2,2-bis(hydroxymethyl)butanoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Only one study was conducted to evaluate the potential of the test item (2,2-bis(hydroxymethyl) butanoic acid,) to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The study was carried out in accordance to the OECD Guideline 421: Reproduction/Developmental Toxicity Screening Test (2016) in GLP condition.

 

The test item (2,2-bis(hydroxymethyl)butanoic acid), in the vehicle distilled water, was administered by gavage once daily to three groups of Sprague-Dawley rats at the doses 200, 500 and 1250 mg/kg bw/day. A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 5 mL/kg bw. Each group consisted of 12 males and 12 females used for dosing and mating. Males were administered 14 daily doses before mating and continuing throughout the mating period for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 12 (for females that delivered) for a total of 48-54 doses; females with no evidence of mating/that failed to deliver were dosed for a total of 54-55 doses.

All animals were observed twice daily for mortality and morbidity and daily during dosing. Detailed clinical observations, body weights, and food consumption were recorded weekly.

All parental females were allowed to deliver and rear their pups until lactation day 13. Born pups were counted and sexed and litters were weighed on post-natal days (PND) 0-1, 4, 7 and 13. The anogenital distance (AGD) of each pup was measured at the end of delivery (on PND 0-1). Any abnormal behavior of the offspring was recorded. The number of nipples/areolae in male pups was counted on PND 13.

Parental males were euthanized following completion of the 28 days dosing period and females that delivered or failed to deliver were euthanized on lactation day 13 (with their litters), or after 54-55 days of dosing, respectively. Complete necropsies were conducted on all parental animals, and selected organs were weighed. Reproductive organs and thyroid glands were examined microscopically from all parental animals in the vehicle control and high-dose groups.

On PND 4, litter sizes were standardized to 5 male and 5 female pups, where possible. Surplus pups were euthanized and blood was sampled for determination of serum thyroxin levels. The remaining F1 pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the external reproductive genitals and with weighing and histology of thyroid glands. Thyroxin level was assayed in adult males and females, and PND 4 and PND 13 pups.

 

There was no morbidity and mortality of animals caused by the test item administration. The test item-related clinical finding was diarrhea in the 1250 mg/kg bw/day dose group. The total body weight gain was slightly decreased in males starting from lower 200 mg/kg bw/day dose; females body weight gain was non-significantly decreased for the gestation and lactation periods on 1250 mg/kg bw/day dose. The food consumption was not changed. Males of the 500 and 1250 mg/kg bw/day dose group had the increased weight of kidneys relative to body weight.

The test item did not affect the reproductive performance of males.

The test item administration has not changed the indices of fertility, conception, and gestation of females. Decrease in the number of implantation sites (1250 mg/kg bw/day), decrease in the number of delivered pups (500 and 1250 mg/kg bw/day), decrease in the mean number of female newborns per litter (1250 mg/kg bw/day), increased pre-natal loss of offspring (500 and 1250 mg/kg bw/day doses), is a female reproductive performance effect.

In the study with DMBA, the control vehicle group had a low prenatal loss value, which gave a statistical increase in the percentage of prenatal loss starting from 500 mg/kg. However, by linking to historical control data, it is possible to conclude that the number of implantation sites on the 500 mg/kg dose was the same as in historical control; the decrease in the mean number of liveborns was only a weak trend, and a slight increase in the post-implantation loss was not statistically changed compared to the historical control value. Considering that other fertility and post-natal development parameters did not change at a dose of 500 mg/kg, a slight increase in the percentage of post-implantation loss, which remained at the level of the historical control value, could be considered as non-adverse.

There were no abnormal pups, changes in F1 offspring postnatal survival and body weight gain, no changes in anogenital distance, and signs of offspring masculinization or feminization. Relative thyroids weight was decreased on postnatal day 13 in the high dose group without clear evidence with the treatment; thyroxin level was not changed; there was no test item related microscopic findings in offspring thyroids.

