2,2-bis(hydroxymethyl)propionic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2013 to 13 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The weight range of the animals at initiation of dosing deviated from the target weight range of 200-240g in the protocol. As all animals were of the correct age at mating, this was considered not to have affected the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis(hydroxymethyl)propionic acid

EC Number:

225-306-3

EC Name:

2,2-bis(hydroxymethyl)propionic acid

Cas Number:

4767-03-7

Molecular formula:

C5H10O4

IUPAC Name:

3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid

Specific details on test material used for the study:

- Name of the test material used in the study report: Bis-MPA
- Batch no.: 521152
- Purity: 98.00%
- Appearance: white crystalline powder
- Expiry date: 01 Dec 2013
- Storage conditions: ambient, dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 9 weeks old at the time of mating
- Weight at study initiation: 218- 317g at the initiation of dosing
- Fasting period before study: No
- Housing: 1 per cage in suspended polycarbonate cages with stainless steel grid tops and solid bottoms, containing an integral food hopper and sterilised white wood shavings bedding material
- Diet (e.g. ad libitum): SDS Rat and Mouse (modified) No. 3 Diet SQC Expanded was provided ad libitum
- Water (e.g. ad libitum): The animals had access to water ad libitum from the public supply
- Acclimation period: from arrival until Day 6 of gestation (3-6 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 53-61%
- Air changes (per hr): A minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 08 Apr 2013 To: 24 Apr 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 0, 20, 50, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): 021M0067V
- Purity: 0.5% methyl cellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis on week 1 and 2 for all groups. Concentration and homogeneity were analysed. Analyses were performed by reverse phase HPLC with ELSD detection using a validated analytical procedure.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

From day 6 to day 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

20 days

No. of animals per sex per dose:

24 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on a 28 day dose range finding study where dose levels of up to 1000 mg/kg bw/day were considered to be well tolerated. Therefore, 1000 mg/kg bw/day was chosen as the high dose level for this study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early morning and as late as possible each day for viability. All the animals were examined for reaction to treatment prior to dosing and regularly throughout the day.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, from the start of dosing

BODY WEIGHT: Yes
- Time schedule for examinations: pretrial once on Day 4 of gestation and then daily during the dosing period Day 6 – 20 of gestation.

FOOD CONSUMPTION: Yes
- Frequency: Daily from Day 4 of gestation (first measured quantity given on Day 3 of gestation).
- Procedure: Food consumption was quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Frequency: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae (size, color or shape) and live and dead foetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined by open dissection for abnormalities of the thoracic and abdominal viscera, and for sex. These viscera were then discarded. Half per litter were examined for soft tissue abnormalities and sex.
- Skeletal examinations: Yes: all per litter
- The body weight of each foetus was recorded

Statistics:

Means and standard deviations were calculated for body weight, food consumption and pregnancy data.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test is significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Foetal weight data was subjected to the Kruskal-Wallis non-parametric analysis.

Indices:

Not relevant

Historical control data:

Historical control data were supplied by the laboratory to clarify the significance of the apparent increase in post-implantation loss

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical observations were considered to be incidental background findings commonly observed in this species at Charles River, Edinburgh. An isolated incident of hunched posture at 500 mg/kg/day could not be positively attributed to treatment due to low incidence and absence from other treatment groups.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths in this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight in treated groups was comparable with controls throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption in treated groups was comparable with controls throughout the study.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross necropsy findings noted in dams and fetuses on this study were of the type and distribution which did not indicate any association with treatment.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Total litter losses by resorption:

effects observed, treatment-related

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Increased numbers of early resorptions at 500 and 1000 mg/kg bw/day, but without any effect on litter size or mean live implants. At 500 and 1000 mg/kg/day there was an increase in total dead implants, when compared with controls, caused by an increase in early embryonic deaths. In both groups, the number of litters affected and number of early embryonic deaths per litter were also increased when compared with controls. However, these appear to be largely due to a high proportion of resorptions in two animals at 1000 mg/kg bw/day (8/16 and 11/14 resorptions, respectively) and in one animal at 500 mg/kg bw/day (12/13). Single incidences of dams with a high proportion of early resorptions were also seen at 200 mg/kg bw/day (6/17) and in controls (7/12). The total number of early embryonic death and litters with early embryonic deaths at 200 mg/kg bw/day was comparable with controls. In the absence of effects on litter size, these singular incidences are not considered to be adverse. The group mean and percentage values of all other pregnancy performance parameters were similar between all groups. Slight inter-group differences were considered to be incidental and too small to be attributed to treatment with Bis-MPA™.

