Sodium formate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Several thorough studies demonstrated clearly that teratogenicity seen with formic acid or sodium formate in Whole Embryo Culture tests in vitro is a secondary effect that is related to non-physiological pH values or osmolarity. Therefore, results from such tests cannot rule out results from valid Guideline studies.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-Jan-09 through 2007-May-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Sodium formate
- Physical state: solid
- Analytical purity: 100%

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: men 108.0 g; Females: mean 96.0 g
- Fasting period before study: ovenight; feed study
- Housing: individually in Macrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²)
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, was available ad libitum
throughout the study.
- Water: tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: food

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): ground standard diet
- Storage temperature: room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Commercial test kit "Ameisensäure"; enzymatic test with UV/vis detection at 340 nm. Details: Study Part III, p. 882

Duration of treatment / exposure:

Exposure period: throughout the study
F0 and F1 generation: 75 days premating exposure; during mating and gestation until post natal day (PND) 21

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Based on diet

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Based on diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Based on diet

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of preceding studies

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily
(Saturday, Sunday or on public holidays).


DETAILED CLINICAL OBSERVATIONS: Yes
- Tim eschedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
testis weight, epididymis weight, sperm count in testes and in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight at birth and weight gain, physical or behavioural abnormalities, sexual maturation (vaginal openng, preputial separation).


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead. Determinationof pup organweight (brain, spleen, thymus).

Postmortem examinations (parental animals):

SACRIFICE
F0 and F1 animals:
- Male animals: All surviving animals after the F1 or F2 pups were weaned (PND 21).
- Maternal animals: All surviving animals after the F1 or F2 pups were weaned (PND 21).


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in section 3.9.2 (page 51) were weighed and those in section 3.9.3 (page 52) were prepared for microscopic examination.
Tissues examined by light microscopy are tabulated in section 3.9.4.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGTHS
Brain, spleen, and thymus were weighed.

Statistics:

Extensive statistical evaaluations included Dunnett test; Fisher's exact test; Wilcoxon test; Kruskal-Wallis test (cf. report, section 3.8.3)

Reproductive indices:

Male mating index; male fertility index. Female mating index; female fertility index; gestation index. Live birth index; postimplantation loss.

Offspring viability indices:

Viability index day 4. Lactation index (viability on day 21)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No findings in F0 and and F1 parental animals noted.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No changes in F0 and and F1 parental animals noted.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean test substance intake was close to the nominal values:
F0 males: 93 / 282 / 939 mg/kg bw/day in treated groups
F0 females: 97 / 291 / 977 mg/kg bw/day in treated groups

F1 males: 91 / 266 / 949 mg/kg bw/day in treated groups
F2 females: 98 / 295 / 980 mg/kg bw/day in treated groups


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effect in F0 females (section 4.2.1.6) or F1 females (section 4.2.36)


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related effct in F0 males (section 4.2.1.8) or F1 males (section 4.2.3.8)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effect in male or female F0 and F1 animals

F0 animals:
Males:
The male mating index varied between 96% in the control and 100% in the test groups.
The male fertility index ranged between 92% and 100% without showing any effect of dosing.
Females:
The female mating index calculated after the mating period for F1 litter was 96% in the control group and 100% in test groups 01-03 (100, 300 and 1000 mg/kg bw/d).
The mean duration until sperm was detected (GD 0) varied between 4.4 and 5.6 days without any relation to dosing. The fertility index was 96% in test group 01, 92% in test group 02 and 100% in test groups 00 and 03. The mean duration of gestation varied between 22.0 and 22.2 days without any relation to dosing.
The gestation index was 100% for all test groups, indicating that all pregnant F0 females had live F1 pups in their litters. There were no statistically significant differences in postimplantation loss between the test groups and the control (see Tab. IA-028). The live birth index was 99%, 99%, 100% and 99% in test groups 00-03.

F1 parental aimals
Males:
The male mating index was 96% in the low-dose group and 100% in the remaining test groups.
The male fertility index ranged between 92% and 100% without showing any effect of dosing.

Females: The female mating index for the F2 litter was 96% in test group 11 (100 mg/kg bw/d) and 100% in test groups 10, 12 and 13 (0, 300 and 1000 mg/kg bw/d).
The fertility index was 92% in the control and 100% in the remaining test groups.
The gestation index was 100% in all test groups, indicating that all pregnant F1 females had live F2 pups in their litters. The mean duration of gestation was very similar in all F1 test groups and varied between 21.8 and 22.0 days, with no relation to dosing.
There were no statistically significant differences of postimplantation loss between the test and the control groups.
The live birth index varied between 99% (test group 10) and 100% (test groups 11, 12 and 13).
ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 animals:
Mean absolute organ weight parameters did not show significant differences compared to the control groups. In females, the mean relative weights of the brain, kidneys, and ovaries significantly changed (up to p<0.01), but were attributed to the slight reduction of the terminal body weight.

F1 animals:
Some significant changes (increase and reduction) of the absolute and relative weight were seen (kidneys, liver, pituitary gland, cauda epididymis affected; cf. section 4.3.2). There was no relation to dose or histopathological correlate.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 animals:
No changes noted (section 4.3.1)
F1 animals:
No changes apart from incidental glandular stomach erosion/ulcer in one control and eight mid dose animals.


OTHER FINDINGS (PARENTAL ANIMALS)

Sperm parameters: unchanged.
Estrous cycle data: no treatment-related change noted.

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on reproductive performance and reproductive organs of F0 animals.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)

F1 pups:
The viability index indicating pup mortality during early lactation (PND 0-4) varied between 98% (test group 03 – 1000 mg/kg bw/d) and 99% (test groups 00, 01 and 02 – 0, 100 and 300 mg/kg bw/d).
The lactation index indicating pup mortality on PND 4-21 varied between 99% (test group 02 – 300 mg/kg bw/d) and 100% (test groups 00, 01 and 03 – 0, 100 and 1000 mg/kg bw/d).

F2 pups:
The viability index indicating pup mortality during early lactation (PND 0-4) varied between 99% (test groups 11, 12 and 13 – 100, 300 and 1000 mg/kg bw/d) and 100% (control group).
The lactation index indicating pup mortality on PND 4-21 varied between 98% (control group) and 100% (test groups 11, 12 and 13 – 100, 300 and 1000 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)
There were no treatment related clinical signs in F0 and F1 pups.

BODY WEIGHT (OFFSPRING)
Mean body weights and mean body weight gain of the male and female F1 (Figure page 69) and F2 pups (Figure page 85) were not influenced by the test substance during the entire lactation period. All values in test groups at 100, 300 and 1000 mg/kg bw/d were comparable with the concurrent control group.