 

Therefore, under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was:

• NOAEL for parental males’ reproductive toxicity: 1250 mg/kg bw/day


• NOAEL for parental females’ reproductive toxicity 200 mg/kg bw/day (based on female reproductive performance effect - decrease in number of implantation site (1250 mg/kg), decrease in number of delivered pups per litter (1250 mg/kg), decrease in number of delivered female pups per litter (1250 mg/kg), increase in pre-natal loss of offspring (500 and 1250 mg/kg))


• NOAEL for F1 postnatal developmental toxicity: 1250 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley (SD), outbreed

Details on species / strain selection:

Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS: Nauki 6, Puschino, Moscow region, Russia 142290
www.spf-animals.ru
Characteristic: Nulliparous and non-pregnant animals with defined microbiological health status received by separate litters to avoid of sibling mating. The animal health monitoring is performed by breeder under FELASA-guidelines quarterly in AnLab, s.r.o. (Czech Republic); the report on health monitoring is attached to the study file.


Age: Approximately 9 weeks old on day of pre-mating oestrous cycle evaluation;
approximately 10-11 weeks old at the initiation of dose administration (day 1);
approximately 12-13 weeks old when paired on study day 14.

Body weight at first day of dosing (MEAN ± SD):
Males: 252-326 g, N = 48
Females: 171-231 g, N = 48

Sex:

male/female

Details on test animals or test system and environmental conditions:

Diet: The animals were fed Standard Diet for laboratory mice and rats Mucedola 4RF21 autoclavabile (Mucedola s.r.l., Via G.Galelei, 4-20019 Settimo Milanese MI, Italy) ad libitum.
Water: Filtered by MilliRO tap water was provided ad libitum in standard water bottles
Environmental Conditions: - Temperature: 20-24 (°C), Humidity: 30 – 70 (%), Air changes: 12 (per hr), Photoperiod: 12 hours dark/12 hours light

Adaptation/Acclimatization
The animals were received from Lab Animals Breeding Center “Pushchino” at the age of 3-4 weeks. Each animal was examined on the day of receipt.
During adaptation/acclimatization, animals were kept in groups (by litter/sex) in the barrier zone of the facility, and the animal’s condition was evaluated daily by cage-side observation. Before pre-dosing oestrus cycle evaluation and groups assignment, all animals were identified by ear punch, weighed, and clinical observations were recorded. For all males, the testes were palpated, and for all females, the opened vagina was inspected visually, and oestrus cyclicity was established. Only females with a regular 4-5 days cycle were taken into experimental groups. Animals considered unsuitable for the study were excluded prior to group assignment.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared from a neat test item like a water solution in calculated concentrations of 40, 100 and 250 mg/mL. For each concentration, the formulation staff weighed the required amount of the test item and dissolved it in a calculated volume of distilled water by stirring. Calculations were documented in Excel spreadsheets and maintained in the study file as printouts.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration, and stored in tightly closed glass jars at room temperature. The required aliquots of distilled water from the total stored volume were placed in labelled jars and stored at room temperature for the control group.
On the day of dosing, the aliquot of each formulation was transferred to the barrier zone.


DIET PREPARATION
The animals were fed Standard Diet for laboratory mice and rats Mucedola 4RF21 autoclavabile (Mucedola s.r.l., Via G.Galelei, 4-20019 Settimo Milanese MI, Italy) ad libitum. This diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by the test facility for microbiological contaminants. Results of analyses are kept in the archive at the test facility. A copy of the latest check is placed in the study file as raw data

VEHICLE
Name: Distilled water
Provider: Tap water filtrated with the MilliRO Millipore system
Storage conditions: Room temperature: 20 ± 5°C

Details on mating procedure:

- M/F ratio per cage: 1:1 basis within each treatment group following 14 days of treatment for the males and females avoiding sibling mating;
- Length of cohabitation: 2 oestrus cycles or two weeks with irregular cycle
- Proof of mating: sperm in vaginal smear or vaginal plug referred to as gestation day 0

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Verification of dose concentrations 40, 100 and 250 mg/mL was carried by validated GC-MS method using a TRACE™ 1310 Gas Chromatograph with MS detector TSQ 9000 (Thermo Fisher Scientific).

Duration of treatment / exposure:

Males were administered 14 daily doses before mating and continuing throughout the mating period for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 12 (for females that delivered) for a total of 48-54 doses; females with no evidence of mating/that failed to deliver were dosed for a total of 54-55 doses.

