3-chloropropane-1,2-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Clear description of method and results in a non-guideline and non-GLP study. No analytical information regarding test substance.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Lack of effect on rat testicular organogenesis after in utero exposure to 3-monochloropropane-1,2-diol (3-MCPD)

Author:

El Ramy R, Elhkim MO, Poul M, Forest MG, Leduque P, and Le Magueresse-Battistoni B

Year:

2006

Bibliographic source:

Reproductive Toxicology 22 (2006) 485-492

Materials and methods

Test guideline

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

Testicular organogenesis during gestation was studied in rats in utero. Pregnant rats were given daily doses of 5, 10 or 25 mg/kg BW of 3-MCPD from days 11.5–18.5 postcoitum (dpc). On 19.5 dpc, testes were removed from fetuses for histological examination and testosterone analysis. Eight genes were selected among the differentiation markers of testicular cell lineages, and their expression was studied by RT-PCR (reverse transcription-polymerase chain reaction) procedure. The genes encode a transcription factor Sox-9 (Sry homebox-like gene 9); hormone receptors including the aryl hydrocarbon receptor (AhR), the estrogen receptor alpha (ER) and the follicule-stimulating hormone receptor (FSHR); the steroidogenic acute regulatory protein (StAR) which controls the rate-limiting step steroidogenesis; steroidogenic enzyme beta-hydroxysteroid dehydrogenase type I (3-HSD); the doublesex and mab3-related transcription factor (DMRT-1) which contributes to testis organogenesis; and the nerve growth factor receptor p75 (NGFR) which is a specific marker of peritubular cells. Cell apoptosis and cell proliferation were analyzed in male fetuses exposed in utero to 3-MCPD using immunohistochemical techniques. The levels of 3-MCPD and its main metabolite, beta-chlorolactic acid, were assayed in fetal tissues and dam plasma.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-chloropropane-1,2-diol

EC Number:

202-492-4

EC Name:

3-chloropropane-1,2-diol

Cas Number:

96-24-2

Molecular formula:

C3H7ClO2

IUPAC Name:

3-chloropropane-1,2-diol

Constituent 2

Reference substance name:

3-monochloropropane-1,2-diol

IUPAC Name:

3-monochloropropane-1,2-diol

Details on test material:

- Name of test material (as cited in study report): 3-monochloropropane-1,2-diol
- Obtained from Sigma-Aldrich Chemical Co., St. Quentin-Fallavier, France

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (le Genest, France)
- Age at study initiation: 12 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: plastic cages in air conditioned room
- Diet (e.g. ad libitum): commercial food ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h light, 12 h dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Doses were adjusted daily to individual body weight.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

The morning after mating was designated as day 0.5 of gestation and was confirmed by the presence of sperm in vaginal saline washes.

Duration of treatment / exposure:

Testicular gene expression: From gestational day 11.5 through day 18.5
Apoptosis and cell proliferation detection: From gestational day 11.5 through day 18.5
3-MCPD and beta-chlorolactic acid levels in dam plasma and in whole fetuses: Day 14.5 postcoitum

Frequency of treatment:

Testicular gene expression: Daily
Apoptosis and cell proliferation detection: Daily
3-MCPD and beta-chlorolactic acid levels in dam plasma and in whole fetuses: Single oral dose

Duration of test:

Testicular gene expression: Pregnant rats were sacrificed on day 19.5 postcoitum.
Apoptosis and cell proliferation detection: Animals were allowed to deliver and assays were performed on 3 to 5 days-old testes.
3-MCPD and beta-chlorolactic acid levels in dam plasma and in whole fetuses: Animals from each group were sacrificed at 30 min, 1, 2, 3, 5 or 8 h after treatment.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 10 or 25 mg/kg bw (Testicular gene expression)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 or 25 mg/kg bw (Apoptosis and cell proliferation detection)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0 or 25 mg/kg bw (3-MCPD and beta-chlorolactic acid levels)
Basis:
actual ingested

No. of animals per sex per dose:

Testicular gene expression: 5 - 6 pregnant female rats per dose
Apoptosis and cell proliferation detection: 5 pregnant female rats per dose
3-MCPD and beta-chlorolactic acid levels in dam plasma and in whole fetuses: 21 pregnant rats (in groups of 3 animals each)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on preliminary experiments, doses of 3-MCPD greater than 50 mg/kg induced an excess of maternal and fetal toxicity. Therefore, exposures ranging from 5 to 25 mg/kg BW were chosen for the current study.

- Groups of 3 to 4 pregnant rats were also treated following the same experimental design and testes from male fetuses were used for the measurement of testosterone levels.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data
- Cage side observations: Mortality

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 19.5 postcoitum by CO2 inhalation
- Organs examined: Plasma was analyzed using gas chromatography for 3-MCPD and beta-chlorolactic acid.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of resorptions: Yes

Fetal examinations:

Testicular gene expression: For RNA analysis, testes from male fetuses of the same litter were pooled. Additional testes were collected at 19.5 days postcoitum for histology. Testes from a further group of male fetuses were used for the measurement of testosterone levels. Sex ratio and body weights of fetuses were also obtained.

Apoptosis and cell proliferation detection assays were performed on 3 to 5 day-old testes.