SEXUAL MATURATION (OFFSPRING)
F1 pups:
Males, preputial separation: the mean number of days to reach the separation was 41.4, 41.5, 41.2, and 42.0 days in controls and 100, 300 and 1000 mg/kg body weight/day test groups, indicating that the test substance did not influence male sexual maturation.
Females, vaginal opening: the mean number of days to reach the criterion amounted to 31.3, 30.8, 31.4, and 31.9 days in controls and 100, 300 and 1000 mg/kg body weight/day test groups, respectively, indicating that female sexual maturation was not influenced by the test substance.

F2 pups: not determined

ORGAN WEIGHTS (OFFSPRING)
F1 pups: mean absolute and relative pup organ weights did not show statistically significant differences to the control group, withth exception of statistically significantly increased absolute brain weights in groups at 100 and 300 mg/kg bw/day. Relative brain weight was not affected, and there was no dose relationship. Therefore, this finding was not considered to be of toxicological relevance.
F2 pups: mean absolute and relative pup organ weights did not show statistically significant differences to the control group, withth exception of statistically significantly decreased absolute and relative spleen weight in high dose males. Females were not affected. This finding was not considered to be of toxicological relevance.


GROSS PATHOLOGY (OFFSPRING)
There were spontaneous findings at necropsy of the F1 pups (Part III, tables IA-039 through IA-042) and F2 pups (Part III, tables IA-084 through IA-086), but these were without relation to dosing and/or could be found in the historical control data at comparable or even higher incidences.


OTHER FINDINGS (OFFSPRING)
Sex ratio was unchanged in the treated F1 and F2 pups.

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on pre- and postnatal survival, body weights, no clinical or necropsy findings.

Reproductive effects observed:

not specified

Conclusions:

No effects were seen in this study at dose levels of up to 1000 mg/kg bw/d

Executive summary:

The reproductive toxicity potential of sodium formate was examined in a 2-Generation feed study using male and female Wistar rats (25/sex and dose) at dose levels of 0, 100, 300, and 1000 mg/kg bw/d. The study was conducted in accordance with current test guidelines including OECD guideline No. 416, and under GLP conditions.

Nominal dose (mg/kg bw/day)	100	300	1000
F0 males	93.2	282	939.1
F0 females - premating - gestation	96.8 105.4	290.9 323.9	976.9 1081.4
F1 males	90.7	265.6	948.7
F1 females - premating - gestation	97.9 108.1	294.8 276.6	980.1 931.3

                                              

There were no clinical signs of toxicity or mortalities in any of the F0 or F1 parental dose groups. Food consumption and body weights were comparable to that of the concurrent controls. Necropsy and pathology revealed no gross findings or organ weight changes that could be treatment-related. Reproduction parameters in F0 and F1 parental animals There were no indications that sodium formate adversely affected fertility or reproductive performance of the F0 and F1 parental animals at dose levels as high as 1000 mg/kg bw/d. Mating behaviour, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross findings of these organs were comparable between the rats of the test substance-treated test groups and the corresponding controls, and ranged within the historical control data of the test facility. There were no effects on male and female reproduction organs. Sperm parameters and oestrous cycle were not affected. Reproduction and developmental parameters in F1 pups. No test substance induced signs of developmental toxicity were noted in the progeny of the F0 and F1 parents at dose levels as high as 1000 mg/kg body weight/day. The number of delivered pups/litter, the sex ratio, their postnatal survival on days 4 and 21 after parturition, their body weights, and their sexual maturation remained unaffected by the test substance. 

Clinical and pathological findings in F1 and F2 pups

-      Clinical and/or gross necropsy examinations of the F1 and F2 pups revealed only findings which were considered to be spontaneous in nature. The type and incidence of findings was within the range of the concurrent and/or the historical controls. (BASF SE, 2008).

Based on the above, the NOAEL values were as follows:

-      NOAEL 1000 mg/kg bw/d for general systemic toxicity for F0 and F1 parental animals

-      NOAEL 1000 mg/kg bw/d for fertility and reproductive performance for the F0 and F1 parental rats

-      NOAEL 1000 mg/kg bw/d for developmental toxicity, in the F1 and F2 progeny.

  

The study is considered to be valid and can be used for assessment of this endpoint.

 For read across purposes, the NOAEL for the formate anion may be calculated, taking into account formula weights. The calculation (1000 mg sodium formate/kg /69 x 45 = 650 mg/kg bw/day) gives a NOAEL of approx. 650 mg formate/kg bw/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A good quality 2-generation study is available for the submission substance

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There were no clinical signs of toxicity or mortalities in any of the F0 or F1 parental dose groups. Food consumption and body weights were comparable to that of the concurrent controls. Necropsy and pathology revealed no gross findings or organ weight changes that could be treatment-related. Reproduction parameters in F0 and F1 parental animals There were no indications that sodium formate adversely affected fertility or reproductive performance of the F0 and F1 parental animals at dose levels as high as 1000 mg/kg bw/d. Mating behaviour, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross findings of these organs were comparable between the rats of the test substance-treated test groups and the corresponding controls, and ranged within the historical control data of the test facility. There were no effects on male and female reproduction organs. Sperm parameters and oestrous cycle were not affected. Reproduction and developmental parameters in F1 pups. No test substance induced signs of developmental toxicity were noted in the progeny of the F0 and F1 parents at dose levels as high as 1000 mg/kg body weight/day. The number of delivered pups/litter, the sex ratio, their postnatal survival on days 4 and 21 after parturition, their body weights, and their sexual maturation remained unaffected by the test substance. 

Clinical and pathological findings in F1 and F2 pups

-      Clinical and/or gross necropsy examinations of the F1 and F2 pups revealed only findings which were considered to be spontaneous in nature. The type and incidence of findings was within the range of the concurrent and/or the historical controls. (BASF SE, 2008).

 Based on the above, the NOAEL values were as follows:

-      NOAEL 1000 mg/kg bw/d for general systemic toxicity for F0 and F1 parental animals

-      NOAEL 1000 mg/kg bw/d for fertility and reproductive performance for the F0 and F1 parental rats

-      NOAEL 1000 mg/kg bw/d for developmental toxicity, in the F1 and F2 progeny.

 

The study is considered to be valid and can be used for assessment of this endpoint.

 

For read across purposes, the NOAEL for the formate anion may be calculated, taking into account formula weights. The calculation (1000 mg sodium formate/kg /69 x 45 = 650 mg/kg bw/day) gives a NOAEL of approx. 650 mg formate/kg bw/day.

Effects on developmental toxicity

Description of key information

Studies in the rat and rabbit are available for sodium formate. Several thorough studies demonstrated clearly that teratogenicity seen with formic acid or sodium formate in Whole Embryo Culture tests in vitro is a secondary effect that is related to non-physiological pH values or osmolarity. Therefore, results from such tests cannot rule out results from valid Guideline studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Sodium formate
- Analytical purity: 100%

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

- Species:  rabbit
- Strain:   Himalayan
- Source:  Charles River Laboratories, Research Models and Services, Germany GmbH (former Charles River Laboratories, Germany)
- Sex: female
- Number: 25 per group
- Age: 13-21 weeks at study initiation
- Body weight (group mean): 2560 g
- Mating: Insemination; day of insemination designated day 0 of the study

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the solutions, an appropriate amount of the test substance was weighed in a graduated measuring flask depending
on the dose group, topped up with doubly distilled water and subsequently thoroughly mixed until it was completely dissolved.