Frequency of treatment:

Daily

Details on study schedule:

The test item (2,2-bis(hydroxymethyl)butanoic acid), in the vehicle distilled water, was administered by gavage once daily to three groups of Sprague-Dawley rats at the doses 200, 500 and 1250 mg/kg bw/day.
A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 5 mL/kg bw.
Each group consisted of 12 males and 12 females used for dosing and mating. Males were administered 14 daily doses before mating and continuing throughout the mating period for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 12 (for females that delivered) for a total of 48-54 doses; females with no evidence of mating/that failed to deliver were dosed for a total of 54-55 doses.

All animals were observed twice daily for mortality and morbidity and daily during dosing. Detailed clinical observations, body weights, and food consumption were recorded weekly.
All parental females were allowed to deliver and rear their pups until lactation day 13. Born pups were counted and sexed and litters were weighed on post-natal days (PND) 0-1, 4, 7 and 13. The anogenital distance (AGD) of each pup was measured at the end of delivery (on PND 0-1). Any abnormal behavior of the offspring was recorded. The number of nipples/areolae in male pups was counted on PND 13.

Parental males were euthanized following completion of the 28 days dosing period and females that delivered or failed to deliver were euthanized on lactation day 13 (with their litters), or after 54-55 days of dosing, respectively. Complete necropsies were conducted on all parental animals, and selected organs were weighed. Reproductive organs and thyroid glands were examined microscopically from all parental animals in the vehicle control and high-dose groups.

On PND 4, litter sizes were standardized to 5 male and 5 female pups, where possible. Surplus pups were euthanized and blood was sampled for determination of serum thyroxin levels. The remaining F1 pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the external reproductive genitals and with weighing and histology of thyroid glands. Thyroxin level was assayed in adult males and females, and PND 4 and PND 13 pups.

Dose / conc.:

1 250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

Each group consisted of 12 males and 12 females used for dosing and mating.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosage levels are based on a dose-range-finding study with the treatment of male and female animals for 14 days at doses of 500 and 1500 mg/kg bw/day. Test item-related gastrointestinal abnormalities (soft stool) were noted in the 1500 mg/kg treated group. The 1500 mg/kg bw/day male group had a slightly decreased mean body weight gain at day 14
Based on the dose-range-finding study results and the data of 28-day repeated dose toxicity study from the sponsor, the 1250 mg/kg bw/day as the maximal dose, and doses of 500 and 200 mg/kg bw/day as medium and minimal doses, respectively, with 2.5-fold interval were chosen for administration to male and female rats in the main study.

- Rationale for animal assignment:
Animals without clinical signs of health abnormalities and with regular oestrus cycle (females) were assigned to groups based on body weight stratification into a block design and taking into account the number of the litter to avoid of sibling mating. Animals not assigned to study were transferred to the stock colony.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Twice a day during study period
DETAILED CLINICAL OBSERVATIONS:
Detailed physical examinations - on a weekly basis; females expected to deliver were also observed twice daily during the period of expected parturition.
BODY WEIGHT:
On the first day of dose administration, and weekly thereafter throughout the study for males and until evidence of copulation was observed for females. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 of lactation), and at day 2 of lactation (for calculation of food consumption), day 4 and day 13 of lactation.
FOOD CONSUMPTION:
Food consumption was recorded for each cage prior to the initiation of dose administration (day 0-1), and weekly until cohabitation. Food consumption was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0-1, 6-7, 13-14, and 19-20 and on lactation days 1-2, 4-5, and 12-13.

Oestrous cyclicity (parental animals):

OESTROUS CYCLICITY:
Oestrous cycle was monitored for two weeks before start of the treatment to select females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for two weeks during the pre-mating period with continued monitoring into the mating period until there was evidence of mating.
PARAMETERS EXAMINED
Pre-coital interval, days
Male Mating Index, %
Female Mating Index, %
Male Fertility Index, %
Male Copulation Index, %
Duration of Oestrus cycle, days
Female Fertility Index, %
Female Conception Index, %
Gestation index (%, N/N)
Duration of gestation, days
Implantation sites count
Pups delivered, liveborns, N
Pre-natal loss of offspring, %
Post-natal loss of offspring, %
Dams with stillborn pups, N, %
Dams with live pups, N, %

Litter observations:

