, and 696) of one dam no. 61 were noted with shortened right hind limb. Four of these 5 fetuses revealed dysplastic limb bones during skeletal evaluation so that a treatment related effects could not be excluded.

At 1000 mg/kg, further malformations were noted as insignificantly increased during skeletal examination as dysplastic tibia, fibula, proximal and distal phalanges, and metatarsals (at the right or left site, or bilateral), two different combined skeletal findings, missing thoracic vertebral arches, one supernumerary lumbar vertebral arch, and missing lumbar vertebral arches. During gross external examination, malformations were noted as a general edema in three fetuses of three litters, and a situs inversus totalis in one fetus. During visceral examination, a ventricular septal defect of the heart and the esophagus lying on the right side of the thoracic cavity were seen in one fetus and in three fetuses of two litters, respectively.These types of malformations are generally representative samples of spontaneous malformations in the rat strain used, and comparable with spontaneous findings in historical control groups.

All remaining findings were isolated findings, revealed no dose dependency and were thus considered as incidental.

Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to 300 mg/kg.

Fetal External and Visceral Deviations

External and visceral deviations comprised general edem edema, domed head, slight edema at the head, eye rudiment flat (not confirmed as malformation), anterior and posterior eye chambers filled with whitish mass, vitreous body disintegrated, thyroid gland reduced in size, thymus gland extended cranially, brown mass in the abdominal cavity, brown spots in the liver, origin of left carotid artery displaced towards left subclavian artery on aortic arch, slight dilation of renal pelvis, slight dilation of ureter, and displaced testes, pale skin, slight edema during visceral examination, and slight dilations of the different brain ventricles (lateral, 3rd, and 4th ventricle).

The external and visceral deviations observed were of common type and comparable with spontaneous findings as seen in the current control group and/or historical control groups and represented the normal range of scattering in the rat strain used in this study.

At 1000 mg/kg bw/day, statistically significantly increased fetal deviations were noted mainly as pale skin, slight edema during visceral examination, and slight dilations of the different brain ventricles (lateral, 3rd, and 4th ventricle).

The other findings were assumed as sign of fetal retardation secondary to the decreased maternal body weight gain and the decreased fetal weight: In line with this interpretation, the highest incidences were seen in the 1000 mg/kg group.

A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg.

Fetal Skeletal Deviations

At the 1000 mg/kg level, fetal examinations for degree of ossification and skeletal variations revealed statistically significant findings at several localizations (retarded ossification of 1st and 5th distal phalanges of digits bilateral, 2nd-5th proximal phalanges of digits bilateral, 5th metacarpals bilateral, 1st and 5th distal phalanges of toes bilateral, 2nd-4th proximal phalanges of toes bilateral , 1st metatarsals bilateral, 3rd-6th sternebrae, l, 1st-7th cervical vertebral bodies, 4th-13th thoracic vertebral bodies, 3rd caudal vertebral arches bilateral, supraoccipital bone as well as increased incidence of wavy ribs (2nd-12th ) and supernumerary 14th ribs (punctiform and/or comma-shaped, sum, right, left, and bilateral), when calculation was done on a fetal basis and mostly also on a litter basis. , Here a treatment related effect is assumed, since findings were seen on a fetal and litter basis, since some values on a fetal basis lay outside the range of historical control data (necropsy on day 21 p.c.) of the rat strain used (see Annex, Section 8.8.12) and since fetal weights were also significantly decreased at this dose level

At the 300 mg/kg level, fetal examinations for degree of ossification and skeletal variations revealed statistically significant findings at several localizations (reduced degree of ossification of 1st distal phalanges of digits right, 2nd, 3rd, and 5th proximal phalanges of digits bilateral, 4th proximal phalanges of toes bilateral and 5th sternebra, as well as less sternebral supernumerary ossification centers, less dumbbell shaped 11th and 12th thoracic vertebral bodies, an increased number of fetuses with 7th caudal vertebral body present as well as an increased number of fetuses with wavy ribs), when calculation was done on a fetal basis. None of these findings was statistically significant on a litter basis. However, a treatment related effect cannot totally be excluded, since fetal weights were also decreased at this dose level, and since a few values on a fetal basis lay outside the range of historical control data of the rat strain used.