VEHICLE
- Amount of vehicle (if gavage): dose volume: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with external calibration. Column Synergi 4µ Hydro-RP 80 A, 250 x 3 mm. Eluant: phsophate buffer pH 2.9. UV-detection at 200 nm (report, page 229 onwards).

Details on mating procedure:

Artificial insemination:
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.
This implied that 0.2 ml of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal®) were
injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination
were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of
the females participating in this study.
The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).

Duration of treatment / exposure:

days 6-28 post implantation

Frequency of treatment:

daily

Duration of test:

22 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Clinical signs: checked each day
- Mortality: checked twice daily on working days, once on Saturday, Sunday, at public holidays


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: recorded regularly throughout the entire study period, i.e. on days 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29 p.i.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Food consumption was recorded daily throughout the entire study period.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated by resorptions, live and dead fetuses) were determined. Number and distribution of implantations sites were classified as live fetuses and dead implantations. Dead implantations comprised early resorptions, late resorptions and dead fetuses.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Placenta weight: yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means * for p < 0.05, ** for p < 0.01: Food consumption, body weight, body weight change, corrected, body weight gain (net maternal, body weight change), carcass, weight, weight of unopened, uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight. 
Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions * for p < 0.05, ** for p < 0.01: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians * for p < 0.05, ** for p < 0.01: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Indices:

Conception rate, pre- and postimplantation loss

Historical control data:

Included in Part III of the report

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No clinical signs of toxicity, no treatement-related mortalities. Food consumption, body weights, necropsy findings not affected.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal weight, sex distribution, placenta weight not affected. Pre- and postimplantation loss not affected. External, soft tissue and skeletal malformations not affected by treatment.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Analysis of Test Substance
By various analyses, the stability of the test substance solutions over a period of 13 days at room temperature was
demonstrated, and the correctness of the prepared concentrations was shown.

EFFECTS
1 - Parental animals

Mortalities: none in any of the groups.
Clinical signs: none in any of the groups (Report, p. 34)
Food consumption: comparable across all groups, no relevant difference in treated groups compared to the control group
(Report, p. 35).
Body weights: comparable across all groups, no difference in treated groups compared to the control group ((Report p. 36).
Corrected body weight gain: did not reveal statistically significant differences between the test substance-treated groups and the control group (Report p. 36)
Uterus weight: no effect; the mean uterus weights were comparable between all groups (Report p. 37)
Necropsy findings: no test-substance related effect noted (Report p. 37)

Table1: Selected results for does in test groups 0 - 3,  i.e. at 0, 100, 300, or 1000 mg/kg bw per day

===========================================================
Parameter        Group 0     Group 1    Group 2     Group 3
-----------------------------------------------------------
Females mated/   
pregnant/       25/24/24   25/23/23    25/22/22   25/23/23
with viable 
fetuses

Food con-
sumtion (g)      100.5       97.8         94.5       94.6
day 0-29                    (97%)         (94%)      (94%)

Body weight      306.0      323.5        260.5      297.7 
gain (g)                    (106%)       (85%)      (97%)
day 0-29

Necropsy         - - No treatment-related findings  - -        
findings 
in dams 

Conception         96          92          88         92
rate (%)

Abortions           0           0           0          0

Corpora lutea    201/24     196/23        193/22    185/23
total/number 
of dams                

Implantations    176/24     185/23        156/22    161/23
total/number 
of dams
        
Resorptions       13/24     16/23          19/22     22/23
total/number 
of dams

total number      163        169            137       139
of fetuses                

total number       24         23             22        23
of litters                

pre-implantation 12.5(19.1) 6.7(11.9)   18.4(24.9) 13.3(16.1)
loss (+/- SD) %                

post-implantation 7.3(10.1) 7.9(9.3)  13.0(16.4) 13.9(16.2)
loss (+/- SD) %

fetuses/litter  6.8(2.25)  7.3(1.7)  6.2(2.5)  6.0(2.0)
(+/- SD)
live fetuses/   92.7(10.1)  92.1(9.3) 87.0(16.4) 86.1(16.2)
litter (+/- SD) %                

placenta weight   4.7        4.5          4.6          4.3
(mean) [g]                  (96%)        (98%)       (92%)

fetus weight     37.8        36.4        36.7        35.2
(mean) [g]                  (96%)        (97%)       (93%)
 
Fetal sex ratio 38.7:61.3  50.9:49.1   54.0:46.0   51.1:48.9        
[ratio m : f]
===========================================================
SD = Standard Deviation


2 - Fetuses
There were no test substance-related effects in any of the test groups at 100, 300, and 1000 mg/kg bw/day. 

Table 2: Selected results for fetuses in test groups 0 - 3,  i.e. from does at 0, 100, 300, or 1000 mg/kg bw per day:

===========================================================
Parameter        Group 0     Group 1    Group 2     Group 3
-----------------------------------------------------------
Total external malformations [%]
Fetuses           0.0         0.0        0.7         0.7
Litter            0.0         0.0        4.5         4.3 
Affect. fetuses   0.0         0.0        0.6         0.5
per litter

Total external variations [%]
Fetuses           4.3         0.6        2.2         1.4
Litter            13          4.3        9.1         8.7
Affect. fetuses   2.3         0.9        2.0         1.4
per litter

Skeletal malformations [%]
Fetuses           1.8         1.8        1.5         3.6
Litter            13          13         9.1         22 
Affect. fetuses   2.3         2.0        1.0         3.4
per litter

Skeletal variations [%]
Fetuses           52          65         60          65
Litter           100          91         95          96
Affect. fetuses   53.5        61.8       62.2        65.7
per litter

Soft tissue malformations [%]
Fetuses           1.2         0.6       2.2         4.3
Litter            8.3         4.3       14          26
Affect. fetuses   1.5         0.5       2.1         4.5
per litter

Soft tissue variations [%]
Fetuses           4.3         11        11          10
Litter            21          48        41          39
Affect. fetuses   4.2        10.9       9.4         8.9
per litter
===========================================================

Conclusions:

No maternal toxicity, embryofoetal toxicity or teratogenicity was seen in this study at any dose level (up to 1000 mg/kg bw/d)

Executive summary:

In a developmental toxicity study performed according to OECD test guideline No. 414 and under GLP conditions, sodium formate (in water) was administered to 25 female Himalayan rabbits by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/d from days 6 through 28 of gestation. There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, caesarean parameters, and terminal necropsy in the does. The maternal NOAEL is therefore 1000 mg/kg bw/d. There were no treatment-related effects in developmental parameters. Foetal weight at birth, sex distribution, placenta weight, pre- and post-implantation loss were not affected. There were no unusual or increased incidences of external, soft tissue or skeletal malformations attributable to the treatment. The developmental NOAEL is therefore 1000 sodium formate mg/kg bw/d. The NOAEL for teratogenicity is also 1000 sodium formate mg/kg bw/d, the highest dose tested (BASF AG, 2008). The developmental toxicity study in the rabbit is classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbits. Generally, formate salts are used as test material in studies requiring repeated dosing, due to the corrosivity of formic acid. NOAEL values obtained in such studies may be used to calculate the NOAEL for the formate anion which may be read across to other salts or formic acid, taking into account stoichiometry and formula weights. For the ease of the reader, this is tabulated below (calculation in brackets).