STANDARDISATION OF LITTERS
Performed on day 4 postpartum: by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter.
PARAMETERS EXAMINED
Number and sex of pups, stillbirths, live births, Postnatal survival, Viability index, presence of gross anomalies, body weight (postnatal days 0-1, 4, 7, 13) and weight gain, physical or behavioural abnormalities, Anogenital distance (absolute and normalized), sex: females (N), males (N, %), the number of nipples/areolae in male pups (postnatal day 13), weight and pathomorphology of thyroid glands (postnatal day 13), serum thyroxin (on postnatal days 4 and 13).
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
Parental males: Following completion of the mating period and 28 days of dose administration.
Maternal females: on lactation day 13 with with their litters (for females that successfully delivered offspring) or day 54-55 of dosing (for females that did not produce offspring).
GROSS NECROPSY AND HISTOPATHOLOGY
Gross necropsy, number of implantation sites, organ weights (brain, ovaries, Cowper’s glands, pituitary, epididymides, prostate, glans penis, seminal vesicles & coagulating glands, kidneys, spleen, levator ani & bulbocavernosus muscles (LABC) complex, testes, liver, thyroid glands, uterus).
Histopathology on testes, epididymides, ovaries.

Postmortem examinations (offspring):

SACRIFICE
The F1 offspring were sacrificed on postnatal 13.
GROSS NECROPSY AND HISTOPATHOLOGY
Number of nipples/areolae, thyroid weight (1 pups/sex/litter), external genitals, gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals, and histopathology of thyroids (1 pups/sex/litter).

Statistics:

All statistical tests were performed separately for each sex using Microsoft Excel (descriptive statistics) and statistical software Statistica for Windows v.7.1 to compare the treated groups to the control group. Descriptive statistics (MEAN, standard deviation (SD), and N) are presented for all measurement data and shown in the summary tables. The litter, rather than the pup, was considered as the experimental unit.

Continuous data variables (mean body weights, body weight gain, and food consumption data) were analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences.
Oestrus cycle length, pre-coital intervals, gestation length, former implantation sites, and thyroxin values were analyzed by a one-way analysis of variance (ANOVA) with Dunnett-test to compare the treated groups to the control group. The t-test was used additionally to compare each dose group with control value. Male copulation, female conception, male and female mating and fertility indices of the treated groups were compared to the control group using the Chi-square test.

Pup viability and sex ratio data were subjected to the Kruskal-Wallis nonparametric ANOVA test with post-hoc Dunn-test to compare the treated groups to the control group. Mean litter weights and litter size were subjected to the ANOVA Dunnett’s test with the litter representing the experimental unit.
Histopathological findings of each treated group were compared to those of the control group by Fisher's Exact test. Organ weights (absolute and relative to body weights and relative to brain weights) were subjected to a parametric ANOVA Dunnett's test. The t-test was used additionally to compare the organ weights of each dose group with the control value.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was no morbidity and mortality of animals caused by the test item administration. The test item related clinical finding was diarrhea in the 1250 mg/kg bw/day dose group (12 of 12 males and 7 of 12 females).

Mortality:

no mortality observed

Description (incidence):

There was no morbidity and mortality of animals caused by the test item administration. The test item related clinical finding was diarrhea in the 1250 mg/kg bw/day dose group (12 of 12 males and 7 of 12 females).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The total body weight gain of test item treated males was decreased compared to the control group. This change was statistically significant (p < 0.05) in the 200 and 1250 mg/kg bw/day dose groups but slight (by 4 % in the high dose group) and without clear dose-dependence.
For the gestation and lactation periods, females' body weight and weight gain in the 1250 mg/kg bw/day dose group was slightly decreased (approximately by 4 % compared to the control group). However, these changes were not significant.