3-MCPD and beta-chlorolactic acid levels: The placental/fetal units were removed from the uterus and the fetuses were separated from the placenta, sampled and weighed. Fetal tissues were analyzed for 3-MCPD and beta-chlorolactic acid.

Statistics:

The data are presented as the mean +/- S.D. Statistical analysis of data was performed by ANOVA, and pairwise comparisons were made using the Dunnett’s test. A p-value of 0.05 or less was determined to be statistically significant.

Indices:

Not applicable

Historical control data:

Not applicable

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Statistically significant decreases in mean body weight gain were observed in pregnant rats treated with 10 mg/kg BW (37%) or 25 mg/kg BW (59%) of 3-MCPD from days 11.5–18.5 of gestation. The body weight gain of pregnant females was also reduced during the treatment period in the 5 mg/kg BW group (11%), but the difference from controls did not reach the level of significance. No death or resorptions were noted.

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

> 5 - <= 10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment related differences were noted with regard to the sex ratio or the body weights of fetuses from females that were dosed with 5, 10 or 25 mg/kg BW of 3-MCPD.

Effects on testicular morphology and on germ cell apoptosis and proliferation: The testicular morphology of fetuses at 19.5 days postcoitum or 3–5 day-old neonates exposed in utero to 3-MCPD was comparable to the concurrent control groups. Neither the proliferative activity, nor the apoptotic process was affected in the testes of neonates from dams that were treated with the highest dose of 3-MCPD. One to four germ cells per seminiferous tubule were stained with anti-BrdU antibody, and 0–2 apoptotic germ cells per tubule were observed in control and treated neonate rats.

Effect on testosterone levles in fetal testes: Intratesticular testosterone levels were not altered in testes exposed in utero to 25 mg/kg of 3-MCPD when compared to untreated testes. Secreted testosterone in testes exposed in utero to 25 mg/kg of 3-MCPD and measured in a culture medium
after 3 h of incubation were not statistically different from those secreted by untreated testes. In addition, 3-MCPD-exposed testes retained their capacity to respond to hCG stimulation as evidenced by significant enhancement (p < 0.05) of the testosterone levels in the culture medium.

Effect on testicular gene expression in the fetus: The RT-PCR band intensity for genes encoding markers of Leydig cells, such as 3-HSD, StAR and ER, indicated no differences in gene expression between the control and 3-MCPD-exposed fetuses. No changes were observed in the levels of Sox-9 or FSHR, markers reflecting Sertoli cell functions. AhR, the transcription factor that potentially regulates/interferes with StAR, was also examined, but its expression was not affected following 3-MCPD administration. Gene encoding markers of peritubular cells (NGFR), Sertoli and germ cells (DMRT-1) remained unaffected by the treatment. In addition, after administration of a higher dose of 3-MCPD (50 mg/kg BW), the expression of additional genes such as ER (Sertoli and germ cells marker) and Dhh (Sertoli cells marker) were also determined. None of the markers had its expression altered by this dose of 3-MCPD.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

> 25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

> 25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Determination of 3-MCPD and beta-chlorolactic acid (the main metabolite of 3 -MCPD) in dam blood and whole fetus: 3-MCPD and beta-chlorolactic acid were detected in maternal plasma and fetal tissues at all sampling times between 30 min and 5 h after administration. However, 5 h after administration, the plasma and tissue levels of 3-MCPD were under the LOQ (2 ug/g). The Cmax of 3-MCPD in dam plasma (16.4 ug/g) and in fetal tissues (14.3 ug/g) was reached 30 min and 1 h after administration, respectively. The Cmax of beta-chlorolactic acid was observed 1 and 2 h after administration in dam plasma (10.0 ug/g) and in fetuses (9.5 ug/g), respectively. The mean fetus-to-dam plasma partition coefficients (using the ratio AUC0-3 h,fetus to AUC0-3 h,plasma for 3-MCPD and the ratio AUC0-5 h,fetus to AUC0-5 h,plasma for beta-chlorolactic acid) were 0.93 and 1.08 for 3-MCPD and beta-chlorolactic acid, respectively. The MRT0−3h of 3-MCPD was similar in plasma and fetus (1.26 and 1.31 h, respectively). The MRT0−5h of beta-chlorolactic acid was slightly higher in fetus than in plasma (2.32 and 2.06 h, respectively). Apart from a slight difference in Tmax, dam plasma and fetal tissue kinetic parameters were broadly similar for both compounds.

Applicant's summary and conclusion

Conclusions:

The results show a statistically significant decrease in the mean body weight gain of pregnant rats treated with 10 and 25 mg/kg BW of 3-MCPD. Fetal testes exposed to 3-MCPD at doses up to 25 mg/kg bw/day exhibited normal histology and produced testosterone at levels that were similar to controls. In addition, 3-MCPD did not alter gene expression in the fetal testes at doses up to 25 mg/kg bw/day. This lack of effect occurred under conditions where 3-MCPD and beta-chlorolactic acid (the main metabolite of 3-MCPD) were found to readily cross the placental barrier and diffuse throughout the fetal tissues. Under the conditions of this study, 3-MCPD has minimal effect on rat testicular organogenesis.