	Formula weight	NOAEL maternal toxicity	NOAEL developmental toxicity	NOAEL teratogenicity
		(mg/kg bw/d)
Sodium formate	69	1000	1000	1000
Formate anion	45	652 (1000/69x45)	652 (1000/69x45)	652 (1000/69x45)
Formic acid	46	667 (1000/69x46)	667 (1000/69x46)	667 (1000/69x46)

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 November 2003 to 23 March 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

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)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): sodium formate
- Physical state: solid/crystalline
- Analytical purity: >99%.
- Storage: at room temperature under nitrogen

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals
- Species: rat.
- Strain: Wistar.
- Sex: female.
- Age: 70-84 days at gestational day 0.
- Body weight: 141-202  grams on gestational day 0.
- Housing: singly in stainless steel wire mesh cages.
- Diet: ground Kliba maintenance diet rat/mouse and water ad libitum.

- Environment: fully air-conditioned rooms.
- Temperature range 20-24°C, relative humidity 30-70%.
- Dark/light cycle: 12/12 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The aqueous test substance solutions were prepared at the beginning of the administration period and thereafter at intervals which
took into account the analytical results of the stability verification . For the preparation of the solutions, an appropriate amount of the
test substance was weighed in a graduated measuring flask, topped up with doubly distilled water and subsequently thoroughly mixed
using a magnetic stirrer .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detection

Details on mating procedure:

- Mating: time-mated by the breeder.

Duration of treatment / exposure:

Gestation Days 6-19

Frequency of treatment:

Daily

Duration of test:

Dams were sacrificed on Gestation Day 20

Dose / conc.:

0 mg/kg bw/day

Remarks:

physiological saline

Dose / conc.:

59 mg/kg bw/day

Dose / conc.:

236 mg/kg bw/day

Dose / conc.:

945 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes

Details on study design:

- Due to technical reasons the study was conducted in three replicates.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical symptoms and mortality: at least daily
- Cage side observations checked were included (Part A, Table IA-001).


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p .c . . The body
weight change of the animals was calculated from these results.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Parameters examined: gross pathology. Determination of corrected body weight

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:
- Placenta weight: Yes
- Number of live and dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Other:
fetal weight
Sex distribution

Statistics:

Statistical evaluation included the use of Dunnett's test, Fisher's exact test, and of the Wilcoxon test.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No clinical signs of toxicty, no mortalities, food consumption and body weight not affected. Mean gravid uterus weight and reproduction data (concenption rate, corpora lutea, pre- and postimplantation loss, number of viable fetuses) not affected at any dose level. No findings at necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

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

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A single malformation (unilateral anopthalmia) observed in one foetus of the high dose group is not considered to be related to treatment.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Placenta weight, fetal weight, sex distribution not affected. External malformations inside historical control range; no soft tissue or skeletal malformations. Incidence and type of variations does not suggest a treatment-related effect at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

EXAMINATION OF THE DAMS
Only pregnant dams were used for calculations. The number of pregnant  animals per group was as follows:

Table 1: dose levels and number of tested animals

Group	Dose (mg/kg bw /day)	Number of pregnant animals
0	0	24
1	59	25
2	236	23
3	945	24

Mortalities:  none in any of the groups.
Clinical signs: none in any of the groups.
Food consumption: similar across all groups, no difference in treated  groups compared to the control group.
Body weights: similar across all groups, no difference in treated groups  compared to the control group.
Corrected body weight gain (terminal body weight minus weight of the  unopened uterus minus body weight on gestational day 6): no difference 

of  biological relevance in treated groups compared to the control group.
Mean carcass weights: similar among all test groups 0-3.

Terminal examinations of dams:
Uterus weight: the mean gravid uterus weight of the treated test groups  1, 2, and 3 was not influenced by the treatment. The differences 

between  these groups and the control group revealed no dose-dependency and were  without biological relevance.

Necropsy findings: none.

Reproduction data: Conception rates were not affected by treatment. 

Table 2: conception rate

 

Group	Dose (mg/kg bw /day)	Conception rate (%)
0	0	96
1	59	100
2	236	92
3	945	96

There were no substance-related and/or biologically significant  differences among the test group in the conception rate,  the mean number  of corpora lutea and implantation sites, or in the values calculated for  the pre- and postimplantation losses  as well as the number of resorptions  and viable fetuses.

EXAMINATION OF THE FETUSES
Sex distribution : nor affected.
Weight of placentae: mean weights in treated groups comparable to the  control group.
Fetal weight: mean weights in treated groups comparable to the control  group.

External examinations
External malformations were within the historical control range. There  was no external variation recorded in any of the fetuses.
Table 3: Incidence of external malformations

Group	Dose (mg/kg bw /day)	External malformations (incidence / %)
0	0	0/123 fetuses / 0%
1	59	0/220 fetuses / 0%
2	236	0/212 fetuses / 0%
3	945	1/212 fetuses / 0.5%

Soft tissue examinations
No soft tissue malformation was recorded for any of the fetuses.
Two soft tissue variations (uni- or bilateral dilatation of the renal  pelvis with or without dilated ureter) were detected in each group  including 

the controls without any dose-relationship.

Table 4: Incidence of soft tisue variations

Group	Dose (mg/kg bw /day)	Soft tissue variations (%)
0	0	5.6
1	59	3.6
2	236	6.1
3	945	1.7

Skeletal examinations
There were no skeletal malformations in treated fetuses.

Table 5: Incidence of skeletal malformations

 

Group	Dose (mg/kg bw /day)	Skeletal malformations (incidence / %)
0	0	1/113 fetuses / 0.9%
1	59	0/116 fetuses / 0%
2	236	0/113 fetuses / 0%
3	945	1/109 fetuses / 0%

The incidence of skeletal variations does not suggest a treatment-related  effect. The observed pattern of skeletal variations was not different from that  seen in the historical controls. The scattered occurrence 

of  statistically  significant differences compared to the control group was  not dose-related and not considered to be of 

toxicological relevance.