TABLE S5-1. SUMMARY DATA OF BODY WEIGHT AND BODY WEIGHT GAIN - MALES

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption in the 200, 500 and 1250 mg/kg bw/day group was similar to that in the vehicle control group during all study days.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related histological alterations were observed in evaluated tissues (testes, epididymides, ovaries, and thyroid glands).
During a qualitative examination of the testes in the 1250 mg/kg bw/day group with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure, no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed.
In a testis of a single male (No. 52) from the high dose group, a focus of degenerative and atrophic seminiferous tubules and tubules with signs of mineralization was revealed. In most of the altered tubules, only Sertoli cells were observed, and single tubules had alteration at 4-5 stages or 8-9 stages of the spermatogenic cycle. Thus, degenerative changes in the seminiferous tubules were not associated with one stage of the spermatogenic cycle, were focal and of minimal grade, and considered incidental, not test related. The reproductive performance of this male was not changed.
In the epididymides, the mononuclear infiltration was noted in the same number of males from the control vehicle and high dose groups. The mononuclear infiltration of tissues can be found as a background finding without changes in the prevalence and severity.
In thyroid glands, ultimobranchial cysts found in some control and test item treated animals, and ectopic lymphoid tissue are congenital findings unrelated to treatment.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The test item did not affect the reproductive performance of males. The pre-coital intervals in the test item groups did not differ from the value in the control group. All males sired a litter in the high dose groups, and indices of fertility and copulation were 100.0 %.
In females, fertility and conception indices did not significantly differ from the control group's values and historical control data. The oestrous cycle duration, being evaluated during the administration period before the onset of pregnancy, was approximately the same in all groups. There were no females with irregular oestrous cycles. The gestation indices in the medium and high dose group were 90.9% and 91.7 %, respectively, which did not differ significantly from a value in the control group.
In the 1250 mg/kg bw/day dose groups, the mean number of delivered pups was lower compared to the control vehicle group and historical control value (9.5 ± 3.6 compared to the 12.3 ± 1.4 in the control group, p < 0.01, and historical control value 12.3 ± 2.4). The decrease in the mean number of delivered pups was dose-dependent and considered test item-related. In the 500 mg/kg bw/day dose group, the mean number of delivered pups was also slightly decreased by 8 % (11.0 ± 2.7). The decrease in the number of newborns correlated with lower implantation sites count in the high dose group (11.0 ± 2.3 compared to the 13.2 ± 0.9 in the control vehicle group, p < 0.01) and with the increased percentage of offspring pre-natal loss in the 500 and 1250 mg/kg bw/day dose groups (14.1 % and 13.6 % compared to the 7.0 % in the control vehicle group, p < 0.05). The mean number of female newborns per litter was decreased in the high dose group (4.5 ± 1.6 compared to the 6.5 ± 2.9 in the control vehicle group and historical control value 6.6 ± 1.6). The post-natal loss of offspring was low in all groups and was not increased by the test item.

Gestation Length and Parturition Observation

Most females had a gestation period of 21 days, and mean gestation lengths were similar between all groups and abnormalities in the parturition were not observed.
One mated female from the 500 mg/kg bw/day did not deliver and had no implantation sites. One mated but non-delivered female from the 1250 mg/kg bw/day dose group had implantation sites revealed during necropsy. So, the gestation indices in the medium and high dose group was 90.9% and 91.7 %, respectively, which did not differ significantly from a value in the control group and corresponded to historical control data

Loss of Offspring

In the 1250 mg/kg bw/day dose groups, the mean number of delivered and liveborn pups was lower compared to the control vehicle group. This decrease was slight but statistically significant (9.5 ± 3.6 compared to the 12.3 ± 1.4 in the control group, and historical control value 12.4 ± 2.4 (p < 0.01) and correlated with lower implantation sites count in the high dose group (by 16.7 % compared to the control vehicle group) and with the percentage offspring pre-natal loss in the 500 and 1250 mg/kg bw/day dose groups (14.1 % and 13.6 % compared to the 7.0 % in the control vehicle group). The decrease in the mean number of delivered pups was dose-dependent and considered to be test item related.
One female (No.80) in the 1250 mg/kg bw/day dose group was with the total loss of implantations; however, the total dams with pre- and post-natal loss of offspring was not significantly changed in test item treated groups.
The post-natal loss of offspring was low in all groups and was not increased by the test item. There were no dams with abnormal pups.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

1 250 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: maximum tested level

Remarks on result:

other: The test item did not affect the reproductive performance of males.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No effects. A decrease in relative thyroid weight in the offspring of the high dose group is considered to be without clear evidence with the treatment

Histopathological findings:

no effects observed

Description (incidence and severity):

Thyroids - No effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: There were no abnormal pups, changes in F1 offspring postnatal survival and body weight gain, no changes in anogenital distance, and signs of offspring masculinization or feminization. Relative thyroids weight was decreased on postnatal day 13 in the high