Thus, a treatment related effect on skeletal deviations (retardations, variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, ribs, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) at the 1000 mg/kg level, and it cannot be excluded for skeletal deviations at few localizations (digits, toes, sternebrae, ribs) as borderline effects at the 300 mg/kg level in relation to dose-dependently statistically significantly decreased fetal weights at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw/day.

Executive summary:

Maternal NOAEL: 100 mg/kg bw/day:

At 1000 mg/kg bw/day, animals were seen with sunken flanks, hypoactive and with decreased water consumption. Mean body weight loss occurred transiently at the 1000 mg/kg dose level. Absolute and corrected body weight gains and carcass weight were also decreased by treatment at dose levels of 300 mg/kg bw/day and above, and resulted in statistically significantly reduced final body weights at 1000 mg/kg bw/day.

Developmental toxicity NOAEL: 100 mg/kg bw/day:

Fetal weights were dose-dependently decreased at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw /day.

A treatment related effect on malformations (incidence or type) was not evident at dose levels up to 300 mg/kg. At the distinctly maternally toxic 1000 mg/kg dose level, a treatment related effect on fetal malformations (mainly dysplastic, fore- and hind limb bones, malformation of eyes and dilation of brain ventricles) is assumed.

A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg. At 1000 mg/kg bw/day, increased fetal deviations were noted as pale skin and slight edema. These findings were assumed as sign of fetal retardation in relation to the decreased maternal weight gain and the decreased fetal weight. A treatment related effect is assumed for the number of slight dilations of the different brain ventricles, taking into account the according malformations and the distinctly reduced fetal weights.

A treatment related effect on skeletal deviations (retardations and variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) and incidence of common skeletal variations (wavy and 14th ribs) at the maternally toxic 1000 mg/kg level, and borderline effects of a few localizations (digits, toes, sternebrae, wavy ribs) at the still maternally toxic 300 mg/kg level in relation to dose-dependently decreased fetal weights at 300 mg/kg and 1000 mg/kg dose levels.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rat

Quality of whole database:

Guideline study according to OECD TG 414 in rats.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD TG 414 Study was conducted in 2013/2014. Twenty-five, respectively thirty (300 mg/kg group, only) inseminated female Wistar rats each were treated daily orally by gavage with Trimethylolpropan (purity 99.0 %) in tap water from day 6 to day 20 p.c. in the following doses: 0, 100, 300, and 1000 mg/kg body weight (bw)/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

At 1000 mg/kg bw/day, animals were seen with sunken flanks, hypoactive and with decreased water consumption. Mean body weight loss occurred transiently at the 1000 mg/kg dose level. Absolute and corrected body weight gains and carcass weight were also decreased by treatment at dose levels of 300 mg/kg bw/day and above, and resulted in statistically significantly reduced final body weights at 1000 mg/kg bw/day.

Food intake was decreased at 1000 mg/kg, whereas water intake and fecal and urinary excretions were mostly unaffected.

Necropsy revealed no treatment related findings up to 1000 mg/kg.

The gestation rate was unaffected by treatment at dose levels up to 1000 mg/kg.

At 1000 mg/kg, placentas were light discolored or greenish yellowish placental borders were noted and placental weight was minimally reduced.

Post-implantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 300 mg/kg. At 1000 mg/kg, the mean post-implantation loss was statistically significantly increased, yet could not be confirmed to a decreased number of living fetuses. The number of fetuses was unaffected by treatment up to 1000 mg/kg bw/day.

Fetal sex distribution was unaffected by treatment at dose levels up to 1000 mg/kg.

Fetal weights were dose-dependently decreased at dose levels of 300 mg/kg bw/day and 1000 mg/kg bw /day.