Table 6: Incidence of skeletal variations

Group	Dose (mg/kg bw /day)	Skeletal variations (%)
0	0	96.9
1	59	91.4
2	236	96.7
3	945	96.7

 

Conclusions:

No maternal toxicity, embryofoetal toxicity or teratogenicity was seen in this study at any dose level (up to 945 mg/kg bw/d)

Executive summary:

In this study, time-mated female rats (25/dose, OECD TG 414; study conducted under GLP conditions) received sodium formate via oral gavage at 0, 59, 236, and 945 mg/kg bw/d during gestation days 6 to 19.  Maternal toxicity was not seen. Gestational parameters were not influenced and there were no effects on the developing foetuses.  No malformations or skeletal variations were seen. The NOAEL for maternal and developmental toxicity was 945 mg sodium formate/kg bw/day, the highest dose tested (BASF AG, 2005). This developmental toxicity study is also classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

 

	Formula weight	NOAEL maternal toxicity	NOAEL developmental toxicity	NOAEL teratogenicity
	(mg/kg bw/day)
Sodium formate	69	945	945	945
Formate anion	45	616 (945/69x45)	616 (945/69x45)	616 (945/69x45)
Formic acid	46	630 (945/69x46)	630 (945/69x46)	630 (945/69x46)

 

Reference 3

Endpoint:

developmental toxicity

Type of information:

not specified

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

embryo in-vitro incubation

GLP compliance:

not specified

Specific details on test material used for the study:

Sodium formate from Fisher Scientific, Fairlawn, NJ, USA.

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

other: whole-embryo culture

Duration of treatment / exposure:

48 hr

Abnormalities:

not specified

Developmental effects observed:

not specified

1) Effect on culture medium pH 
The effect of the addition of formate and of the starting pH
on culture medium pH measured after 48 h was low.

===========================================================
Start.        pH-values at formate conc. (mg/ml)
pH      0*      0     0.2     0.4     0.8    1.2    1.6
-----------------------------------------------------------
8.13   7.71    7.51   7.54    7.50    7.49   7.51   7.52
7.75   7.69    7.41   7.42    7.42    7.49   7.43   7.45
7.00   7.50    7.21   7.27    7.21    7.29   7.24   7.30
6.50   7.26    7.02   7.04    6.99    7.03   7.07   7.11 
===========================================================
Mean values (n= 8-14/group)
* = control flask without embryos


2) Effect on embryo growth
Numerous embryonic developmental parameters showed that both
the decreasing pH and the increasing formate concentration
had an influence on embryonic development in this test
system. 

At the highest concentration, the parameters crown-rump
length, head length, somite developmental and protein
concentration were significantly reduced (p<0.05) regardless
of the pH. At formate concentrations of 0.8 and 1.2 mg/ml,
these parameters were
significantly reduced at a low pH. 

At formate concentrations of 0.4 and 0.2 mg/ml, crown-rump
length, head length and the protein concentration were still
significantly reduced at a pH of 6.5 in the medium. 
However, at pH 6.5, lethality was already marked (27%) in
the absence of formate, and increased to 50 and 92% at the
highest concentrations. At pH 6.0, lethality was complete,
i.e. 100%, irrespective of the formate concentration. 

===========================================================
       Start.        Values at formate conc. (mg/ml)
        pH         0     0.2     0.4     0.8    1.2    1.6
-----------------------------------------------------------
Crown-rump length (mm)
       8.13        3.8   3.8     3.8     3.7    3.6    3.3*
       7.75        3.9   4.0     3.9     3.6    3.5*   3.7
       7.00        3.7   3.8     3.8     3.6    3.6    3.4*
       6.50        3.5   3.4*    3.3*    3.4*   3.0*     -
Head length (mm)
       8.13       1.91  1.95    1.90    1.82   1.68   1.48*
       7.75       2.03  2.03    1.90    1.76   1.77   1.66*
       7.00       1.86  2.00    1.77    1.68   1.67*  1.50*
       6.50       1.76* 1.61*   1.61*   1.57*  1.38*  1.15*
Somite development
       8.13      25.7   25.4    25.8    25.4   24.7   21.4*
       7.75      26.3   26.9    25.5    25.1   23.9   23.7*
       7.00      24.9   26.3    25.7    23.7*  24.5   20.8*
       6.50      25.5   24.1    24.6    22.7*  21.2*  20.1*
Protein content (µg/embryo)
       8.13       117    119    123      120     94     78*
       7.75       144    164    122       96     93     92
       7.00       121    135    105       88*   100     71*
       6.50        95     79*    88*      72*    42*    37* 
Embryo lethality (% dead) 
       8.13         0      0      0        0      0      8
       7.75         0      0      0        9     13     18
       7.00         0      0      0        8     25     42*
       6.50        27     13      8       25     50*    92* 
       6.00      100*    100*   100*     100*   100*   100* 
===========================================================
Mean values (n= 8-14/group)
* = p<0.05

Conclusions:

Both high concentrations of sodium formate and low pH-values of the culture medium influenced the rat embryo development and survival in vitro.
The significance of this to in vivo developmental toxicity after exposure to formate is not known.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

embryo in-vitro incubation

The study of Andrews and coworkers (1993) was selected as representative for several other in vitro studies of sodium formate and formic acid (and also of the precursors methanol and formaldehyde) on developing rat or mouse embryos that have been published. Summaries of some of these studies are included in the formic acid IUCLID document.  It was generally shown that high concentrations of sodium formate and/or low pH-values have effects on the embryo in vitro, but the significance of this to in vivo developmental toxicity after exposure to formate is not known. For the sake of clarity of the IUCLID document only a few representative study summaries were generated, while other studies were identified and listed in a separate entry under the heading of the "Whole Embryo Culture Test".

GLP compliance:

no

Species:

other: rat and/or mouse

Route of administration:

other: whole-embryo culture

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Teratogenicity seen with formic acid or sodium formate in Whole Embryo Culture tests in vitro is a secondary effect that is related to unphysiological pH values or osmolarity.

Abnormalities:

not specified

Developmental effects observed:

not specified

Further Whole-Embryo Culture Tests

===========================================================
Species     TS    Conc. (mg/ml)    RS           RE
-----------------------------------------------------------
rat&       NaF     0.2 - 3         +      Andrews et al.
mouse      FA      0.14 - 2        +      (1995);
                                          Ebron-McCoy et
                                          al. (1994) Abs
-----------------------------------------------------------
rat        MeOH    0 - 10.3   combination Andrews et al.
           FA      0 - 2      less toxic  (1995) Abs
-----------------------------------------------------------
rat        pH     5.75 - 8.23      +      Ebron-McCoy et
                                          al. (1994) Abs
-----------------------------------------------------------
mouse      2-MAA      5    inhibition of  Stedman & Welsch  
                          DNA synthesis  (1989) 
                           >55%                   
           NaF        1    inhibition 6%; not significant
       2-MAA+NaF    5 + 1  attenuation of DNA synthesis: 
                           inhibition approx. 11%
-----------------------------------------------------------



===========================================================

TS = test substance (individual substances given,
combinations not listed here)
RS = result; + = findings similar to Andrews (1993);
RE = reference
Abs =abstract

2-MAA = 2-methoxyacetic acid
FA = formic acid
Form = formaldehyde
NaF = sodium formate
MeOH = methanol

Conclusions:

Several thorough studies demonstrated clearly that teratogenicity seen with formic acid or sodium formate in Whole Embryo Culture tests in vitro is a secondary effect that is related to unphysiological pH values or osmolarity. Therefore, results fromsuch tests cannot rule out results from valid Guideline studies.