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Findings	Historic Control value	0 mg/kg bw		200 mg/kg bw		500 mg/kg bw		1250 mg/kg bw
		M	F	M	F	M	F	M	F
Clinical signs
Diarrhea		0 / 12	0 / 12	0 / 12	0 / 12	1 / 12	0 / 12	12 / 12**	7 / 12**
Body weight gain, total, % ^(a)
MEAN		18.9	55.6	15.5 *	54.1	16.2	55.1	15.0 *	51.7
SD		2.1	7	3.8	9.0	2.8	6.5	3.7	5.8
Kidney: Relative weight, g/100 g body weight
MEAN		0.629		0.646		0.674 *		0.666 *
SD		0.044		0.056		0.055		0.041
Kidney: Relative weight, g/100 g brain weight
MEAN		115.3		114.4		122.5		120.3
SD		11.1		10.0		12.6		11.8
Reproductive performance
Number of delivered  pups
MEAN	12.4		12.3		11.5		11.0		9.5**
SD	2.4		1.4		3.4		2.7		3.6
Number of liveborn pups
MEAN	11.7		12.3		11.5		11.0		9.5**
SD	2.7		1.4		3.4		2.7		3.6
Number of implantation sites
MEAN	12.8		13.2		12.0		12.8		11.0**
SD	2.9		0.9		3.5		1.2		2.3
Pre-natal loss of offspring	11.8 %		7.0 %		4.5 %		14.1 %*		13.6 %*
Number of delivered female pups per litter, N
MEAN	6.3		6.6		6.5		5.5		4.5*
SD	2.1		1.6		2.9		2.2		1.6
Male %	391/791 (49.4%)		68/147 (46.3%)		54/126 (42.9%)		55/110 (50.0%)		65/114 (57.0%)

^(a) Females: for the gestation period,

*  = Significantly different from 0 mg/kg bw/day  at 0.05

**  = Significantly different from 0 mg/kg bw/day  at 0.01

Conclusions:

There was no morbidity and mortality of animals caused by the test item administration. The test item-related clinical finding was diarrhea in the 1250 mg/kg bw/day dose group. The total body weight gain was slightly decreased in males starting from lower 200 mg/kg bw/day dose; females body weight gain was non-significantly decreased for the gestation and lactation periods on 1250 mg/kg bw/day dose. The food consumption was not changed. Males of the 500 and 1250 mg/kg bw/day dose group had the increased weight of kidneys relative to body weight.
The test item did not affect the reproductive performance of males.
The test item administration has not changed the indices of fertility, conception, and gestation of females. Decrease in the number of implantation sites (1250 mg/kg bw/day), decrease in the number of delivered pups (500 and 1250 mg/kg bw/day), decrease in the mean number of female newborns per litter (1250 mg/kg bw/day), increased pre-natal loss of offspring (500 and 1250 mg/kg bw/day doses), is a female reproductive performance effect.
There were no abnormal pups, changes in F1 offspring postnatal survival and body weight gain, no changes in anogenital distance, and signs of offspring masculinization or feminization. Relative thyroids weight was decreased on postnatal day 13 in the high dose group without clear evidence with the treatment; thyroxin level was not changed; there was no test item related microscopic findings in offspring thyroids.
Therefore, under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was 1250 mg/kg bw/day for parental males, 200 mg/kg bw/day for parental females reproductive toxicity, and 1250 mg/kg bw/day for F1 postnatal developmental toxicity.

Executive summary:

This study was designed to evaluate the potential of the test item (2,2-bis(hydroxymethyl)butanoic acid (DMBA) to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The study was carried out in accordance to the OECD Guideline 421 in GLP conditions.

 

Method

Dosage levels are based on a dose-range-finding study with the treatment of male and female animals for 14 days at doses of 500 and 1500 mg/kg bw/day.

The test item (2,2-bis(hydroxymethyl)butanoic acid), in the vehicle distilled water, was administered by gavage once daily to three groups of Sprague-Dawley rats at the doses 200, 500 and 1250 mg/kg bw/day. A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 5 mL/kg bw. Each group consisted of 12 males and 12 females used for dosing and mating. Males were administered 14 daily doses before mating and continuing throughout the mating period for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 12 (for females that delivered) for a total of 48-54 doses; females with no evidence of mating/that failed to deliver were dosed for a total of 54-55 doses.

All animals were observed twice daily for mortality and morbidity and daily during dosing. Detailed clinical observations, body weights, and food consumption were recorded weekly.