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no maternal toxic effects. There were no premature deaths. All clinical observations were considered to be incidental background findings commonly observed in this species. Group mean body weight and food consumption in treated groups was comparable with controls. All gross necropsy findings noted in dams and fetuses on this study were of the type and distribution which did not indicate any association with treatment.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Refer to

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weights were similar between all groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

The group mean of fetal weights were similar between all groups. The type and distribution of major and minor fetal abnormalities and skeletal ossification parameters did not indicate any association with treatment. Slight inter-group differences were considered to be incidental and unrelated to treatment with Bis-MPA™.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Refer to

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Refer to

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary of litter parameters

 

Parameter	Dose level (mg/kg bw/d)
	0	200	500	1000
Corpora lutea (#)	13.6	14.1	15.6	14.8
Pre-implantation loss (%)	6	5	4	3
Implantations (#)	12.8	13.4	15.0	14.4
Post-implantation loss (%)	7	8	10	12
Early resorption (%)	7	8	10	12
Late resorption (%)	0.3	0.3	0	0
Litter size (#)	11.9	12.4	13.5	12.6
Foetal death (#)	0	0	0	0
Gravid uterus weight (g)	71	76	81	75
Foetal weight (g)	3.73	3.80	3.85	3.74

Applicant's summary and conclusion

Conclusions:

In a developmental toxicity study conducted according to OECD 414, treatment with Bis-MPA™ at dose levels up to 1000 mg/kg bw/day was not associated with any treatment effects or findings in dams at any dose level. At 500 and 1000 mg/kg bw/day, there was an increase in early embryonic deaths when compared with controls and 200 mg/kg bw/day. These findings appear largely due to a high proportion of resorptions in two animals at 1000 mg/kg bw/day (8/16 and 11/14 resorptions, respectively) and in one animal at 500 mg/kg bw/day (12/13). Single incidences of dams with a high proportion of early resorptions were also seen at 200 mg/kg bw/day (6/17) and in controls (7/12). There was no effect on litter size and therefore these singular incidences are not considered adverse. All other pregnancy performance parameters and fetal weights were comparable with controls in all treated groups, and therefore showed no treatment-related effects of findings. Based on the results of this study, the maternal No Observed Effect Level (NOEL) was considered to be 1000 mg/kg bw/day and the foetal NOEL was considered to be 200 mg/kg bw/day.

Executive summary:

In a developmental toxicity study (conducted according to OECD TG 414), Bis-MPA™ (purity 98.0%) was administered to 24 female Sprague-Dawley rats/dose by gavage at dose levels of 200, 500, or 1000 mg/kg bw/day from day 6 through 19 of gestation, where the day of detection of mating was designated Day 0. There were no treatment-related effects in clinical signs, body weight, food consumption or gross necropsy in dams at any dose level up to 1000 mg/kg bw/day. The maternal No Observed Effect Level (NOEL) is 1000 mg/kg bw/day. At 500 and 1000 mg/kg bw/day, there was a slight increase in early embryonic deaths when compared with controls and 200 mg/kg bw/day. These singular incidences in the absence of effect on litter size are not considered adverse. All other pregnancy performance parameters and foetal weights were comparable with controls in all treated groups, and the type and distribution of foetal major abnormalities, minor visceral and skeletal abnormalities and skeletal ossification parameters did not indicate any treatment related effect. The foetal NOEL is 200 mg/kg bw/day. The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.