Executive summary:

Several thorough studies demonstrated clearly  that teratogenicity seen with formic acid or sodium formate in Whole Embryo Culture tests in vitro is a secondary effect that is related to unphysiological pH values or osmolarity. Therefore, results fromsuch tests cannot rule out results from valid Guideline studies.

Reference 5

Endpoint:

developmental toxicity

Type of information:

not specified

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dissociation of the teratogenic properties of methanol and formate in mice by combining in-vivo examinations including pharmacokinetics with the in-vitro Whole Embryo Culture test.

Specific details on test material used for the study:

Sodium formate from Aldrich Chemical Co. and methanol (HPLC-grade) from Sigma Chemical Co.

Species:

mouse

Strain:

CD-1

Route of administration:

oral: gavage

Frequency of treatment:

single dose on gestational day 8

Details on study design:

Sex: female

Abnormalities:

not specified

Developmental effects observed:

not specified

RS-Freetext:
1. IN-VIVO STUDIES

i) Pharmacokinetic: Oral gavage of MeOH

1 hour after a single oral dose of 1.5 g MeOH/kg bw, MeOH
levels had reached 50.3 mM in maternal plasma and declined
linearly to normal levels in the log concentration-time plot
within 24 hr. Pretreatment with 4-methylpyrozole (4-MP)
caused a slower elimination of MeOH.
Deciduas' MeOH concentration reached 33.3 mM at 1 h and
tended to be lower than in maternal plasma. 
Pretreatment with 4-MP increased the MeOH concentration in
maternal plasma, decidua, and the Area Under the Curve (AUC)
following the 1.5g/kg bw exposure.

Pretreatment with 4-MP had no significant effect on the
maximal formate concentrations were noted in maternal plasma
or decidua:

===========================================================
                             Formate concentration
Pretreatment  Dose          Plasma         Decidua
              MeOH           (mM)           (mM)
              (mg/kg bw)
-----------------------------------------------------
saline        1500         0.75+/-0.08     0.64+/-0.27 
4-MP          1500         0.93+/-0.26     0.62+/-0.08 
===========================================================

ii) Pharmacokinetic: MeOH inhalation 
The mean end-of-exposure plasma levels of MeOH were 65 and
223 mM following the exposure to 10000 or 15000 ppm,
respectively. Similar concentrations were reached in
decidua. The pretreatment with 4-MP had little effect on the
peak levels and the elimination of MeOH from plasma. 

The peak plasma and decidua formate levels following 10000
ppm MeOH inhalation were comparable to the levels found in
the MeOH gavage experiment, and the pretreatment with 4-MP
had no significant effect:

===========================================================
                             Formate concentration
Pretreatment  MeOH dose      Plasma         Decidua
              10000           (mM)           (mM)
              (ppm) 
-----------------------------------------------------
saline                     0.62+/-0.06     2.1+/-1.0 
4-MP                       0.50+/-0.07     1.8+/-0.4 
===========================================================


iii) Developmental outcome

The incidence of open neural tubes was significantly
increased in embryos from dams exposed to 10000 ppm,
compared to control mice exposed to HEPA-filtered air. A
lower increase that did not reach the level of statistical
significance (p>0.05) was noted in the group pretreated with
4-MP. Head and body lengths were not affected.


In embryos from dams that had received sodium formate at 750
mg/kg bw the incidence of open neural tubes was not
statistically different from controls. Head and body lengths
were also not affected.

===========================================================
                   GD 10                    GD 18
             --------------------   -----------------------
Treat-       No. of   Open neural   No. of      Open neural
ment         litters   tubes (%)    litters      tubes (%)
-----------------------------------------------------------
INHALATION
MeOH, saline   12    9.65+/-3.13*    -              
MeOH, 4-MP     12    7.21+/-2.65     -               
HEPA-Air,      14    2.29+/-1.01     -
saline        

GAVAGE
Formate        14    2.02+/-1.08     8           0+/-0
Water          12      0+/-0         3           0+/-0
===========================================================
* p<0.05



2. WHOLE EMBRYO CULTURE TESTS

Morphological findings in embryos exposed on gestational day
8 tabulated indicate that both methanol and formate
increased the incidence of dysmorphogenic effects in a
dose-related manner.

===========================================================
                                 Number of
              ---------------------------------------------
Group   (mM)  Life/      Cephalic     Prosence-  Branchial
              abnormal   dysraphism,  phalic     arch
              embryos    total        lesions    hypoplasia
-----------------------------------------------------------
Vehicle   0    20/3        2            2           0 
MeOH     62    13/1        0            1           0
        125    14/5        2            4           1
        187    13/7        6            4           1 
        250    15/7        7            8           6* 
        375    12/7       11*          10*          8*

Formate   4    12/2        0            2           1
          8    13/5        6            6           0 
         12     9/5        5            3           0 
         20    16/7        7            3           1
         40    16/14*     14*           8          13*
===========================================================
* p<0.05

It was additionally reported that there were dose-related
delays in the embryo development noted (reduction in yolk
sac diameter, reduced crown-rump length, delayed rotation)
with both methanol and formate.

Conclusions:

The authors concluded that exencephaly results from the parent compound methanol, administered at high concentrations, rather than the accumulation of formate, i.e. that methanol is the teratogen, and not formate.
This conclusion was based on the following findings:
i) no significant formate accumulation was seen in the MeOH-treated mice.
ii) exencephaly occurred without elevated maternal plasma or decidual tissue formate levels.
iii) treatment of mice with a high oral formate dose did not result in exencephaly, even with formate tissue levels
comparable to those observed after a high concentration inhalation exposure.
iv) methanol, but not formate, induced neural tube closure defects in cultured mouse embryos at media concentrations
comparable to those observed in decidual swelling after maternal exposure to methanol.

Reference 6

Endpoint:

developmental toxicity

Type of information:

not specified

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment. However, the unclear relevance to in-vivo developmental toxicity after exposure to formate should be considered.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Exposure of developing embryos in the Whole Embryo culture Test, using direct exposure in the culture medium and microinjection into amniotic fluid.