All parental females were allowed to deliver and rear their pups until lactation day 13. Born pups were counted and sexed and litters were weighed on post-natal days (PND) 0-1, 4, 7 and 13. The anogenital distance (AGD) of each pup was measured at the end of delivery (on PND 0-1). Any abnormal behavior of the offspring was recorded. The number of nipples/areolae in male pups was counted on PND 13.

Parental males were euthanized following completion of the 28 days dosing period and females that delivered or failed to deliver were euthanized on lactation day 13 (with their litters), or after 54-55 days of dosing, respectively. Complete necropsies were conducted on all parental animals, and selected organs were weighed. Reproductive organs and thyroid glands were examined microscopically from all parental animals in the vehicle control and high-dose groups.

On PND 4, litter sizes were standardized to 5 male and 5 female pups, where possible. Surplus pups were euthanized and blood was sampled for determination of serum thyroxin levels. The remaining F1 pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the external reproductive genitals and with weighing and histology of thyroid glands. Thyroxin level was assayed in adult males and females, and PND 4 and PND 13 pups.

 

Conclusion

There was no morbidity and mortality of animals caused by the test item administration. The test item-related clinical finding was diarrhea in the 1250 mg/kg bw/day dose group. The total body weight gain was slightly decreased in males starting from lower 200 mg/kg bw/day dose; females body weight gain was non-significantly decreased for the gestation and lactation periods on 1250 mg/kg bw/day dose. The food consumption was not changed. Males of the 500 and 1250 mg/kg bw/day dose group had the increased weight of kidneys relative to body weight.

The test item did not affect the reproductive performance of males.

The test item administration has not changed the indices of fertility, conception, and gestation of females. Decrease in the number of implantation sites (1250 mg/kg bw/day), decrease in the number of delivered pups (500 and 1250 mg/kg bw/day), decrease in the mean number of female newborns per litter (1250 mg/kg bw/day), increased pre-natal loss of offspring (500 and 1250 mg/kg bw/day doses), is a female reproductive performance effect.

In the study with DMBA, the control vehicle group had a low prenatal loss value, which gave a statistical increase in the percentage of prenatal loss starting from 500 mg/kg. However, by linking to historical control data, it is possible to conclude that the number of implantation sites on the 500 mg/kg dose was the same as in historical control; the decrease in the mean number of liveborns was only a weak trend, and a slight increase in the post-implantation loss was not statistically changed compared to the historical control value. Considering that other fertility and post-natal development parameters did not change at a dose of 500 mg/kg, a slight increase in the percentage of post-implantation loss, which remained at the level of the historical control value, could be considered as non-adverse.

There were no abnormal pups, changes in F1 offspring postnatal survival and body weight gain, no changes in anogenital distance, and signs of offspring masculinization or feminization. Relative thyroids weight was decreased on postnatal day 13 in the high dose group without clear evidence with the treatment; thyroxin level was not changed; there was no test item related microscopic findings in offspring thyroids.

Therefore, under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was 1250 mg/kg bw/day for parental males, 200 mg/kg bw/day for parental females’ reproductive toxicity, and 1250 mg/kg bw/day for F1 postnatal developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Mode of Action Analysis / Human Relevance Framework

Reproduction/developmental toxicity screening test (according to OECD 421) was performed, where DMBA was administered by gavage once daily to rats (3 groups á 24 subjects, 50:50 male:female) at the doses 200, 500 and 1250 mg/kg bw/day. Control group received the vehicle on a comparable regiment. The NOAEL was considered to be 200 mg/kg bw/day. No other reproduction toxicity study is available for DMBA.

There was no morbidity and mortality of animals during the study caused by DMBA administration. There were no abnormal pups, changes in F1 offspring postnatal survival and body weight gain, no changes in anogenital distance and no signs of offspring masulinisation or feminization. No effect on reproductive function of males was found. The test item administration has not changed the indices of fertility, conception, and gestation of females. Decrease in the number of implantation sites (1250 mg/kg bw/day), decrease in the number of delivered pups (500 and 1250 mg/kg bw/day), decrease in the mean number of female newborns per litter (1250 mg/kg bw/day), increased pre-natal loss of offspring (500 and 1250 mg/kg bw/day doses), is a female reproductive performance effect.

 

Justification for classification or non-classification

Data conclusive but not sufficient for classification.