A treatment related effect on malformations (incidence or type) was not evident at dose levels up to 300 mg/kg.

At the distinctly maternally toxic 1000 mg/kg dose level, a treatment related effect on fetal malformations (mainly dysplastic, fore- and hind limb bones, malformation of eyes and dilation of brain ventricles) is assumed.

A treatment related effect on external and visceral deviations was not evident at dose levels up to 300 mg/kg. At 1000 mg/kg bw/day, increased fetal deviations were noted as pale skin and slight edema. These findings were assumed as sign of fetal retardation in relation to the decreased maternal weight gain and the decreased fetal weight. A treatment related effect is assumed for the number of slight dilations of the different brain ventricles, taking into account the according malformations and the distinctly reduced fetal weights.

A treatment related effect on skeletal deviations (retardations and variations) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, thoracic vertebral bodies, caudal vertebral arches, supra¬occipital bone) and incidence of common skeletal variations (wavy and 14th ribs) at the maternally toxic 1000 mg/kg level, and borderline effects of a few localizations (digits, toes, sternebrae, wavy ribs) at the still maternally toxic 300 mg/kg level in relation to dose-dependently decreased fetal weights at 300 mg/kg and 1000 mg/kg dose levels.

Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined in rats:

Maternal toxicity: 100 mg/kg bw/day

Developmental toxicity: 100 mg/kg bw/day

Eighty-eight mated female New Zealand White rabbits were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 50, 150 and 450 mg/kg/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

Maternal findings

A dose-related effect on food consumption and body weight gain was seen at 150 and 450 mg/kg/day. When compared to the control group, reduced mean values for food intake and body weight gain were seen during the first 10 days of treatment (i.e. from Days 6-16 post-coitum). Changes were relatively slight and did not always reach statistical significance. Furthermore, as recovery towards control values was seen during the remainder of the treatment period, the decreases in food intake and body weight gain were not considered to be adverse. Terminal body weights corrected for (gravid) uterine weight were unaffected by treatment.

No effects on food consumption and body weight were seen after treatment at 50 mg/kg/day.

Remaining maternal parameters (i.e. mortality, clinical signs, macroscopic appearance at necropsy and maternal pregnancy data) were unaffected in the 50, 150 and 450 mg/kg/day groups.

Developmental findings

No developmental toxicity was observed after treatment up to 450 mg/kg/day.

The trend towards slightly lower male and female fetal body weights noted at 150 and 450 mg/kg/day was most likely secondary to the lower body weight gains observed in maternal animals from Days 6-16 post-coitum. As changes were only slight (not reaching statistical significance) and all values remained within normal limits, it was not considered to be toxicologically relevant.

Litter size and sex ratio were unaffected by treatment up to 450 mg/kg/day.

There were no treatment-related external, visceral and skeletal variations or malformations.

Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined in rabbits:

Maternal toxicity: 450 mg/kg bw/day

Developmental toxicity: 450 mg/kg bw/day

In conclusion, the NOAELs for maternal and developmental toxicity in rabbits are higher than in rats.

Therefore the NOAELs in the rat study are used for the endpoint conclusion and selection.

Justification for selection of Effect on developmental toxicity: via oral route:
Two Guideline study according to OECD TG 414 in rats and rabbits are available.

Toxicity to reproduction: other studies

Description of key information

After feeding of trimethylolpropane to young male and female Wistar rats at 0, 0.03, 0.1, 0.3, 1.0 % in diet (corresponding to 20, 67, 200 or 667 mg/kg bw/d) for three months no adverse effects on reproductive organs of male Wistar rats were found; the significantly increased average relative weight of ovars in female rats occurred at clear toxic dose and was without pathological or histopathological correlate. Thus, this finding cannot be evaluated as specific effect due to reproductive toxicity.

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification as Repr. 2 ( H361df: Suspected of damaging fertility. Suspected of damaging the unborn child.) is justified.