GLP compliance:

not specified

Specific details on test material used for the study:

Methanol, formaldehyde, and sodium formate.

Species:

other: mouse and rat

Route of administration:

other: whole-embryo culture

Control animals:

yes

Abnormalities:

not specified

Developmental effects observed:

not specified

RS-Freetext:
1.) Embryo exposure in culture medium
==========================================================
Conc.    viability   normal rotation    neural tube closure 
(mg/ml)     (%)          (%)                  (%)
----------------------------------------------------------
Mouse
0.0         94           93                   92
0.5         94           76*                  88
1.0        100           94                   88
2.0         90           70*                  60*
4.0         93           54*                  85

Rat
0.0        100           92                  100
0.5         97           93                  100
1.0         87           85                  100
2.0         60*         100                  100
4.0         47*          78*                 100
8.0          0*          NA                   NA
==========================================================


2.) Embryo exposure via microinjection
==========================================================
Conc.    viability   normal rotation    neural tube closure 
(µg)        (%)          (%)                  (%)
----------------------------------------------------------
Mouse
0            92          94                   91  
0.03-0.5    100          73*                  91
0.51-1.0     82          21*                  79
1.01-1.5     65*         27*                  55*
1.51-4.0      0*         NA                   NA

Rat
0.0         100          89                  100
0.03-0.5     95          93                  100
0.51-1.0     90          11*                 100
1.01-1.5     78           0*                  25
1.51-4.0      0*         NA                   NA
==========================================================
* = p<0.05
NA = nort applicable

Conclusions:

Key findings of earlier studies (Contreras et al., 1995) were confirmed:
- mouse embryos are more susceptible than rat embryos regarding embryo lethality and development (neural tube closure).
- direct addition of the test substances to the culture medium is less toxic than injection into the amniotic fluid,
suggesting that the visceral yolk sac provides protection against toxicity.
- embryolethal concentrations of formaldehyde were approx. 1000-fold lower than those of methanol or sodium formate
(data not contained in the study summary).

Reference 7

Endpoint:

developmental toxicity

Type of information:

not specified

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

GLP compliance:

not specified

Specific details on test material used for the study:

Methanol, formaldehyde, and sodium formate.

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

other: whole-embryo culture

Control animals:

yes

Abnormalities:

not specified

Developmental effects observed:

not specified

RS-Freetext:
1) Methanol exposure (12 and 24 mg/ml) produced a decrease
in normal growth parameters and a dose-dependent loss of
viability. 

2) Formaldehyde (3 and 6 µg/ml) had deleterious effects on
embryo growth and viability. 

3) Sodium formate exposure (0.5 and 2.0 mg/ml) resulted in a
high mortality rate but viable embryos did not manifest any
abnormalities. 

4) Glutathione (GSH) depletion
Methanol, formaldehyde, and formate all produced a
significant depletion of GSH in both embryo and visceral
yolk sac. 

5) Inhibition of GSH synthesis 
Inhibition of GSH synthesis by L-buthionine-S,R-sulfoximine
(BSO) treatment exacerbated methanol, formaldehyde, and
formate embryotoxicity. 
Interestingly, only methanol/BSO and formaldehyde/BSO
co-treatments caused increased malformation, while embryos
treated with formate/BSO did not produce any developmental
deformities. 

Rat embryo treatment with formate, with or without BSO, for
24 in vitro
==========================================================
Group         viabi-   rotation   closure  malformations    
             lity
(mg/ml)        (%)       (%)      (%)      (n/out of)  
----------------------------------------------------------
Control      100         100      92          0/26

NaF 
0.5           84*        100      94     blisters 1/16
2.0           40*        100     100     necrotic 2/15

BSO 2.0       95          95      95     blisters 1/22

BSO 2.0/      71          83     100     blisters 1/17
NaF 0.5                                  necrotic 5/17

BSO 2.0/      27**       100     100     necrotic
NaF 2.0                                 
==========================================================

NaF = sodium formate
BSO = L-buthionine-S,R-sulfoximine

* = p<0.05 compared to control
** = p<0.01, compared to NaF only

Conclusions:

Sodium formate was selectively embryolethal and did not produce dysmorphogenesis. The results implicate formaldehyde as the most embryotoxic methanol metabolite, on a molar basis, in terms of causing dysmorphogenesis, alterations of normal growth parameters and embryolethality. Formaldehyde toxicity is potentiated by GSH depletion, indicating that GSH may be more directly involved in its detoxication in the embryo.

Reference 8

Endpoint:

developmental toxicity

Type of information:

other: Abstract

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

Microinjections of test substance into the amniotic fluid.

GLP compliance:

no

Specific details on test material used for the study:

Methanol, formaldehyde, and sodium formate.

Species:

rat

Route of administration:

other: whole-embryo culture

Abnormalities:

not specified

Developmental effects observed:

not specified

RS-Freetext:
1) Methanol
Microinjection of methanol at doses in excess of 175 µg
failed to reduce viability below 80% or cause embryonic
protein to be reduced by more than 20%, although significant
increases in abnormal axial rotation and decreases in crown
rump length were observed. Higher doses of methanol were not
possible due to severe distension of the amnion. 

2) Formaldehyde 
Formaldehyde was found to be the most toxic of all compounds
tested, producing decreases in viability to 42% and 8% of
controls following microinjections of 1.5 and 3.0 µg of
formaldehyde, respectively. Low doses of formaldehyde
(0.2-0.5 µg) resulted in an apparent increase in embryonic
protein (358.6 ± 13.3 µg for formaldehyde and 286.4 ± 14.3
µg for controls) and crown rump length (3.5 ± 0.2 mm for
formaldehyde and 3.37 ± 0.2 mm for controls), but these
parameters decreased significantly below 50% of control
values at higher doses.

3) Sodium formate 
Sodium formate elicited a similar decrease in viability as
formaldehyde, beginning at a dose of 5.0 µg. 

4) Calculated toxic concentrations 
Calculated toxic concentrations of methanol, formaldehyde
and sodium formate, based on an estimated volume of
intraamniotic fluid of 500 nl, produced final concentrations
in the amniotic fluid :
methanol 350 mg/ml, formaldehyde 4 mg/ml, and sodium formate
10 mg/ml.

According to the authors these results indicate that the
metabolites of methanol produce embryotoxicity at
concentrations similar to those reported to elicit similar
effects when added directly to the culture medium. The
100-fold excess of methanol required to elicit any similar
effects suggests that this compound requires significant
visceral yolk sac metabolism in order to produce
embryotoxicity.

Conclusions:

Following microinjection into the amniotic fluid, the calculated toxic concentrations were as follows: methanol 350 mg/ml, formaldehyde 4 mg/ml, and sodium formate 10 mg/ml.

The authors concluded that these values were similar to those which elicit toxicity when the test chemicals were directly added to the culture medium.

Reference 9

Endpoint:

developmental toxicity

Type of information:

other: Abstract

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

Exposure of developing embryos in the Whole Embryo culture Test, using direct exposure in the culture medium and microinjection into amniotic fluid.

Specific details on test material used for the study:

Methanol, formaldehyde, and sodium formate.

Species:

other: mouse and rat

Route of administration:

other: whole-embryo culture

Abnormalities:

not specified

Developmental effects observed:

not specified

1) Methanol 
Microinjection in 4-10 somite CD-1 mouse embryos resulted in
a 75% decrease in viability at doses as low as 45 µg,
whereas amounts in excess of 178 µg were required at the
same stage to produce an equivalent effect in the rat. 

2) Formaldehyde
Four-fold lower amounts (0.26-0.50 and 1.51-2.00 µg in mouse
and rat, respectively) of formaldehyde were required in the
mouse to produce a 76% decrease in viability, a 50%
incidence of incomplete neural tube closure and a
significant increase in axial rotation defects. 

3) Sodium formate
Intraamniotic exposure to sodium formate resulted in
negligible differences between species for any parameter
measured.

Conclusions:

Differences observed in embryos undergoing intramniotic
exposure:

1) Species differences: -mouse embryos were more susceptible than rat embryos. Viability of mouse embryos was decreased by ca. 75% by methanol and formaldehyde at lower concentrations compared to rats where 4-fold higher concentrations were needed to produce the same effect.

-No species difference was seen with sodium formate.

2) Toxicity of chemicals: Formaldehyde was approx. 100-fold more toxic compared to methanol in both rat and mouse embryos.

Reference 10

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

Examination of toxicity and malformations in embryos following injection of test material into hen's eggs at 48 or 96 hrs after hatching.

GLP compliance:

no

Specific details on test material used for the study:

Sodium formate

Species:

other: hen

Route of administration:

other: injection into hen's egg

Control animals:

other: historical and positive (0.025 mg hydrocortisone /egg)

Abnormalities:

not specified

Developmental effects observed:

not specified

Overall result: negative

(1) The injection of 5, 10, or 20 mg calcium formate (CaF)
into eggs did not increase the incidence of malformations
compared to the untreated controls (n=1051), nor was there a
malformation that had not been noted in the untreated
control embryos.

(2) It was demonstrated that after injection of another TS,
10 mg NaF, formic acid was formed, and that formic acid was
completely eliminated from the egg within 8-10 days. The
mean concentrations were as follows:

=====================================================
Day after           Formic acid          Controls,   
injection            (mg/egg;           upper boarder     
                 approx. means, n=74)    (mg/egg)   
-----------------------------------------------------
 1                     6
 4                     5.75
 6                     4.5
 7                     2.25
 8                     1.4                  1.4         
12                     0.5 
=====================================================

Conclusions:

Formic acid was found at elevated concentrations in the hen's egg following the injection of a 10 mg sodium formate per egg. It is assumed that
this also applies to other formate salts.
The formic acid was eliminated and reached control values within 8 to 10 days.
Injected calcium formate (5, 10, and 20 mg/egg) did not increase the incidence or change the pattern of malformations seen in
controls, i.e. sodium formate was not teratogenic under these conditions.

Executive summary:

Formic acid was found at elevated concentrations in the hen's egg following the injection of a 10 mg sodium formate per egg. It is assumed that this also applies to other formate salts. The formic acid was eliminated and reached control values within 8 to 10 days. Injected calcium formate (5, 10, and 20 mg/egg) did not increase the incidence or change the pattern of malformations seen in controls, i.e. sodium formate was not teratogenic under these conditions.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Quality of whole database:

Good quality studies are available for the rat and rabbit and are supported by in vitro studies in whole embryo cultures.

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

Sodium formate showed no developmental toxicity in GLP guideline studies using rabbits and rats.

Studies in vivo

In a developmental toxicity study performed according to OECD test guideline No. 414 and under GLP conditions, sodium formate (in water) was administered to25 female Himalayan rabbits by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/day from days 6 through 28 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, caesarean parameters, and terminal necropsy in the does. The maternal NOAEL is therefore 1000 mg/kg bw/d. Likewise, there were no treatment-related effects in developmental parameters. Foetal weight at birth, sex distribution, placenta weight, pre- and post-implantation loss were not affected. There were no unusual or increased incidences of external, soft tissue or skeletal malformations attributable to the treatment. The developmental NOAEL is therefore 1000 mg/kg bw/d. The NOAEL for teratogenicity is also 1000 mg/kg bw/d, the highest dose tested (BASF, 2008). The developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbits.

The above is supported by a rat developmental toxicity study. Time-mated female rats (25/dose, OECD TG 414; study conducted under GLP conditions) received sodium formate via oral gavage at 0, 59, 236, and 945 mg/kg bw/d during gestation days 6 to 19.  Maternal toxicity was not seen. Gestational parameters were not influenced and there were no effects on the developing foetuses.  No malformations or skeletal variations were seen. The NOAEL for maternal and developmental toxicity was 945 mg/kg bw/d, the highest dose tested (BASF AG, 2005). This developmental toxicity study is also classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Studies in vitro

The in vivo studies clearly indicate that sodium formate lacks developmental toxicity and overrule several in vitro Whole Embryo Culture tests. Some of these reported positive findings following exposure to methanol, formaldehyde, or formic acid and sodium formate. It was, however, shown that confounding factors (pH value, osmolarity) rather than formic acid or formate caused positive responses in vitro.

Read across to related chemicals

Generally, formate salts are used as test material in studies requiring repeated dosing, due to the corrosivity of formic acid. NOAEL values obtained in such studies may be used to calculate the NOAEL for the formate anion which may be read across to other salts or to formic acid, taking into account stoichiometry and formula weights. For the ease of the reader, this is tabulated below (calculation in brackets).

Table: calculation of NOAEL values

	Formula weight	NOAEL maternal toxicity	NOAEL developmental toxicity	NOAEL teratogenicity
		(mg/kg bw/day)
Sodium formate	69	1000	1000	1000
Formate anion	45	652 (1000/69x45)	652 (1000/69x45)	652 (1000/69x45)
Formic acid	46	667 (1000/69x46)	667 (1000/69x46)	667 (1000/69x46)

Toxicity to reproduction: other studies

Additional information

While several studies reported foetotoxicity and teratogenicity following exposure of embryos against methanol, formaldehyde, formic acid, or sodium formate in vitro in the Whole Embryo Culture test (WEC). However, Andrews and others clearly demonstrated that such findings are secondary to non-physiological pH values or osmolarity. Therefore, results from such WEC tests cannot rule out results obtained in valid guideline studies.

Justification for classification or non-classification

There is no indication for the developmental or reproductive potential of sodium formate from the available in vivo studies. Sodium formate is therefore not classified for reproducitve toxicity according to the CLP criteria.